The Centrosome and Its Role in the Organization of Microtubules

INTERNATIONAL REVIEW OF C Y I O L O G Y . VOL.. loh

The Centrosome and Its Role in the Organization of Microtubules I . A.

VOROBJEV A N D

E.

s. NADEZHDINA

A. N . Bidoziwky Ltrhortitoty oj’Mokiwrlmr Biology Litid Bioorganic ChiJniistry, Moscow State University, M o s c m i 119899, USSR

I. Introduction The present review deals with the centriole and associated structures. We shall consider its tine structure and function in the cell. By the function of the centrosome, we imply the organization of microtubules (MTs), for no other functions have been detected as yet (even though they are presumed). A number of reviews have already been published on the subject (de Harven, 1968; Brown et al., 1983; Fulton, 1971; Mclntosh, 1983; Peterson and Berns. 1980; Pitelka, 1974; Stubblefield and Brinkley, 1967; Tucker, 1983; Wolfe, 1972) and even a monograph has appeared (Wheatley, 1982), along with a large number of reviews and mini reviews on contiguous problems, such as the role of MTs in cells, assembly of MTs and its regulation, etc. However, the past few years have yielded many important experimental data directly related to the topic indicated in the title. These data prompt us to query some of the earlier notions. We have tried to emphasize these new data by comparing them both with the well-known facts and with those which have received less attention, though they might have been obtained long ago. At present, various investigators use different terms to denote the same structures. So to avoid confusion, we shall define our terminology first. Pickett-Heaps (Pickett-Heaps, 1969) formulated the concept of a microtubule-organizing center (MTOC). According to this concept, MTOCs are the structures from which or on which MTs start their assembly. Subsequent studies have revealed that the centrioles and the structures surrounding them (the ccntrosome) operate as MTOCs (we shall not consider the role of kinetochores in the organization of MTs). Electron microscopy has shown that MTs entering the cell center may be attached there to electron-dense structures-the “microcenters.” The size of the microcenters correlates with that of the MTs. Therefore, Borisy and Could (1978) designated these microcenters as microtubule-nucleating centers (MTNCs). For clarity, we shall henceforth use these two terms: MTOC-a center, mainly observed in a light microscope by using the immunofluorescence 227 Copyright 0 1987 by Academic Press, Inc. All righla of reproduction in any form reberved.

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technique with antitubulin antibodies. It is the area of cytoplasm from which MTs grow. MTNC-an electron-dense body (usually too small to be visible in a light microscope) to which MTs are directly attached. We shall apply the term “centrosome” to centrioles and specific structures surrounding them (MTNC in particular) located in a definite region of the cytoplasm-devoid of ribosomes and membranes. The definition of “centriole” does not appear to cause controversy. The centriole is a cylindrical structure in the cytoplasm whose wall consists of MTs and interconnecting structures. A similar structure, lying near the plasma membrane and forming a kinocilium, is called a basal body. The main part of the centriole and basal body is the centriolar cylinder, the radially symmetric structure consisting of nine interconnected triplets of MTs. But it must be stressed that the centriolar cylinder is not an equivalent of the centriole or the basal body, because the latter two organelles may contain some other important structures (sattelites, connectives, etc.) We shall use the term “pericentriolar satellite” the way Bessis (1964) understood it; he was the first to describe satellites as dense “massules” attached to the centriole. It would be premature to define other centriolerelated structures; their description will be given later. The centrosome can organize MTs in four different patterns (Fulton. 1971):as a mitotic spindle, as an interphase network, as axonemes (ciliary or flagellar), or as a new centriole (basal body). We shall concentrate below on the role of the centrosome for assembly of MTs in mitosis and in interphase, since it is in this field that the greatest progress has been made in the last few years. Other completely different functions are attributed to centrioles: perception of external signals (Albrecht-Buehler, 1981), regulation of cell metabolism (as a “pacemaker”) (Bornens, 1979), etc. These speculations may contain some rational grains, but, at the present time, their discussion appears premature to us due to the lack of experimental data for or against them. Yet it becomes clear that the role of centrosomes in the cell is not limited to a mere formation of MTs. Organizing the MT network, the centrosome is involved in creating a spatially organized asymmetric form of the cell (Solomon, 1980; Tucker, 1983; Mitchison and Kirschner, 1984a). Centrioles perform other functions (to be considered in the final section) that can hardly be related to MT organization. It is not accidental that the centriole has been named as the central enigma of cell biology (Wheatley, 1982). We have tried our best to adhere to facts only with a minimum of speculations. Our review considers a variety of subjects: the ultrastructure of centrioles and MTNCs, the genesis of centrioles and basal bodies in cells,

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the biochemistry of centrioles, its role in the assembly of MTs, and its properties as an particularly independent organelle. 11. Ultrastructure of Centrioles and Basal Bodies

The fine structure of centrioles and basal bodies was described in the 1950s, at the height of electron microscopic investigations of cell organelles (de Harven and Bernhard, 1956; Burgos and Fawcett, 1956; Bessis and Breton-Gorius, 1957; Yamasa, 1956; Amano, 1957; Fawcett and Porter, 1954; Manton et al., 1956). As it became clear from the very first works, the ultrastructure of these organelles is quite similar: this fact confirmed the Henneguy and Lenhossek hypothesis on their homology. New circumstantial and substantive descriptions appeared in the 1960s (reviews: de Harven, 1968; Fulton, 1971; Wolfe, 19721, after the introduction of aldehyde fixation (Sabatini et al., 1963). Centrioles proved to be relatively constant in form and size: they are cylindrical structures, about 0.2 pm in diameter and 0.2-0.5 pm in length. Basal bodies have the same diameter as centrioles, but they are somewhat longer. In some cases (spermatids of insects), a basal body may be as long as 8 pm (Friedlander and Wahrman, 1970). Most of the information on the fine structure of both centrioles and basal bodies may be obtained from the ultrathin sections perpendicular to the centriolar cylinder axis (Fig. 1). In this case, the structure itself turns to be radially symmetric (it has a symmetry axis of the ninth order), which makes it possible to obtain rotation images according to the method of Markham and co-workers (Markham et d.,1963). These images show the radial-symmetrical structures in greater contrast against the nonsymmetrical details of the negative. The first three-dimensional centriole model based on rotation images was suggested in 1967 (Stubblefield and Brinkley, 1967).The reconstruction of a basal body on the basis of a complete series of cross-sections was accomplished in 1972 (Anderson, 1972) (Fig. 21, and a similar reconstruction of the centriole was accomplished in 1980 (Vorobjev and Chentsov, 1980) (Fig. 3). The ultrastructure of centrioles and basal bodies abounds in minute details; unfortunately, these details have received no comprehensive and systematic description. Here we shall not enumerate all the observations bearing on the subtle structural features of centrioles and basal bodies, for this in the main has been done in previous reviews (Stubbletield and Brinkley. 1967; Fulton, 1971; Wolfe, 19721, but rather we will consider an overall centriolar structural model and structural distinctions of centrioles and basal bodies.

FIG. I . (A-C) Serial sections of the centriole in an interphase pig embryo (PE) cell. Sections 1, 5 , and 6 (first, proximal; sixth, distal end). ap. Appendages; con, connectives (A-C links); h, hub; rl, ring of links (A-A links);tb, triplet bases; st. pericentriolar satellite. Bar, 50 nm.

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FIG.2. Model of the basal body. (Reproduced from The Jorrrnd c$Ce// Eicdogy, 1972. 54, 260 by copyright permission of the Rockefeller University Press and courtesy o f R. G . W. Anderson.)

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A FIG.3. Model of the centriole from a mammalian cell. (From Vorobjev and C h e n t w v .

1980.) ( A ) Three-dimentional reconstruction. (B-D) Cross sections of proxinial. middle. and

distal parts of the centriole, respectively.

Already Bernhard and de Harven (1960) demonstrated that the centriole is a polar structure, i.e., it has nonequivalent ends. A daughter centriole is formed at one end, while a ciliary axoneme grows from the other. Since in basal bodies the end from which the cilium grows is oriented to the outer surface of the cell, it has been named distal. In centrioles, the distal end is one from which a stereocilium or a primary cilium grows. Correspondingly, the end where the founding of a daughter centriole takes place is named proximal. The plus ends of centriolar microtubules are at the 1974). A three-dimensional distal end of the centriolar cylinder (Snell r t d., reconstruction of centrioles and basal bodies revealed differences in their fine structure along the cylinder; these differences followed a regular pattern from one end to the other (Stubblefield and Brinkley. 1967: Anderson, 1972; Vorobjev and Chentsov. 1980). A general description of the centriolar

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ultrastructure in the cells of vertebrates proceeding mainly from the data of Vorobjev and Chentsov (1980) follows. The list of other references is given at the end of the description. The most conspicuous component of the centriole is nine MT triplets stretching along the full length of the centriolar cylinder. The slope of the triplets to the radius decreases from 70-80" to 50-55" from the distal on to the proximal end; the diameter of the internal lumen of the centriolar cylinder does not change, and its external diameter at the proximal end increases somewhat. The middle and outer microtubules of the triplets may have an incomplete wall at the distal end, and the outer MTs may not be present at all. The data in the literature on centrioles with double MT are usually based on photographs obtained from sections of the centriolar cylinder's distal part. The triplets of MTs are interlinked by different kinds of connectives: in the proximal part, these are the connectives between the inner and outer MTs of neighboring triplets, and in the distal part, they form a continuous ring linking the inner MTs of all the triplets. The photographic images of the system of connectives may create an illusion of a fourth MT adjacent to the triplet, which is not the case. In the lumen of the centriolar cylinder closely to its distal end, is found an amorphous hub. This hub is attached by its short protrusions to the ring of connectives linking the triplets. The protrusions are not organized in a strictly symmetric pattern. At the distal end ofthe centriolar cylinder, the hub is replaced by accumulations of electron-dense material. This material may be girdled by a 5-nm-thick fiber in the form of an incomplete circumference. Sometimes, the hub also occupies the distal part of the centrioles. In the proximal part, the lumen of the centriolar cylinder is usually free of the electron-dense material. The proximal part of the cylinder has triplet bases located along the triplets, inside the cylinder; in the middle part and at the distal end, there remain only columns of electron-dense material at the junctions of the inner and middle MTs of the neighboring triplets. Along the full length of the centriole, the triplets are surrounded from the outside by a "rim" of amorphous material. the matrix. The matrix is most clearly distinct in the proximal part of the centriole. The matrix is a constant centriolar Component persisting during the whole cell cycle. It differs from the mitotic halo by higher electron density and smaller diameter. There exists yet another structure, which may be located in the luman of the centriolar cylinder, in its proximal part. This is the pinwheel (for a detailed description, see Section 111). In contrast to the amorphous hub. the pinwheel is a strictly radial-symmetric structure, and it has adorned

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many rotation photographs. Yet in vertebrate cell centrioles, the pinwheel occurs only at their initial formative stages and disappears later. It may remain in mature organelles in the fungi and mosses, and in some invertebrates, insects in particular. The distal end of the centriole has appendages attached to the triplets from the outside. The appendages are not an obligatory centriolar component (thus they may be absent in some of Chinese hamster cell lines), but they are common for most cell types. Their position does not follow a completely regular pattern with respect to the axis of symmetry, and therefore, appendages are weakly contrasted on rotation images. Should the centriole form a stereocilium or a primary cilium, its appendages are practically symmetric, the way they are in basal bodies (see below). Immature daughter centrioles do not have appendages (see Sections 111 and IV), and so only one of the two centrioles in an interphase cell carries them, while the other does not. [Triplet MTs have been described by Ringo (1967). Ross (1968), Brinkley and Stubblefield (1970), Heath and Greenwood (1970), Kiefer (1970), Wolfe (19701, and McNitt (1974). The hub, triplet bases, and triplet interconnectives have been described by Dahl(1963), Murray et al. (1965), Dingle and Fulton (1966), Stubblefield and Brinkley (l967), de Harven (l968), Perkins (l970), Konishi et al. (l973), and Rattner and Phillips (1973). Appendages have been described by Cachet and Thiery (1964), Murray et af. (196% Doolin and Birge (1966), and Stubblefield and Brinkley (1967).] Besides the structures listed above, one may come across others, i.e., nonobligatory ones, in the lumen of the centriolar cylinder; these are the membrane-bound vesicle or dense granule (Sorokin, 1962; Doolin and Birge, 1966; Vorobjev and Chentsov, 1977). In these cases, the contents of even two centrioles in a cell are different. Unfortunately, the fine structure of centrioles has been studied only for a small number of plant and animal species and more often than not. the data relate to spermatogenic cells alone, where the ultrastructure may exhibit considerable aberrations (see below). Yet, even n from the available scant evidence, it is clear that the centriolar structure may vary for different species. The known distinctions in the centriolar structure may pertain to the structure (the length of the amorphous hub, the length of the unclosed part of triplets of microtubules, to certain details in the organization of the system of connectives, the presence or absence of appendages) of the centriolar cylinder’s distal part. The structure of the cylinder’s proximal part is more conserved. There exist two variants only, with an empty lumen or with a pinwheel connected by the radial spokes to the triplets. It should be emphasized once again that the basic centriolar structure, a cylinder composed of 9 MT triplets is the most universal one, and ab-

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errations are exceptionally rare. Centrioles, composed of 60-80 single MTs, were described for spermatogenic cells of the fly Sciuru (Phillips, 1967) and so were the centrioles and basal bodies of the coccids Eirnerci intrstitiu/i.s and Eirneru mugnu, consisting of nine single MTs (Chesin. 1967). Nevertheless, somatic cells of many plants, fungi, and of some animals may contain MTOCs other than centrioles. Usually these are balls, disks. or plates of electron-dense material (Friedlander and Wahrman. 1970; Zickler. 1970: Aist and Williams, 1972; Franke and Reau, 1973; McDonald t>t d . , 1977; Gifford and Larson, 1980; Dave and Godward, 1982). Zoospores or spermatozoa with flagella in such species may have centrioles (basal bodies) of usual structure (Renaud and Shift, 1964; Reichle, 1969; Dingle and Fulton, 1966). If flagella or cilia are never formed, e.g., in angiosperms, diatoms, or higher fungi, then the centrioles are not observed. Pickett-Heaps (1969) suggested that the centriolar cylinder is only a template for casting the axonemes of cilia and flagella, i.e., it is a potential basal body incorporated within MTOC for equivalent distribution among daughter cells in mitosis. In a way, the Pickett-Heaps’ hypothesis develops the Henneguy and Lenhossek suggestion on the homology of centriole and basal bodies (Henneguy, 1898; Lenhossek, 1898). Indeed, centrioles in vertebrate cells may form a primary cilia. Such cilia are partially or completely immersed in the cytoplasm; they may be several micrometers long or quite short. Only one centriole of the pair (the mother centriole) is capable to form a cilium (Bernhard and de Harven. 1960; Sorokin, 1962; Albrecht-Buehler and Bushnell, 1979; Rieder and Borisy, 1982). A cross section of primary cilia may show a random disposition of MTs, even though they may form something like type 9 + 0 axonemes (Jensen et al., 1979; Albrecht-Buehler and Bushnell, 1979; Vorobjev and Chentsov, 1982). At first, these cilia were considered a curiosity belonging to certain cell types (Barnes, 1961; Sorokin, 1962; Dahl, 1963; Doolin and Birge, 1966; Allenspach and Roth, 1967; Dingemans. 1969; Rash et d., 1969; Wheatley, 1969; Fonte et u/., 1971; Chung and Keefer, 1976). Yet it is clear now that this phenomenon is a rule rather than an exception especially for cultured cells. The primary cilia disappear from cultured cells before mitosis or at its beginning (Fonte et a / . , 1971; Archer and Wheatley. 1971; Rieder el d.,1979; Tucker et ( I / . , 1979a). The disappearance of primary cilia may occur immediately after the cell receives a proliferation signal (Tucker rt d . , 1979b; Lockwood and Pendergast, 1980). Probably, the primary cilia are most typical of cells in the G,,period. Centrioles are also responsible for the formation of single stereocilia of sensory cells (von Narnack, 1963; Flock and Duvall, 1965; Schmidt, 1969; Wolff, 1969; Tilney Pt a / . , 1980). In stereocilia as in primary cilia, axonemes may be significantly modified or may be absent altogether (Tilney et d . ,

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1980; Matera and Davis, 1982), but they may be of a regular structure as well (Wolff, 1969). As a stereocilium is formed, the pair of centrioles migrates to the cell surface; one (the mother centriole) attaches to the plasmalemma and forms an axoneme, while the other (the daughter centriole) lies nearby and has no axoneme (Bernhard and de Harven, 1960). In either case, the centrioles contact the membranes (the plasmalemma if a stereocilium is formed, and the intracellular vesicle if it is a primary cilium). Their appendages become more sturdy and symmetrical and are attached to the membrane (Vorobjev and Chentsov, 1982). Centrioles lying at the base of stereocilia are traditionally regarded as basal bodies, and this is a clear example for tracing the homology of these two organelles. Sometimes (for example in sea urchin blastomeres) the kinocilia are formed in the same way as stereocilia: their basal bodies simultaneously act as a pair of centrioles, which are the only MTOCs of these cells (Tilney and Goddard, 1970). Both centrioles and basal bodies are not the obligatory cell organelles. Although they are widespread in eukaryotes, there are organisms whose cells are devoid of them (angiosperms). In some cases, the cells may have only basal bodies and no centrioles (for example, Flagellata and Infusoria). Others, however, have only centrioles and no basal bodies (some fungi and mosses). Only vertebrates, insects, and echinoderms reliably have both these organelles. With animals, centrioles are present in most of the cells. Basal bodies are present only in special cases: in multiciliate cells of ciliated epithelia, in single-cilium sensory cells, and in spermatozoa. The centrosome restructuring during spermatozoon flagellum formation is an independent area of research to which a large number of workers have been devoted (reviews: Phillips, 1970; Danylova, 1982). Spermatozoa are the example of the cell’s uttermost specialization which applies to all the intracellular structures without exception. Changes of the centrioles in spermatozoa and the structure of the basal bodies thus obtained are highly variable and species specific. Frequently, the spermatozoon basal body is replaced by oddly packed electron-dense material (Phillips, 1970). The question is to what extent the changes in the basal bodies of spermatozoa are reversible and what happens to them after fertilization (in the zygote); we shall consider this in Section VIII. On the whole, however, we shall try not to refer to works on spermatogenesis and spermatozoa because of the highly specific nature of the subject. So, the homology and structural similarity of centrioles and basal bodies may be of practical interest only in the case of ciliated epithelia of different animals (vertebrates and invertebrates). Basal body triplets of MTs, unlike centriolar ones, are hardly detectable

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on cross-sections simultaneously. To explain this phenomenon, it was suggested that the triplets are coiled axially in the centriolar cylinder (Gibbons, 1961; Fawcett. 1966; Phillips, 1970; Anderson, 1972). As in the centriole, the lumen of the basal body cylinder may have an amorphous hub at the distal end (Reese, 1965; Anderson, 1972). or a pinwheel at the proximal end (Friedlander and Wahrman, 1970), but it does not have membranebound vesicles and a fine fiber in the distal part, which are often described in centrioles. The system of triplet connectives has not been studied in basal bodies in such detail as in centrioles. The total length of the basal body exceeds, as a rule, that of the centriole (Wolfe, 1972). To some extent, this is due to a special transition zone, the necklace, which, in basal bodies, is located at the distal end after the appendages (Thornhill, 1967; Gilula and Satir, 1972). Only doublets remain in the transition zone instead of the triplets of MTs (the triplet's outer MT is absent); these doublets are arranged strictly in a circle. Each one is attached to the plasmalemma via a system of connectives. A basal plate is found in kinocilia, above the transition zone, to separate the basal body from the axonema (Perkins and Amon, 1969; Wolfe, 1972; Rubin and Cunningham, 1973). The basal body appendages (often designated in the literature as transition fibers) diverge from the outer surface of triplets and are attached to the membrane (Szollosi, 1964; Reese, 1965; Anderson, 1972). They are arranged strictly symmetrically around the centriolar cylinder of the basal body and are similar in size to the appendages of centrioles (the basal body appendages may be somewhat larger). In the proximal part, striated rootlets diverge from the basal body (one or several in different cell types). Each rootlet is a bundle of thin (about 5 nm) fibers with transverse periodic striations. In multicellular animal cells, the striation period of the rootlets is equal to 60-70 nrn (Dahl, 1963; von Narnack, 1963; Doolin and Birge, 1966; Matsusaka. 1967; Schmidt, 1969; Olson and Rattner, 1975; Stephens, 1973, and in Protozoa, the striation period of the rootlets is equal to 12-78 nm (Dingle and Fulton, 1966; Dingle and Larson, 1981; Simpson and Dingle, 1971; Brown et d., 1976; Amos et a / . , 1979; Larson and Dingle, 1981; Salisbury, 1982):Striated rootlets around centrioles have been described as well (Sakaguchi, 1965; Loweryns and Boussaw, 1973; Wachmann and Hennig, 1974; Vorobjev and Chentsov, 1977). They are much thinner than those of basal bodies; as a rule, they are not directly linked to the centriolar cylinder. On the contrary, the basal bodies are immured into a rootlet by their proximal end (Anderson, 1972). The number of pericentriolar rootlets varies; there may be as many as 10 and more per centriole (Loweryns and Boussaw, 1973).

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In many cells, the centrioles are surrounded by dense masses attached to them, i.e., the pericentriolar satellites (Bessis et a l . , 1958; Bernhard and de Harven, 1960; David-Ferreira, 1962; Bessis, 1964; Vorobjev and Chentsov, 1977). Satellite counts on serial cross sections of centrioles in axolotl hemopoietic tissue cells point to the variable number of satellites (from two to thirteen, and more often, from four to seven); they may be located along the full length of the centriolar cylinder (Vorobjev and Chentsov, 1977). Tissue culture cells contain fewer satellites (one to five per centriole), but their number varies as well (Vorobjev and Chentsov, 1982). The satellites are composed of a head which serves as a MT-nucleating center (MTNC), and of a foot, fixing the head to the centriolar cylinder (Bessis et al., 1958; Dalcq, 1964; de The, 1964; Szollosi, 1964; Vorobjev and Chentsov, 1977). The satellite foot is conical in shape, and it often exhibits transverse striation. The satellite is attached to two or three triplets of the centriolar MTs (Szollosi, 1964; Vorobjev and Chentsov, 1977).

Satellites are found in basal bodies as well. Commonly designated as basal feet, they have the same structure as the centriolar satellites. The basal bodies and the centrioles differ in the number of satellites. Most of the investigated basal bodies in ciliated epithelia contain only a single satellite (Fawcett and Porter, 1954; Gibbons, 1961; Szollosi. 1964; Anderson, 1972; Afzelius, 1980). The basal body satellites are positioned in a definite manner-in the cilium effective stroke plane (Gibbons, 1961; Afzelius, 1980). Thus, all the satellites are oriented in the same direction within a cell (Gibbons. 1961; Afzelius, 1980). Under diseases resulting in immotile cilia syndrome, the satellites of ciliated epithelium cells show random disposition (Afzelius, 1980). So, unlike the centrioles, the basal bodies have a bilateral symmetry, sometimes, a functionally conditioned one. Unfortunately, no comparative studies have been made on the structure of centrioles and basal bodies taken from the same object. Nor is it known how the structure of a centriole forming a single cilium changes (besides the changes in the position of appendages). The available data on the ultrastructure of centrioles and basal bodies enable us to make the following important conclusion: in the cells of multicellular animals, the basal body is an organelle with a more narrow specialization than that of the centriole. The centriole may have all the basal body structures, but the variability of the centriolar structure within one and the same organism is higher than that of the basal bodies. So, we conclude that the centriole in the cells of multicellular animals is a primary structure capable of different functional restructurings, while the basal body is just the result of one of them. This presumption is largely at var-

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iance with the Pickett-Heaps’ hypothesis about centriolar “passivity.” (Pickett-Heaps. 1969). It should be noted that in Protozoa, too, the organization of basal bodies is rigidly determined to a much greater extent than the organization of the centriolar apparatus of multicellular organisms. Possibly, the rigid organization of basal bodies is due to the necessity of spatial orientation of kinocilial beating. The ultrastructure of centrioles and basal bodies may reveal mutant deviations. Thus, in certain lines of Chinese hamster cells, the centrioles lack some of the triplets (McGill el al., 1976; Vorobjev, 1979); triplets of MTs, different in length, are found in M I 0 green monkey cells (1. A. Vorobjev, personal communication), etc. Yet, no centriolar structure in mutant cell lines have been obtained so far. Chlamydomonas mutants may exhibit motility disturbances and changes in the connectives linking the basal bodies, but they reveal no changes in the basal bodies themselves (Wright et al., 1983). So, mutations in the centriolar structure are either lethal or affect its functions and thus cannot be analyzed.

Ill. The Ontogenesis of Basal Bodies and Centrioles The principal stages of the formation of centrioles and basal bodies are much similar for different groups of eukaryotes. New centriolar cylinders are formed in four modes: I . Next to the preexisting centrioles, - end-to-end and in direct contact with them (Heath and Greenwood, 1970; Moser and Kreitner, 1970; Heath, 1974); 2. Near and perpendicular to the lateral surface of the mother centriole or the basal body (Bernhard and de Harven, 1960; Gall, 1961; Murray et d.,1965; Sorokin, 1968; Allen, 1969; Millecchia and Rudzinska, 1970, etc.). Although this process is traditionally named replication, it has nothing in common with DNA replication; 3. Around the aggregations of fibrogranular material, the deuterosomes (Stockinger and Cireli, 1965; Dirksen and Crocker, 1966; Steinman. 1968; Sorokin. 1968; Dirksen, 1971) or blepharoplasts (Mizukami and Gall, 1%6). Both, in the second and in the third case, the daughter structures (procentrioles) seems not to be directly linked either to the mother centrioles or to the deuterosomes; 4. Finally, centrioles may be formed de novo, when the nascent centriolar cylinders are not associated with any special intracellular structures.

Dr now formation of centrioles has been described for sea urchin eggs both for parthenogenesis and for artificially activated eggs (Dirksen, 1961;

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Koichi and Masao, 1971 ; Kato and Sugiyama, 1971; Miki-Noumurd, 1977; Kallenbach and Mazia, 1982), and it has also been described for dividing mouse blastomeres (to a stage of 8-16 blastomeres) (Szollosi rt d.,1972). Probably, it takes place under karyoplast regeneration (Zorn rt (11.. 1979). An attempt at a more in-depth analysis into the finding of centrioles during their de n o w formation yielded no definite result (Kallenbach, 19831, i.e.. it was not possible to identify procentrioles at their early formative stages. The second mode applies to centriole and basal body formation in the cells of both multicellular animals and Protozoa; in some cases, the basal bodies may be formed by the third mode (in multiciliate cells as a rule). Ciliogenesis in multiciliate cells enables one to trace the formation of a multitude of basal bodies. In each cell, there are many centriolar cylinders at different formative stages; therefore, this process is easily and sufficiently well investigated (Reese, 1965; Steinman, 1968; Sorokin, 1968; Kalnins and Porter, 1969; Anderson and Brenner, 1971; Jirsova et d., 1974) (Fig. 4). At the earliest discernible stage, the basal bodies are shaped like short cylinders, 65-120 nm long and about 160 nm in diameter. The walls of the cylinders are composed of amorphous substance, and the inner lumen of the cylinder is equal to 80-85 nm. A cartwheel, about 30 nm in diameter with nine symmetric pins, appears in the lumen. Longitudinal sections show that the pins are located in rows, one over another. Then MTs appear at the pin ends (the inner or the A tubules of future triplets), parallel to the central axis. As the number of the MTs reaches nine (according to the number of pins), middle (B) and outer (C) tubules of the triplets are added in succession from the outside. Connectives are formed between the A and the C tubules of neighboring triplets. At this stage, the pin ends are interconnected by a solid electron-dense ring. The triplets in the juvenile basal body are oriented practically radially (Fig. 5). Later they turn around to form an angle of about 50 with the radius. Then the basal body cylinder is elongated, mainly at the expense of the growth of the triplet microtubules. The cartwheel grows to reach half of the length of a normal basal body (or even less). N o cartwheel is present, as a rule, in the mature basal bodies of multiciliate cells (see Wolfe, 1972, for review), but it may occur fairly often in basal bodies of the Protozoa (Allen, 1969; Tamm, 1972; Wright et al., 1979). The ultrastructure of replicating centrioles has been investigated in one work only (Vorobjev and Chentsov, 1982) (Fig. 5). On the whole, the centriolar foundation and growth resemble that of the basal bodies with the exception that the incipient stages, i.e., the amorphous cylinder and the cartwheel without MTs, are not detectable. The minimal centriolar structure includes both the cartwheel and the single or double MTs of future triplets attached to the pin ends. Such a procentriole is about 100

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FIG.4. A diagrammatic interpretation of the development of the basal body. (a) Earliest stage of formation. Neither the annulus, nor the cartwheel is completed. (b) The cartwheel is complete, and the first A tubule is completely initiated. (c) The triplet tubules are growing. The tubules tend to be progressively less complete from the base to the apex. (Reproduced from 7 7 1 Jorrrncrl ~ c~f’CdlBio/ogy, 1971. 50, 18, 22, 26 by copyright permission of the Rockefeller University Press and courtesy of R. G. W. Anderson.)

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FIG.5. Cross section of the procentriole. a, Axis; sp. spokes. Note incomplete triplets of MTs attached to the ends of the spokes (arrowheads). Bar, 50 nm.

nm in length and about 150 nm in diameter. The MTs are not quite symmetric about the central axis. Subsequently, complete MT triplets are formed, grow, and thereby cause centriolar cylinder elongation. The cartwheel grows to reach only half of the centriolar length at most. During the initial stages, the triplets are located almost on a radius, but then they turn through at an angle of 55-70'. The cartwheel disappears in a mature daughter centriole. The MT triplets become interconnected immediately upon their formation. Yet, the final system of triplet connectives takes shape only as the cartwheel has disappeared. The formation of mature basal bodies is completed upon their departure from the mother centriole (basal body) or from the deuterosome (Reese, 1965; Sorokin, 1968; Anderson and Brenner, 1971). Likewise, complete maturation of the centrioles occurs as the daughter centriole departs from the mother centriole (after the disruption of the diplosome) in a cell cycle subsequent to its foundation (Vorobjev and Chentsov, 1982; Rieder and Borisy, 1982). Thus, centriole and basal body formation is composed of three stages: foundation of the axial structure and MT triplets on it; elongation of the

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triplets and formation of a centriolar cylinder; and final restructuring of the centriolar cylinder, corresponding to the functional maturation of the organelle. Centriolar maturation consists of some structural changes. First, the cartwheel in the proximal part of the cylinder disappears and the amorphous hub appears in the distal part. The absence of a ring of connectives and the presence of a cartwheel are certain indications that the centriole is immature (in vertebrate cells). Yet in invertebrate cells, it may persist even in a mature centriole (Lutz and Huebner, 1980). Second, connectives between the inner MTs of the triplets are formed. Third, appendages appear at the distal end of the centriole. Prior to that, the daughter centrioles have only outer projections, ribs, along the triplets in the distal part. The appendages appear during the centriole’s second mitosis (Vorobjev and Chentsov. 1982). And finally, as we have noted above, the mature centriole is surrounded by MTNCs, i.e., pericentriolar satellites, mitotic halo, and free microtubular convergence foci. The immature centriole has few, if any, such structures around it. Centriole formation by the end-to-end replication mode has been described for some of the fungi and mosses (Heath and Greenwood, 1970; Moser and Kreitner, 1970; Heath, 1974). At the initial stage and in this case, the cartwheel outgrows the mother centriole. Then, at the pin ends, triplets of MTs of the daughter centriole are formed. However, the triplets of the mother and daughter centrioles are coiled in different directions, so the entire diplosome shows central symmetry. The triplets become elongated and form a centriolar cylinder. The cartwheel does not grow. As a result, the daughter centriole thus formed is connected with the mother via a common axis. The two centrioles diverge only as the cell prepares for subsequent division. In all the cases described, the radial symmetry of the centriolar cylinder is determined at its earliest formative stage: the cartwheel has a symmetry of the ninth order. The MTs formed at the pin ends may not be quite symmetric at first. The triplets become linked not only with the pins but with each other as well, and the structure of the centriolar cylinder attains to strict symmetry. It is suggested that the cartwheel symmetry is determined, in turn, by some finer structure within the wheel (Satir and Satir, 1964); but there is no evidence in favor of this suggestion. The morphological similarity of nascent centrioles and basal bodies does not mean, however, that the processes of their formation are set off and controlled by some common mechanisms. Quite the contrary, the regulation of centriolar replication is cardinally different from that of ciliogenesis. It has been proved that the duplication (replication) of centrioles sets

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is in the S period, though not necessarily at its beginning (Robbins et al., 1968; Wheatley, 1968; Erlandson and de Harven, 1971; Rattner and Phillips, 1973; Vorobjev and Chentsov, 1982). Replication of the two centrioles in a cell is strictly simultaneous, and so it may be touched off by a signal from a common source. Of major significance in this connection would be data on centriolar duplication in cells with multiple centrioles, but such data are poorly represented in the literature: we have found only two descriptions of possible simultaneous centriolar replication in young megakaryocytes and in regenerating liver cells (Wheatley, 1968; MoskvinTarkhanov and Onischenko, 1978). Elongation of the daughter centrioles is completed in mitosis, and their divergence takes place at the beginning of interphase. Complete maturation includes the whole subsequent cell cycle (Vorobjev and Chentsov, 1982; Rieder and Borisy, 1982). In cells retiring from the cell cycle in the G, period, no maturation of the daughter centriole occurs, i.e., it does not form appendages and can form no cilia (Vorobjev and Chentsov, 1982). Ciliogenesis in the monkey and rabbit oviduct is hormone induced (Anderson and Brenner, 1971; Anderson, 1974) and thus does not directly depend on the cell cycle. Replication of basal bodies in Protozoa is not coupled to DNA synthesis also (Younger et al., 1972). The basal body formation process is asynchronous within a single cell, i.e., one can frequently see basal bodies at different formative stages (Reese, 1965; Sorokin, 1968; Kalnins and Porter, 1%9; Anderson and Brenner, 1971). Thus centriolar replication is closely related to the cell mitotic cycle, while basal body formation is only related to cell differentiation. Unfortunately, we do not know whether the typical centrioles and basal bodies can coexist in one cell. Mammalian ciliated epithelium in which the cell multiplication zone is separated from the ciliary formation zone in space or in time, e.g., in the trachea or the oviduct, may not have such cells; earthworm olfactory epithelium has no centrioles in the ciliated cells (Rhein et al., 1981). Yet, it has been proved that ciliated cells of the epithelia of certain invertebrates can enter the mitotic cycle, i.e., they incorporate [3H]thymidine, and mitotic cells are found among them (Kaganovskaya, 1976; Punin, 1981; Zavarsin et al., 1984). Centrioles and basal bodies ought to coexist in such cells, and replication of both is of undeniable interest for study. IV. The Organization of the Centrosome and Its Behavior in a Cell Cycle

We shall consider centrosome dynamics in the cell cycle by beginning with metaphase, i.e., the key moment when one morphologically integral unit divides into two separate cells. In mitosis, the centrosome forms mi-

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totic spindle poles. In metaphase, each pole has a pair of mutually perpendicular centrioles, i.e., the diplosome (Robbins and Gonatas, 1964; Krishan and Buck, 1965; Murray et al., 1965; Roos, 1973). The mother and the daughter centrioles are clearly distinct in the diplosome (Murray et al., 1965; Robbins et al., 1968). The mother centriole has both ends free and is surrounded laterally by fine fibrillar material, the halo, which becomes particularly distinct upon detergent treatment of the cells. The halo is 150-200 nm wide, and the diameter of its fibers fluctuates between 3 and 8 nm. Most of the fibers are about 5 nm thick (Vorobjev and Chentsov, 1982). The second daughter centriole has its proximal end immersed in the halo. This centriole is at the proximal end of the mother centriole, perpendicular to its axis. The halo is a convergence focus of spindle MTs. In some cases, the MTs, radiating from the halo, may get into the lumen of the daughter centriole (Vorobjev and Chentsov, 1982). In metaphase, the daughter centriole is equal in length to the mother centriole or may be slightly shorter (Robbins and Gonatas, 1964; Krishan and Buck, 1965; Roos, 1973); the fine structure of the two centrioles is different (Vorobjev and Chentsov, 1982; see Section 111). In some cell lines (most frequently, in Chinese hamster cells), spherical electron-dense bodies, otherwise known as pericentriolar satellites, may surround the diplosome (Starosvetskaya, 1969; Gould and Borisy, 1977; the Peterson and Berns review, 1980; Rieder and Borisy, 1982). But, since these structures are not attached to the centrioles and do not make contact with the MTs, it would be wrong, we believe, to designate them as satellites. The chemical nature of the bodies is obscure. It is presumed they may be viral particles in some cases (Wheatley, 1974). In metaphase, the location of the mother centrioles in pig embryo (PE) cells and in Chinese hamster cells, as well as in PE cells in anaphase, follows a regular pattern, i.e., the centrioles are predominantly perpendicular to the spindle axis and this orientation is statistically reliable (Vorobjev and Chentsov, 1982). The daughter centrioles are scattered at random with respect to the spindle. The mother and the daughter centrioles retain their mutually perpendicular position throughout mitosis until mid telophase (Robbins and Gonatas, 1964; Krishan and Buck, 1965; Murray et al., 1965; Allenspach and Roth, 1967; Robbins and Jentzsch, 1969; Roos, 1973; Ates and Sentein, 1977; Vorobjev and Chentsov, 1982). According to some data, the fibrillar halo may be lost in anaphase (Robbins and Gonatas, 1964; Robbins and Jentzsch, 1969), or it may persist to telophase (Roos, 1973; Vorobjev and Chentsov, 1982). In all the cases, the number of MTs radiating from the mother centrioles successively decreases in the second half of mitosis, and only few MTs remain at the pole in telophase (Robbins and Gonatas,

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1964; Robbins and Jentzsch, 1969; Ates and Sentein, 1977; Vorobjev and Chentsov, 1982). The centrosome undergoes a profound restructuring during cell transition into interphase. First, the mitotic halo disappears, and the two centrioles retain only a thin rim of electron-dense material (the centriolar matrix), which surrounds them during the entire cell cycle and must be a structural component of the centrioles. Second, the mother and the daughter centrioles lose their mutually perpendicular orientation and the diplosome disintegrates. This may also occur late in the telophase (Rattner and Phillips, 1973). Moreover, the two centrioles may depart from each other as far as 2-3 p m (Rattner and Phillips, 1973; Vorobjev and Chentsov, 1982). After the initial departure of the centrioles early in the G, period, they come back together and, prior to mitosis, do not lie farther than I pm from each other. Thus, a single centrosome, comprising the two centrioles and the surrounding structures, is formed in most interphase cells. Yet the mutual disposition of the two centrioles in interphase has nothing to do with their disposition in mitosis, when a diplosome exists. In interphase cells, the two centrioles are not perpendicular to each other as a rule (Vorobjev and Chentsov, 1977; Gudima et al., 1983a,b; de The, 1964; Schaffer, 1969; Albrecht-Buehler and Bushnell, 1980; Rieder and Borisy, 1982) (Fig. 6). Pericentriolar satellites appear on the mother centriole. The heads of the satellites function as MTNCs. In addition to the satellites in interphase, many cell lines (3T3, CHO, PtK2, and L), as well as hemopoietic tissue cells and blood cells, contain dense convergence foci of microtubules not attached to the centrioles; these are so-called free MTNCs (Rattner and Phillips, 1973; Vorobjev and Chentsov, 1977; the McIntosh review, 1983). Both the satellites and the free MTNCs persist during most of the interphase and disappear as the cells prepare for mitosis (Erlandson and de Harven, 1971; Vorobjev and Chentsov, 1982). The other centriole usually has no satellites or other MTNCs in interphase; the mother centriole has several times more microtubules around it. Consequently, only one of the two centrioles is active in interphase in the same way as it is in mitosis. Dalcq (1964) was the first to point out the difference between the two centrioles in an interphase cell. One had ignored this distinction for a long time until a close study of the centriolar maturation process during the cell cycle made it possible to explain the phenomenon: the centrioles differ in interphase because one of them (the daughter centriole) has no appendages at this time. Its structure becomes completely identical only in the next mitosis (in proliferating cells).' The absence of appendages may ac'The differences between the two centrioles may or may not persist in nonproliferating cells (like neurons). This question needs special study.

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FIG. 6. Scheme of the centrosome in interphase mammalian cell. m, Mother (active) centriole with pericentriolar satellites; d. daughter (inactive) centriole; b. electron-dense bodies; s, striated rootlet. MTs radiate in the main from satellites and free MT convergence foci. Some M’I’ may be attached to the centriolar matrix.

count for the fact that the daughter centriole does not form a cilium (Vorobjev and Chentsov, 1982). The doubling (replication) of centrioles takes place in the S period. This fact, independently verified in a number of mammalian cell cultures, has in effect been proved (Robbins ef al., 1968; Erlandson and de Harven. 1971 ; Rattner and Phillips, 1973; Vorobjev and Chentsov, 1982). However, a more delicate question has yet to be answered: at what moment of the S period does centriolar replication set in? Probably, this event takes place at different times in different cell types. The finding of procentrioles is synchronous at both centrioles. They are found near the proximal end of the mother centrioles in direct contact with their matrix. These two, now mother, centrioles preserve their structural and functional differences in the S period and early in the G, period (Vorobjev and Chentsov, 1982; Rieder and Borisy, 1982): only one of them carries appendages and is capable of forming a cilium. This centriole also has pericentriolar satellites (though this does not apply to all the cell types). As the cell prepares for division, centrosome restructuring takes place (in the G, period). The pericentriolar satellites disappear and so do most of the MTs, radiating from the cell center. This happens in the middle of the G, period (Vorobjev and Chentsov, 1982). At the end of the G, period, numerous small membrane vesicles appear around the centrioles, and their number reaches a maximum in the late prophase. The vesicles disappear rapidly in prophase-prometaphase transition (Robbins et al., 1968; Vorobjev and Chentsov, 1982). Late in the G , period, MTs radiate from both mother centrioles and asters appear. The MTs are attached directly to the

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centriolar matrix. The matrix rim becomes larger, and it transforms into the mitotic halo, surrounding the mother centrioles. The well-defined mitotic halo is observed in late prophase. The daughter centrioles elongates during the G, period, and they may attain up to Y4 of the length of the mother centrioles toward the end of this period. The elongation of the daughter centrioles is completed in mid or late mitosis (Krishan and Buck, 1965; Stubblefield and Brinkley, 1967; Vorobjev and Chentsov, 1982). As shown recently, the structural differences between the centrioles of a dividing cell may persist as late as anaphase, i.e., only one centriole has appendages, while the other has no appendages (Vorobjev and Chentsov, 1982). So, the cell always has only one completely developed (mature) centriole. The centriolar formation process, beginning from the foundation, thus takes a 1 '/z cell cycle (Vorobjev and Chentsov, 1982) (Fig. 7). Consequently, in typical mammalian cells, MTOCs consist of a pair of centrioles surrounded by MTNC structures. In mitosis, there is one such structure, the mitotic halo. The pattern is different in interphase. In some cells, the centriolar surfaces, the matrix, act as interphase MTNCs; in others, the matrix is supplemented with pericentriolar satellites. Furthermore, MTOCs may include MTNCs without structural links to the centrioles. These MTNCs resemble dense granules or small balls, 30-70 nm in diameter (Stubblefield and Brinkley, 1967; Starosvetskaya and Kazanjev, 1977; Peterson and Berns, 1980). In other cells, the balls are interlinked by connectives of less dense substances (de The, 1964; Vorobjev and Chentsov, 1977, 1983). In either case, the MTs approach the balls only; and in the case involving satellites, the microtubules approach the satellite head. Finally, clouds of electron-dense fibrillar material with radiating MTs may be found in the centriolar region (Gould and Borisy, 1977; Schliwa et al., 1978, 1979). The latter case is most typical of fish melanophores in which as many as loo0 MTs may radiate from one MTOC (Schliwa et al., 1978). Cells without centrioles but with basal bodies may exhibit a highly diversified MTNC structure. The basal body satellite heads act as MTNCs (Tilney and Goddard, 1970) and so do the connectives between the basal bodies and their rootlets (Bouck and Brown, 1973; Hyams and Chasey, 1974; Wright et al., 1979; Roobol et al., 1982). In Protozoa, a pair of basal bodies may carry several rootlet-type MTNCs, with a definite number of microtubules radiating from each (Bouck and Brown, 1973; Brown et ul., 1976; Wright et al., 1979). In cells without basal bodies, various plates, disks, or electron-dense balls operate as MTOCs (reviews. Raykov, 1978; Heath, 1980). In all the cases, MTOCs undergo corresponding restructuring in the mitotic cycle.

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FIG. 7. Generd scheme of the centriole cycle in PE cells ( A ) Metatelophase, ( B ) the beginning of interphase. (C) G , period. (D)second half of the S period to the first half of the G , period. and ( E ) the end of the G, period to prophase,. (From The, Jorrrirtrl of’ Cell B i d ~ g . 1982, ~ , 93, 949 by copyright permission of the Rockefeller University Press.)

V. The Biochemistry of Centrioles and Basal Bodies

The present knowledge of the centriolar structure at the molecular level is strikingly poor, especially compared to what has been achieved with respect to such cell components as ribosomes or mitochondria. We do not even know the rough composition of centriolar proteins and MTNC material, let alone their molecular structure. We know very little about the chemistry of basal bodies, though the first steps in this direction have already been made. The reason for this lag lies to some extent in the insufficient attention given to the problem. Yet researchers confront formidable objective difficulties as well. The critical stage in the study of the biochemistry of any cell organelle is its isolation as an individual fraction. This stage has not yet been reached for centrioles, and only a few research teams have succeeded in obtaining basal body preparations suitable for biochemical in-

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vestigations (Snell et al., 1974; Gould, 1975; Anderson, 1977; Stearns and Brown, 1979; Anderson and Floyd, 1980). To isolate some component as a fraction, one begins by selecting an adequate model object, i.e., one readily available for experimental use and containing the required component in a sufficiently high concentration. Such objects are the heart muscle (for mitochondria) or the brain (for microtubules). After the isolation procedures have been practiced on the model object, it becomes possible to obtain the desired fraction from other sources as well. Yet this technique is not good for centrioles. The main obstacle for centriole isolation is that normal cells have only two centrioles. They make up about 0.01% of the average cell volume. So even if the centriolar yield is loo%, large quantities of the starting material are needed for obtaining a sufficient amount of centriolar fraction. No model objects have been found for centrioles as yet. Neuroblastoma-a cell culture with multiple centrioles-appears to hold promise in this respect (Sharp et al., 1981) and recently a centriolar preparation has been obtained from it (Ring et al., 1980; Mitchison and Kirschner, 1984a). To test fractions of isolated cell components, one determines the activity of marker enzymes or specific biological activity; one may also perform direct analyses to detect specific proteins or nucleic acids, etc. None of these procedures was suitable for detecting centrioles. Electron microscopy remained for a long time the only method for assaying the centriolar content in preparations. It is a labor-consuming procedure, which requires comparatively large amounts of material. This drawback made it difficult to apply various linear gradients or chromatographies for isolating centrioles. Certain headway has been made by using specific autoimmune antibodies against centrioles, i.e., in this case, it becomes possible to detect centrioles in fractions by immunocytochemical procedures (Maro and Bornens, 1980; Ring et al., 1980; Mitchison and Kirschner, 1984a). There is also some progress in evolving the assay for biological activity of centriole assembly of MTs in an asterlike pattern (see Section VI). Only a few communications on enriched centriolar preparations have been published (Blackburn et al., 1978; Nadezhdina et al., 1979; Mar0 and Bornens, 1980; Mitchison and Kirschner, 1984a); yet no pure preparation is available. All we have said about centriolar isolation pertains in a way to basal bodies. Yet, there is a good object for isolating basal bodies, i.e., the unicellular flagellated algae. Basal bodies make up a significant part of their small cells. Pure preparations of basal bodies were isolated from Chlamydomonas (Snell et al., 1974; Gould, 1975) and from Polytomella (Stearns et al., 1976). Spermatozoa seem to be likewise appropriate for basal body isolation, but preparations obtained from them are not free of

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contaminations (Maller er al., 1976; Ishikawa er al., 1979; Esponda and A h a , 1983). A major difficulty in isolating both centrioles and basal bodies is to separate the required organelles from the surrounding fibrillar structures. In the case of centrioles, these are mainly the intermediate filaments; in the case of basal bodies, these are mainly a complex of striated rootlets, linking fibers, and special connectives. It is only after several years of purposeful work that Anderson and Floyd succeeded in isolating a pure basal body fraction from ciliated epitchelium of rabbit oviduct (Anderson, 1974, 1977; Anderson and Floyd, 1980). No pure basal bodies have been isolated from Infusoria, though some crude preparations of cortex were obtained (Hufnagel, 1969; Gavin. 1980, etc.). The tight connections of centrioles and basal bodies with filaments may be useful at the initial stages of isolation. In softly homogenized cells, the centrioles remain bound to the nuclei (via the fibrillar structures, see also Section VIII) and may be isolated in a complex (Bornens, 1977; Nadezhdina et al., 1978; Mar0 and Bornens, 1980). The centriolenucleus complex can be used as an intermediate stage in centriolar purification, since in effect, all other cytoplasmic structures can be removed during its isolation. Furthermore, the centrioles can be separated from the nuclei upon additional homogenization and centrifugation (Nadezhdina er d.,1978, 1979; Maro and Bornens, 1980). In a similar way, cortex preparations of multiciliate cells may be an intermediate step in isolating basal bodies (Anderson, 1977). On the other hand, in order to avoid mechanical homogenization leading to disruption of MTNC substance, Ring et al. (1980) and Mitchison and Kirschner (1984a) proposed to lyse the cells in a very lowionic-strength solution. Such lysis releases the centrioles into the solution. Thus, concerning centriole and basal body isolation, we may conclude that 15-min centrifugation at 20,000 g is necessary to precipitate these organoids. Their buoyant density is fairly high and equals about 1.30 g/ cm3, which excludes the possibility of equilibrium density centrifugation in sucrose solution for their purification. Yet, purification is possible by means of short-term centrifugation ( I hour at 70,000-100,000 g ) in a 55% layer of sucrose gradient. Centrioles and basal bodies are not destroyed by EDTA and Triton X-100, and so these substances can be used to remove chromatin and cell membrane components. Centrioles may be highly sensitive to proteases (Snyder, 1980; Kuriyama, 1984), and hence it is recommended that they be isolated in the presence of protease inhibitors, phenylmethylsulfonyl fluoride (PMSF) in particular. A homogenization regime should likewise be carefully selected. Thus, isolation procedures for centrioles and basal bodies involve centrifugation in various sucrose gradients.

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Since the MTNC fibrogranular material is labile to mechanical destruction and is readily detached from the centrioles in cell homogenization (even if it is gentle homogenization), it has not been possible to obtain its preparation so far, and the prospects appear to be bleak. It seems that Mitchison and Kirschner (1984a) alone have succeeded in obtaining MTNC-bound centriolar fractions by dispensing with all mechanical homogenization procedures. Proceeding from direct assays of basal body preparations and from the data of histochemistry, immunocytochemistry, autoradiography , as well as cell inhibitor effects, it has been established that the centrosome consists predominantly of protein; the presence of nucleic acids (RNAs) is not excluded either. Yet in speaking of the biochemical composition of centrioles, it should be kept in mind that the ultrastructure of these organelles is an extremely complex one (see Sections I1 and III), and different structural elements may perform completely different functions. So it is essential to find out not only what kind of protein is in the whole composition of the centrioles, but also where this protein is localized. It has been shown that in salt solutions (NaCI and KCl) over 0.2 M and at an acid or alkaline pH in vitro, selective destruction of centriolar microtubules and destruction of basal bodies takes place (Nadezhdina et d . , 1980; Gavin, 1977, 1980). The matrix layer and certain cylinder lumen structures forming a characteristic centriolar “rim” remain intact (Nadezhdina et al., 1980). This indicates that the MTs and the matrix are comparatively independent elements of centrioles and basal bodies, which is in good agreement with the different functions of these elements: the MTs of centrioles and basal bodies organize a ciliary axoneme, while the cytoplasmic microtubules may radiate from the matrix. It has been found recently that the cartwheel, an obligatory structure of juvenile centrioles, is capable of self-assembly in vitro from a homogenate of Tetrahymena basal bodies (Gavin, 1984). Tetrahymena basal bodies were dissolved in 1 M KCI. On dialyzing the solution against a diluted KCI solution, cartwheel-type structures were reconstructed in it. Possibly, the cartwheel is yet another independent structure of centrioles and basal bodies required for their assembly only. A direct analysis of basal body and centriole preparations by SDSPAGE has shown them to contain dozens of polypeptides (Wolfe, 1972; Gould, 1975; Anderson and Floyd, 1980; Cavin, 1984). Tubulin is found in significant amounts among basal body proteins: it has the same electrophoretic motility and divides into a- and p-subunits as the tubulin of cytoplasmic MTs or cilia (Wolfe, 1972; Gould, 1975; Anderson and Floyd, 1980). This tubulin is capable of assembling into MTs at about the same conditions as other types of tubulin. It can bind to colchicine in such a

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way that M colchicine completely blocks the formation of basal bodies during mass ciliogenesis in frog embryo (Steinman, 1970).Colchicine and other MT poisons have no effect on the basal bodies, an added proof that their MTs, like those of axonemes, are not in equilibrium with the dissolved tubulin pool. Basal body MTs can be disassembled in concentrated salt solutions (Nadezhdina et al., 1980; Gavin, 1980, 1984) or by changing the pH of solutions. In this case, the basal MTs are less stable in high-ionicstrength solutions than the axoneme or the cortex MTs (Gavin, 1984, 1980). A detailed investigation of basal body tubulins has revealed that their peptide maps differ from those of ciliary tubulins, even though they have the same isoelectric point (Anderson and Floyd, 1980). The presence of tubulin in centrioles and basal bodies has also been demonstrated by immunofluorescent and immunoelectron staining by tubulin antibodies (Gordon et al., 1977; Mitchison and Kirschner, 1984a). Yet, these antibodies bind worse to centrioles and basal bodies than to other MT structures. Some explanation is that the centriolar cylinder MTs are covered with a matrix layer which may impede the access of the antibodies. It is more probable, however, that a set of antigenic determinants of tubulin of centrioles and basal bodies differs from that of other varieties of tubulin; this is quite consistent with their distinctions in peptide maps. It is obvious that centriolar MTs and basal bodies consist of tubulin. According to some authors, tubulin may be contained in the oligomeric form in MTNC material, notably, in the mitotic halo (Pepper and Brinkley, 1977, 1979; Dustin, 1983). Indeed, the halo can bind antitubulin antibodies. Under the reconstitution of a net of cell MTs destroyed by cold or colcemid, the immunofluorescent staining by antitubulin antibodies shows MTOC as a fluorescent ring (Bershadsky et al., 1979b), i.e., the antibodies bind to the MTNC material surrounding the centrioles rather than to the centrioles themselves. It is presumed that tubulin oligomers may serve as specific primers for the assembly of MTs on MTNC, and if so, this is a cytoplasmic, and not a centriolar, tubulin. On the other hand, even though it is presumed that MTNC should contain many MAPs (microtubular-associated proteins), antibodies against MAPs do not bind to MTOC as a rule (Kuznetsov et al., 1980; Thompson et al., 1983; Vallee and Bloom, 1983; Bloom et al., 1984), with the exception of the only sample of antibodies against protein MAP-I, which Sherline and Mascardo use for immunofluorescent staining of centrioles (Sherline and Mascardo, 1982a). No cilium ATPase (dynein) has been found in basal bodies (Anderson and Floyd, 1980). Yet an ATP cytochemical reaction reveals significant ATPase activity in centrioles and basal bodies (Matsusaka, 1967; Abel et al., 1972; Nayak, 1972; Dentler, 1977; Anderson, 1977; Ohta and Ishikawa,

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1979; Burnasheva and Solovjeva, 1980). At the electron microscopic level, the reaction product is localized in the centriolar cylinder matrix, in the appendages, in the striated rootlets, and around the centriolar microtubules (Matsusaka, 1967; Anderson, 1977; Burnasheva and Solovjeva, 1980). Anderson (1977) described this enzyme (ATPase) in basal body preparations isolated from rabbit oviducts. ATPase of basal bodies proved to be Ca2+-and Mg“-dependent, and the optimal Ca” concentration is 2 mM; 6 mM Ca2+inhibits the enzyme effect. The optimum pH for basal body ATPase is about 8.5. Dynein has somewhat different characteristics, i.e., it is not inhibited by Ca” and its optimum pH is about 10. The molecular weight and functions of centriolar and basal body ATPase are not yet known. Cytochemical investigations, as well as those involving monospecific antibodies, allowed the detection of purine phosphorylase in centrioles, i.e., an enzyme catalyzing the reversible reaction: purine nucleoside (gua@I purine base + ribose-I-P(EC nosine, inosine, xanthosine) 2.4.2.1). In human leukocytes and fibroblasts, this enzyme is localized in small concentrations in the nucleus and throughout the cytoplasm with the exception of mitochondria. Its concentration is very significant in centrioles, both in interphase and in mitosis, though it is not clear how the centrioles are related to purine exchange (Oliver et al., 1981). Monospecifc (Welsh et al., 1978; Gordon et al., 1982) and some samples of monoclonal (Pardue et al., 1983; Deery et al., 1984) antibodies against calmodulin likewise bind to basal bodies and to centrioles, especially to the mitotic spindle poles. It was demonstrated that hamster trachea basal bodies contain calmodulin on the exterior surface of the triplets, i.e., in the matrix (Gordon et al., 1982). There can be no doubt that calmodulin may play a significant role in MTOC functioning: it is involved in Ca”dependent regulation of assembly and disassembly of MTs, so its presence in MTOC is no cause for surprise. Work is now in progress to obtain monoclonal antibodies against mitotic apparatus proteins. Clevenger and Epstein (1984) obtained antibodies against 100-kDa protein, localized in mitotic poles and in active chromatin of the interphase nucleus. Several other samples of monoclonal antibodies were obtained by Vandre and co-workers (1984a,b). In most cases, their antigens appeared to be phosphoproteins associated with spindle poles. These proteins are specifically phosphorylated in mitosis and probably participate in spindle MTs nucleation. Centrioles and basal bodies also contain a specific antigen, which no other cell components (notably, cytoplasmic MTs and cilia) have. In mammals, autoimmune antibodies are often generated against this antigen (Connoly and Kalnins, 1978; Maunory, 1979). The titer of such antibodies

+

-

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is particularly high in the blood of patients suffering from autoimmune diseases (Brenner and Brinkley, 1982; Tuffanelli et al., 1983; Hyams, 1984). The antigen is localized in the MTNC material (Maro and Bornens, 1980; Calarco-Gillam et al., 1983; Sauron et al., 1984) and must be either the 50- or the 75-kDa protein (Turksen et al., 1982; Sauron et al., 1984). This is an evolutionary conserved antigen, since antibodies against it are bound to centrioles and basal bodies from many objects. The antigen disappears from MTOC upon destruction of the pericentriolar material caused by superheating of the cells (Malawista et al., 1983). The basal bodies do not contain collagen, desmin, myosin, and actin (Anderson and Floyd, 1980). It is not so difficult to isolate striated rootlets bound to the basal bodies, i.e., they have a significant mass and are resistant to salt extractions (Stephens, 1975) and thus have been studied sufficiently well. In each of the investigated objects, the striated rootlets are composed of a protein specific to a given species. In mollusk gill epithelium, this protein (anchorin) has a molecular weight of 230-25OK (Stephens, 1979, about 250K in Tefruhymena (Williams et al., 19791, 170K in Naegleria gruberi (Larson and Dingle, 1981), 90K in Trichomonas (Amos et al., 1979), etc. So far, we have scant data on centrosome proteins. Even less is known about other possible components of the centrosome. Many investigators are certain that the centrosome contains nucleic acids, for formation of new centrioles (see Section 111) is much similar to template-guided replication, a process which must involve nucleic acids. In the course of the first investigations to isolate basal bodies (largely from Infusoria pellicles), significant amounts of DNA were detected by direct assay (Child and Mazia, 1956), by autoradiography (Smith-Sonneborn and Plaut, 1%9), and by acridine orange staining (Smith-Sonneborn and Plaut, 1967; Randall and Disbrey, 1965). Yet it was natural that less DNA was detected as the purity of the preparations increased. Finally, a thorough investigation revealed that the DNA of Infusoria pellicles belongs to the nuclear, mitochondrial, and bacterial fractions (the latter is from the bacterial feed for Infusoria) and has no fractions which could be basal body DNA proper (Hufnagel, 1969; Flavell and Jones, 1971). Inhibitors of DNA synthesis, such as fluorodesoxyuridine, arabinosylcytosine, and amethopterin, have no effect on the foundation of new centrioles in tissue-culture cells (Rattner and Phillips, 1973; De Foor and Stubblefield, 1974), while ethidium bromide, which suppresses DNA synthesis in Stentor, does not inhibit formation of new basal bodies (Younger et al., 1972). Even a prolonged effect of DNase causes no changes in the ultrastructure of basal bodies from Paramecium (Dippell, 1976). DNase treatment of lysed cells has no effect on the assembly of MTs from exogenous tubulin on the mitotic halo (Pepper and Brinkley, 1980).

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Thus, there is no evidence to support DNA presence within centrioles, basal bodies, or in the mitotic halo. Yet there are quite a few facts in favor of RNA there, though it is too early to draw final conclusions. RNA is always found in basal body preparations obtained from various objects, and its amount tends to increase as thorough purification procedures are applied (Hoffman, 1965; Hartman et al., 1974; Heidemann et al., 1977). Unfortunately, no published data are available on RNA in purest basal body preparations, isolated in recent investigations (Anderson and g of RNA Floyd, 1980). Heidemann et al. (1977) determined 2-8 x per basal body in a sufficiently pure preparation of basal bodies from Chlamydomonas. Tetrahymena pellicles, isolated in the presence of a detergent, were found to contain RNA which, hybridized with the cell DNA, is 35% noncompetitive with the ribosomal RNA, does not contain 4 S RNA, and has a sedimentation constant of 17 to 25 S (Hartman et al., 1974). An inhibitor of RNA synthesis, actinomycin D, blocked the finding of centrioles in tissue culture cells at 4 pg/ml (De Foor and Stubblefield, 1974) and the formation of new mitotic centers in dividing sea urchin zygotes (Went, 1977). Upon RNase treatment, Chlamydomonas basal bodies lost the ability to induce cytasters in nonfertilized Xenopus eggs (Heidemann et al., 1977), while basal bodies of mouse spermatozoa have a diminished capacity to induce assembly of MTs in vitro (Esponda and A h a , 1983). Considerable data have been accumulated on the presence of RNA in the mitotic halo. In newt tissue-culture cells, mitotic halo staining by the method of Bernhard, i.e., by uranyl acetate-EDTA on EM preparations, indicated RNA (Rieder, 1979). In order to obtain a truly positive staining, it was necessary to make use of the “thick,” instead of the conventional ultrathin, sections with subsequent observing in a I-MV EM. This positive staining (according to the Bernhard method) was removed by pretreatment with RNase (Rieder, 1979). In other investigations, the mitotic halo was shown to be reduced upon RNase treatment (Pepper and Brinkley, 1980), with the assembly of exogenous tubulin on it being strongly inhibited (Snyder, 1980). To verify the latter case, special assays were performed to exclude the possible protease effect on the halo, and various RNases were tested. It should be noticed, however, that Kuriyama (1984) denies that RNase has an inhibitory effect upon MT assembly on the mitotic halo. Berns and co-workers obtained interesting data on RNA role in the mitotic halo in experiments involving laser microbeam study of living cells (Peterson and Berns, 1978; Berns et al., 1977). If, prior to the microbeam study, the cells were treated with a dye which specifically binds to RNA,

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i.e., psoralen (4-aminomethyl-4,5,8-trimethylpsoralen), and the prophasic or prometaphasic spindle pole was irradiated by a beam with the wavelength corresponding to the dye absorption maximum, destructive changes would take place in the halo. The migration of the chromosomes to the irradiated pole in anaphase would be inhibited, and near the irradiated pole, there would be few, if any, MTs (Peterson and Berns, 1978).If another psoralen, binding to DNA but not to RNA, is used or if the pole is exposed to radiation in the same dose without prior staining of the cell, no changes in the halo would occur, and mitosis would proceed normally. Thereby some damages of the centriolar cylinder may be observed; but they exert no effect on mitosis (Peterson and Berns, 1978). Similar evidence was reported by Berns et al. (1976,1977)and Berns and Richardson (1977) in earlier studies when cells were treated (sensitized) by acridine orange instead of psoralen. The role of the centriolar RNA is quite obscure. Its amount is sufficient for coding several dozens of polypeptides. However, should we suppose that centriolar RNA is a centriolar genome, we should also suppose that RNA-dependent DNA polymerase or RNA replicase is present in the centriole. Another possibility is that the centriolar RNA may serve as a structural matrix, be it only at the early stages of centriole foundation, the way it is in the ribosome. The same role could be attributed to RNA in the mitotic halo as well, i.e., it may operate as a matrix in organizing the proteins which subsequently participate in the assembly or anchoring of MTs. Yet, the cell RNA inhibits MT polymerization in vitro (Zackroff et al., 1976; Meza et al., 1975). The mechanism of MT assembly on the centers must be a highly complex one and demands careful study. VI. Assembly of Microtubules on Microtubule-Organizing Centers (MTOCs) in Vitn,

Organization of a MT system is perhaps the most accessible function of the centrosome for recent methods of research. An asterlike growth of MTs from the centriolar region may be observed in living cells in mitosis or after the removal of MT poisons. We shall consider this question in Section VII. An “asterlike” assembly of MTs on the cell centers can be reproduced in vitro with the exogenous MT proteins and centrioles of detergent-permeabilized cells (McGill and Brinkley, 1975;Snyder and McIntosh, 1975; Gould and Borisy, 1977,etc.) or with preparations of isolated basal bodies and centrioles (Snell et al., 1974;Stearns and Brown, 1979;Telzer and Rosenbaum, 1979;Mitchison and Kirschner, 1984a;Kuriyama, 1984).Be-

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fore describing these experiments, it would be advisable to give a general view on cytoplasmic MTs and their properties, specifically, those which make an asterlike assembly on the centers possible. Tubulin, an essential protein for microtubular wall construction (Herzog and Weber, 1977; Rodionov et al., 1978; Stephens, 19821, constitutes 80% of the MT composition (Mohri, 1968). The other 20% is under MT-associated proteins (MAPS),a highly heterogeneous group of proteins with a molecular weight from 20 to 310 (Dentler et al., 1975; Murphy and Borisy, 1975; Cleveland et al., 1977; Berkowitz et al., 1977; Runge et al., 1979; Black and Kurdyla, 1983). A remarkable property of cytoplasmic MTs is their lability exhibited both in cells and in isolated preparations: the MTs may disassemble into subunits and then repolymerize from them. Axonemal and centriolar MTs are stable; they polymerize once and for all. Investigations in several laboratories on MTs from mammalian brain (a traditional object of biochemical research) revealed that, in the presence of MAPS, the optimal conditions for polymerization are as follows: the ionic strength should not exceed 100 mM, the pH should range from 6.7 to 6.9, the Mg2+concentration should range from 0.01 to 1 mM. the Ca2+ concentration should not exceed not above 0.01 mM, and the presence of nucleosidetriphosphates is also required (preferably GTP). The temperature interval of the reaction should be between 25 and 42°C. Under these conditions, the minimal protein concentration for polymerization should be lo-' mg/ml; it shows variations with different authors. The reaction of MT assembly proceeds in two stages: at first, high molecular primers, the tubulin oligomers, are formed, followed by elongation of the MTs from these primers. Formation of the primers is the slowest, rate-limiting step of the assembly (Bordas et al., 1983). The elongation proceeds until the MTs and the dissolved tubulin are in equilibrium, i.e., steady-state conditions. If the MAP fraction is removed from the MT preparation by ion-exchange chromatography, tubulin may still be polymerized into MTs, though the assembly requirements become more rigorous. For instance, it is possible to achieve polymerization of purified tubulin by increasing its concentration in the solution to 10 mg/ml (Sloboda and Rosenbaum, 1978) or by adding MT-stabilizing substances, such as sucrose, glycerol, polyethyleneglycol, or dimethylsulfoxide (Himes et al., 1977; Herzog and Weber, 1977, 1978), to the incubation mixture or by returning some of the MAP fractions (Cleveland et al., 1977; Kuznetsov et al., 1978). Purified tubulin may also be polymerized on exogenous stabile primers, for instance, on axonemal fragments (Allen and Borisy, 1974; Bergen and Borisy, 1980) or on prepolymerized MTs (Carlier et al., 1984; Mitchison and Kirschner, 1984b).

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Investigations of MT assembly on exogenous stable or labile MTs showed that MTs are polar structures: assembly of tubulin at one end of the MT proceeds faster than at the other end (Allen and Borisy, 1974; Dentler et al., 1974; Olmsted et al., 1974; Binder et al., 1975). In addition, there are such concentrations of tubulin when its polymerization takes places only at one end of the stable primer (Olmsted et al., 1974; Binder et al., 1975; Bergen et al., 1980). A thorough investigation of polymerization and depolymerization kinetics likewise confirmed the fact that the two MT ends are different: in labile MTs, the tubulin molecule association constant is higher at the plus end than at the minus end (Margolis and Wilson, 1978; Kirschner, 1980) (Fig. 8). Blockage of either end may cause drastic changes in the MT properties in the assembly systems (Bergen and Borisy, 1980; Mitchison and Kirschner, 1984b). The process of depolymerization of MTs, as shown by recent studies (Carlier et al., 1984; Hill and Chen, 1984; Mitchison and Kirschner, 1984b), is more complex. After dilution suspension of MTs in the steady state, the number of MTs decreases, though the median length of the remaining

B

I i

l-

a

NH

wn

I:

1;

0 0

c

O

PROTEIN CONCENTRATION

W

5U

FIG . Plot of :.I rate of tubulin polymerization versus monomer concentration. The shaded area denotes the concentration range in which -end-capped MTs remains stable and free MTs depolymerize. Cr , Critical concentration for +end; C;, critical concentration for -end; 6,critical concentration for free MTs. (A) Plot according to the data obtained by Bergen and Borisy (1980) for tubulin with MAPS. (8)The plot according to the data obtained by Mitchison and Kirschner (1984a) and Hill and Chen (1984) for the phosphocellulose, purified tubulin.

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ones may increase initially. The same appears to hold for the steady state as well, i.e., some MTs are depolymerized, but their disassembly is compensated by elongation of the remaining ones (Mitchison and Kirschner, 1984b). To study MTOC polymerization activity, almost all the authors used tubulin preparations that either were uncapable of spontaneous polymerization or that were able to polymerize sufficiently slow. If a suspension of isolated basal bodies is added to purified tubulin, and then the mixture is heated to 37°C in the presence of GTP, bundles of MTs grow from the ends of the basal bodies, i.e., the way they would have grown from the ends of axonemal fragments. Obviously, the MTs of basal bodies can operate as exogenous primers for tubulin assembly (Snell et al., 1974; Kuriyama and Kanatani, 1981; Esponda and A h a , 1983). Microtubules may grow also from the MTNCs associated with basal bodies; they may grow from the striated rootlets (Stearns and Brown, 1979) or from the connective between the two basal bodies in the pair (Roobol et al., 1982; Stearns and Brown, 1981; Esponda and Aliva, 1983). Stearns and Brown (1979) showed that incubation of Polytomella basal bodies in 1 mM Tris, pH 8.0, and 0.1 M EDTA deprives their ability to induce assembly of MTs from the rootlets. In the process of this extraction, four proteins with a molecular mass from 190 to 210 kDa are washed from the basal bodies into the solution. In solution, these proteins also can induce assembly of the purified tubulin into MTs and are incorporated into them, i.e., the proteins act as specific MAPS within MTNC. The centrioles of cultured cells are usually surrounded by MTNC fibrogranular substance. If, in the process of centriole isolation or construction of permeable cell models, this substance is washed away from the centrioles, exogenous tubulin MTs may grow from the ends of the centriolar cylinders, the way they do from the basal body ends (Gould and Borisy, 1977; Schliwa et al., 1979), and may grow separately, i.e., on MTNC substance fragment (Gould and Borisy, 1977). Yet if the centriole remains surrounded by MTNC substance, the MTs grow only from MTNC and form asterlike structures (McGill and Brinkley, 1975; Snyder and McIntosh, 1975; Telzer and Rosenbaum, 1979; Pepper and Brinkley, 1979; Schliwa et al., 1979; Bergen et al., 1980, etc.). Several experimental models were employed to study the polymerization capacity of centrioles. The simplest one involved detergent-permeabilized cells in which their MTs were destroyed by colcemid, with the centrioles being assessable to exogenous tubulin. Such a model ensures the optimal intactness of the centrioles and the surrounding structures. However, the samples thus obtained can be studied only on ultrathin sections or by the immunofluorescent microscopy (Snyder and McIntosh, 1975; McGill and

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26 1

Brinkley, 1975; Brinkley et al., 1981), and this causes difficulties for obtaining quantitative data on the number and length of polymerizing MTs. The next step toward a noncellular system for studying MTOC functioning was made by Gould and Borisy (1977), who developed procedures in accordance with which a suspension of detergent-lysed and softly homogenized cells was sedimented on grids for EM. This preparation was composed mainly of nuclear-centriolar complexes (Gould and Borisy , 1977; Kuriyama and Borisy, 1981a), and it was possible to perform polymerization of MTs on the centrioles with subsequent EM after negative staining (Gould and Borisy, 1977; Kuriyama and Borisy, 1981b). However, in this case the nuclei interfered and made it difficult to observe the MTs, and so this method is suitable for mitotic cells (Telzer and Rosenbaum, 1979; Kuriyama, 1984). The culminating step for the present level of investigations was made by Mitchison and Kirschner (1984a). They isolated centrioles from interphase CHO cells and neuroblastoma in the enriched fraction and then polymerized tubulin on the centrioles in suspension. They then sedimented the preparations thus obtained on grids for EM. Special investigations showed that the number of MTs polymerized on each MTOC is proportional to the concentration of tubulin solution: a stronger concentration gave a larger number of polymerized MTs. Some degree of saturation was always achieved if, with the increase in the protein concentration, the number of microtubules on MTOC remained invariable or even slightly decreased (Brinkley et al., 1981; Kuriyama, 1984; Mitchison and Kirschner, 1984a). If a suspension with MTs already assembled on the centrioles was diluted, the number of MTs on each MTOC significantly decreased, though their length might increase (Mitchison and Kirschner, 1984a). On the other hand, the number of MTs assembling on each MTOC at optimal conditions also varies depending on MTOC properties. Several times as many MTs are polymerized on mitotic centrioles as on interphase centrioles (Snyder and McIntosh, 1975; Telzer and Rosenbaum, 1979; Kuriyama and Borisy, 1981b). This may be directly related to the presence of the mitotic halo. In general, the number of MTs formed on MTOC in vitro correlates with that of the MTs radiating from the same MTOC in vivo. Thus, Brinkley et al. (1981) demonstrated that, in detergent-permeabilized 3T3 and SV-3T3 cells, the purified tubulin is polymerized on one to two centers in each cell, with an average of twenty-four MTs radiating from each MTOC in 3T3 cells, and about nine MTs radiating from each MTOC in SV-3T3 cells. The same number of MTs was formed on MTOC in these cells in vivo, after the cells had been washed free of colcemid.

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It was shown (Schliwa et al., 1979) that, in fish melanophores, the number of MTs, polymerized from exogenous tubulin on one MTOC, depends on the amount of MTNC substance contained in it. Schweitzer and Brown (1984) also demonstrated that the number of MTs radiating from the centrosome in Con A-stimulated lymphocytes is 2-3 times higher than that of unstimulated cells. The same correlation was obtained by these authors after polymerization of tubulin on the centrosomes of lysed lymphocytes in vitro. A MTOC of the dispersed melanophore pigment granules contained much MTNC substance and as many as 388 f 70 MTs radiated from it in vivo. Yet in the case of pigment aggregation, its MTOC had much less MTNC substance and fewer MTs radiating from MTOC (60 k 7). After destruction of the MTs by colcemid and detergent lysis, the amount of MTNC substance in melanophore MTOC did not change. On these MTOCs were assembled about the same number of MTs from exogenous tubulin as in vivo for a given disposition of pigment granules (348 +: 65 in the dispersed and 28 f 8 in the aggregated state, respectively). Schweitzer and Brown (1984) also demonstrated that the number of MTs radiating from the centrosome in Con A-stimulated lymphocytes is 2-3 times higher than that of unstimulated cells. The same correlation was obtained by these authors after in vitro polymerization of tubulin on the centrosomes of lysed lymphocytes. Mitotic centers from CHO cells, containing different numbers of centrioles (one to four), had the number of polymerizing MTs directly proportional to the number of centrioles (Kuriyama, 1984).It should be noted that, in the preparation used, as a result of colcemid treatment of the cells, the mitotic halo must have surrounded each centriole and not only the mother centrioles. Consequently, the number of MTs correlated with the halo volume (the amount of MTNC material) in a given MTOC. A critical tubulin concentration at which it is polymerized into MTs on MTOC is very low, much less than a critical concentration for the assembly of MTs from purified tubulin (Kuriyama, 1984; Hill and Chen, 1984; Mitchison and Kirschner, 1984a). This concentration, however, corresponds to the one needed for tubulin assembly on stable fragments of axonemal MTs (Bergen et al., 1980; Mitchison and Kirschner, 1984a). At low tubulin concentrations, the growth of axonemes proceeds only from the plus end, and under the same conditions the MTs grow from MTOC (Bergen et al., 1980) (Fig. 9). No one has ever directly determined the polarity of MTs assembled on MTOC in vitro; however, by decoration of native MTs in the mitotic spindle with dynein or tubulin “hooks,” it was demonstrated that, in vivo, the plus end of the MTs is a distal one with respect to MTOCs (centrioles) (Haimo et al., 1979; Heidemann and McIntosh, 1980).

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FIG.9. Comparison of centrosome and flagellar seed-initiated MTs. C, Centrosome. Note that the polymerized tubules at the plus end ( + ) of the flagellar seed (arrows) are approximately of the same length as the centrosomal MTs. Bar, 2 pm. (From The Journal of Cell Biology, 1980, 84, I55 by copyright permission of the Rockefeller University Press and courtesy of Bergen el a / . )

It may thus be suggested that MTOC is a set of stable primers (MTNCs) for the growth of MTs, i.e., in the simplest case, of very short fragments of MTs or tubulin oligomers. Here the minus end of a MT is blocked in MTNC substance. Indeed, after short-term incubations of cells in the presence of colcemid or nocodazol, an aster of radiating MTs persists around MTOC, even though free MTs become completely destroyed (We-

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ber and Osborn, 1981). In the case of assembly of MTs on isolated yeast MTOCs in vitro, which are disks of electron-dense material, a regular number of microtubules is likewise formed, and the ends of the MTs are immersed in the MTOC substance and closed (Hyams and Borisy, 1978; Byers et al., 1978). The fact that MTNC primers are in existence is also proved by the blockage of assembly of exogenous MTs on mitotic centrioles by tubulin antibodies (Pepper and Brinkley, 1977, 1979). But biochemical analysis of MTOC is needed for the final conclusion about the presence of MT assembly primers and about the mechanisms of assembly on MTNC. Some data on the biochemical properties of MTOCs have already been obtained. Their polymerization activity is inhibited by 0.2-0.5 M KCI and KI, at pH of the solution over 7.5, after ultrasound treatment, and in heparin. All these cases involved MTOC treatment prior to the addition of exogenous tubulin (Kuriyama and Borisy, 1981b; Kuriyama, 1984; Mitchison and Kirschner, 1984a). Mitotic MTOCs exhibited high-endogenous protease activity, which completely suppressed MTOC polymerization activity in 3 minutes at 35°C or in 2 hour at 5°C (Snyder, 1980; Kuriyama, 1984). This protease was inhibited only at high concentrations of PMSF (1 mg/ml) or in 50% glycerol (Kuriyama, 1984). These observations are at variance with the results of the previous investigations in which assembly of MTs on the mitotic halo was inhibited by RNase (Pepper and Brinkley, 1980). At any rate, Kuriyama (1984) denies the inhibitory effect of RNase on MT assembly on the halo, but Snyder (1980) confirms it. Such high sensitivity to endogenous protease has not been shown for interphasic MTOCs. Probably, endogenous protease is involved in mitotic halo disassembly, which takes place in late mitosis. Despite significant achievements in purifying the system for polymerization of MTs on MTOC in vitro, one should remember that the reaction of polymerization of MTs is a very complex one and can be regulated by many independent factors in such a way that departure of some of the conditions from the optimal ones could be redressed by the others. Assembly of brain tubulin on MTOC at conditions from the optimal ones could be redressed by the others. Assembly of brain tubulin on MTOC at conditions selected for spontaneous assembly of MTs, most of which are not attached to MTOC in vivo either (Chalfie and Thomson, 1979; Tsukita and Ishikawa, 1980), is possibly not a sufficiently adequate model for studying MTOC performance, even though it was the only available one until recently. Entirely different observations were reported by Deery and Brinkley (1982, 1983) for the assembly of endogenous tubulin in 3T3 and SV-3T3 cells. As established by these authors, upon permeabilization of the cells by Brij 58, the tubulin is not washed away and can be assembled

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and disassembled at controlable ionic conditions. If, prior to detergent treatment, the MTs were destroyed by preincubation of the cells in the presence of colcemid, then at “normal” polymerization conditions, i.e., at 3 7 T , pH 6.9, and with the addition of 1 mM GTP, randomly scattered microtubules, about 5 pm long, were formed (though the length of the native MTs was 34 pm). An essential condition for assembly of normal length MTs, and only from MTOCs, was pH 7.6, and addition of GDP and ATP, as well as of 8-bromo-CAMP. If an aster of the remaining short MTs persisted in the permeabilized cells, “normal” conditions of assembly and even the presence of GDP instead of GTP in the system were sufficient for their elongation. The results of this investigation show that optimal conditions for initiating assembly of MTs on MTOCs may differ significantly from those selected for self-assembly of isolated MTs in vitro. The present data on MTOC polymerization activity in vitro enable us to draw the following conclusions: 1. MTOC is capable of inducing assembly of MTs under conditions excluding their spontaneous polymerization; 2. MTOC must contain MT growth primers. The number of primers is limited, depending on the physiological state of the cell; 3. The biochemical composition of the primers is not clear. RNA participation is believed probable but not proved unambiguously. They do not contain DNA. The primers may differ from MT fragments in some properties.

VII. Assembly of Microtubules on Microtubule-Organizing Centers in Vivo

Microtubules were discovered by EM. Yet it was difficult to study a MT pattern in cells on ultrathin sections because of their large extension. After specific antibodies against tubulin had been obtained (Fuller et al., 1975; Weber et al., 1975), it became possible to trace MT localization by light microscopy (by indirect immunofluorescence). It was immediately found that, in interphase cells, MTs form a fairly dense network; so the genesis of this network attracted attention next. It should be noted that, even before the immunofluorescent studies, Tihey and Goddard ( 1970) demonstrated that, in sea urchin blastula after colcemid has been washed away, MTs grow from MTNC surrounding the basal bodies in each blastula cell. To study the origin of MTs, a simple procedure of their preliminary destruction by colcemid or cold in tissue-culture cells was employed.

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Brinkley et al. (1976) and Osborn and Weber (1976)first showed that after 3T3 cells had been washed free of colcemid, MTs began to grow in an asterlike fashion from one (sometimes from two) centers. Then the number of MTs sharply increased and individual MTs in the central part of the cells became invisible. Gradually the system of MTs spread to the cell’s periphery and lost its distinct center, similar to the way it occurs in intact cells. Frankel (1976) obtained similar results by studying L cells and macrophages, though the distinct center persists in macrophages even after a complete reconstitution of the MT network. Background fluorescence of colcemid-depolymerized tubulin interfered with observations. Pretreatment of the cells with the detergent, Triton X100, in a solution protecting MTs before furation gave more distinct images (Osborn and Weber, 1977; Bershadsky et al., 1978b). They proved to be similar to the ones described above, yet short MTs, not bound to the center, became visible in most cases at the beginning of reconstitution (Spiegelman et al., 1979a; Bershadsky and Gelfand, 1981; de Brabander et al., 1982). These free MTs disappeared subsequently and all of the cytoplasm became filled with a network of long MTs. Moreover, coldresistant MTs, not bound to the center, were detected (Bershadsky et al., 1979b). But as a rule, most of the MTs during their reconstitution in the cells are bound to the common center (Fig. 10). A supposition was expressed already in the early studies that the centriolar region is the center from which MTs begin their growth, for it is there that a cilium emerged (Brinkely et al., 1975; Bershadsky et al., 1978a). Correspondence of MTOCs to centrioles was particularly proved convincingly in neuroblastoma cells in which multiple MT growth centers were detected (Spiegelman et al., 1979b); it was then demonstrated that the number of these centers correlated with the number of centrioles (Sharp et al., 1981). A comparison of immunofluorescent and EM images of the same cell also indicated the location of the MT growth center, the centnolar region (Sharp et al., 1981). Karsenti and coauthors furnished the most cogent evidence of the role of MTOC (Karsenti et al., 1984a). They succeeded in performing enucleation of the cells and also in removing centrioles from some of them. Reconstitution of the MT system in the cytoplasts with centrioles proceeded normally, and as in normal cells, a network of MTs was formed; but only few MTs were restored at the periphery of the cytoplasts without centrioles. Cold treatment ( +2-O°C, for 1.5-3 hour) and mild doses of colcemid (0.4-1.0 pg/ml, for 8-40 hour), destroying a system of cytoplasmic MTs, caused blobs of electron-dense material to appear around the centrioles; these blobs resembled MTNC material (Vorobjev and Chentsov, 1983,

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FIG. 10. MTs radiating from the centrosome. Cells were cooled (0°C for 2 hours) and then warmed to 37°C for 7 minutes (A) and 8 minutes ( B ) . Indirect immunofluorescence with antitubulin antibodies.

1985).As the cells were transferred to 37°C or after colcemid was washed away, MTs began their growth not only from the surface of the centrioles or the heads of the pericentriolar satellites, but also from the blobs. The cell center at that time showed many short, randomly scattered MTs (Vorobjev and Chentsov, 1983). Then the electron-dense blobs vanished, and the number of MTs, radiating from the center, decreased correspondingly back to the “normal” level; short MTs disappeared (Vorobjev and Chentsov, 1983). A similar picture showing the appearance of short disorderly MTs around the cell center was observed under a light microscope (de Brabander el al., 1982). Thus, colcemid and cold treatments induce a restructuring of the cell center: extra MTNCs are formed (possibly, under the effect of free tubulin). In mitotic cells, kinetochores also serve as a MT polymerization center after colcemid is washed away or after the cold-treated cells are heated (Witt and Borisy, 1980; Rieder and Borisy, 1981). The question of how MTOC works in the living cell is rather difficult and involves several aspects: first, the dynamics of MT growth (temporal order); second, how MTs can grow preferably in one direction (spatial

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order); and the last problem is why MTs start their polymerization only from MTOC and do not polymerize throughout the cytoplasm. The main part of experimental data and speculations available so far concerns the latter aspect. Experiments showed that the preferential growth of MTs from MTOC in vitro takes place when tubulin concentration is sufficient for MT elongation from the plus end, but too small for spontaneous polymerization. We do not know whether such a situation occurs in the living cell. But it was found that MTs are in dynamic equilibrium with tubulin monomer in vivo (Salmon et al., 1984; Saxton et al., 1984) and their turnover is rather quick (especially during mitosis) (Salmon et al., 1984; Saxton et al., 1984). Though the amount of MTs and the whole content of tubulin per cell protein may vary (Waterhouse et al., 1983), a definite concentration of the tubulin monomer is sustained (Ben-ZeCv et al., 1979; Cleveland et al., 1981, 1983). The simplest suggestion about the mechanism of MTOC operation boils down to the following: there exist MT growth primers within MTOC, and assembly conditions in the cell are such that MTs can grow only on primers of MTNC. If so, MTOC must have a definite number of such primers, each giving rise to one MT only (Borisy and Gould, 1978). In that case, the number of MTs and the direction of their growth depend only on MTOC. This idea agrees well with the results of MT assembly on the centers in vitro, when a definite number of MTs is formed, depending on the state of the cell. Yet at the beginning of MT reconstitution, free MTs also appear in the cell (Spiegelman et al., 1979a; Bershadsky and Gelfand, 1981; de Brabander et al., 1982). A possible explanation is that the system may not be in equilibrium during reconstitution of MTs, i.e., spontaneous polymerization of MTs may take place. A more complex suggestion implies that the minus ends of the MTs are blocked on MTOC (Kirschner, 1980; Tucker, 1984). As we have said in Section VII, if the free tubulin pool is limited, the MTs with the blocked minus end prove to be more competetive than the MTs with both ends free: after all, the former MTs (with blocked minus ends) grow, while the latter MTs (with both ends free) disassemble. Really, the free MTs in the cell, which are formed after colcemid or nocodazole has been washed out, do gradually disassemble (de Brabander et al., 1982). MTs with the free ends also disassemble in fragments excised from fibroblasts (Gelfand et al., 1984) and after laser-caused destruction of the spindle pole (Berns et al., 1977). But they do not disassemble in excised fragments of melanophores, where a new MTOC is formed (McNiven et al., 1984). The hypothesis advanced by de Brabander et al. (1980, 1982an-) alternative to the one on assembly in MTOC on primers-presumes that,

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in MTOC, a selective anchoring of the minus ends of the growing MTs takes place. According to this hypothesis, special condition MTOCs facilitate nucleation of MTs, around MTOCs. Then some of these MTs are anchored and acquire advantages for growth, while those not anchored disassemble, sustaining thereby the required tubulin concentration for the growing MTs (de Brabander, 1982; de Brabander et al., 1982). The action of taxol-a preparation that lowers the critical concentration for assembly-causes MTs of the cell to lose their links with the center and form a chaotic network (Schiff and Horwitz, 1980), since the anchored MTs are deprived thereby of their advantage for growth. As nocodazole has been removed from the cells with a diminished ATP level (under the effect of respiration inhibitors) in which the disassembly, but not the assembly, of MTs is slowed down (Bershadsky and Gelfand, 1981), the MTs likewise form a random network, for the MTs, not linked to the center, are not disassembled (de Brabander et al., 1982). It appears that special ionic conditions are obtained in the MTOC region which favor the assembly of MTs; thus Caz+concentration may decrease due to the operation of membrane transport systems (Silver et a / . , 1980; Aguas and Nickerson, 1981; Kierhart, 1981). How does the MTOC in a cell with assembled MTs maintain their spatial and temporal order? Under normal conditions, a system of cellular MTs is formed “from zero” only twice in a cell cycle: immediately before mitosis and after it (review, Brinkley et d . , 1980). Otherwise, the cell center only sustains the MT system and participates in its renewal. A thorough analysis of reconstitution of a MT system in interphase cells after cooling shows that the number of MTs bound to the centrosome in the beginning of reconstitution is much above the normal level. This observation led Vorobjev and Chentsov (1983) to suggest an idea that two systems of MTs exist in a cell. One forms a network in the cytoplasm and has no definite convergence foci. The other radiates from the centrioles. It is presumed that the radiating MTs persist bound to the cell center only for a definite time interval during their growth. After their growth is over, the MTs disengage and form a cytoplasmic network slowly departing from the center. New MTs may start growing on MTNCs, which the full-grown MTs have left. Consequently, the entire cell center operates as a conveyer feeding MTs into the cytoplasm (Fig. 11). The existence of the two MT systems in cultured cells has been demonstrated (Karsenti et al., 1984b). It was found that the peripheral network of MTs is less resistant to depolymerization by nocodazole than are the center-bound MTs. So the authors concluded that their observations corroborated the idea that, in the cell center, the MTs are anchored by one of their ends, while the MTs not bound to the center have both ends free.

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FIG. I t . The scheme of a conveyor assembly of MTs on the centrosome. MT starts growing from MTNC with its plus end. Then, after a certain period of time, it is detached from MTNC and may be depolymerized. for its minus end becomes free. The next MT starts growing from the same MTNC.

On the contrary, according to some other observations, in nocodazoletreated abnormally large Chinese hamster cells, MTs associated with MTOC are disassembled first and then only the peripheral ones disappear (Raes er al., 1984). This is attributed to the decreased functional activity of the centrioles in such cells. If the suggestion on the attachment of the MT minus end in the cell center is valid, then, in the steady-state condition of the cell, the MTs with both ends free should disassemble, and those with one free plus end should grow or remain in the same condition (Kirschner, 1980). The centrosome apparently exerts some effect on the number of MTs in the cellthe critical concentration of tubulin for the MTs bound to it is lower than for free ones. In the absence of the centrosome, as shown by Karsenti et al. (1984a) on centriole-free cytoplasts, the number of MTs is smaller. It is most probable that the number of MTs in centriole-free cytoplasts, is directly lined to the effective concentration of tubulin in them. The dependence is more complicated in cells and cytoplasts with centrioles. Since MTs with both ends free should disassemble, it is obvious that the time of their existence should be inversely proportional to the depolymerization rate. A more or less dense network of MTs may thus be formed only in cells where the time of growth of MTs radiating from the center is significantly less than the time of disassembly of the free MTs, or if the minus-end is protected, against disassembly. One may point to two extreme variants: neutrophils, where no free MTs are practically detected (Schliwa er al., 1982;Anderson et al., 1982),and fibroblasts, where

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cold-resistant MTs, not bound to the center, are found (Bershadsky et al., 1979a). In the latter case, an excised cell fragment with no center retains MTs for several hours (Gelfand et al., 1984). Moreover, reconstitution of a MT system in the axon of the neuron in Caenorhabditis elegans showed it to have relatively short MTs not bound to any common center (Chalfie and Thomson, 1979).Axons of other animals possibly contain such systems of MTs as well (Tsukita and Ishikawa, 1980). Regulation of the depolymerization rate of free MTs is a subject beyond the topic of the present work, and so we shall point to most probable options only. First, the rate may be dependent on MAPS (Jameson and Caplow, 1981); second, disassembly may be inhibited by modification of the MT ends (Mitchison and Kirschner, 1984b; Chalfie, 1982). In our opinion, the conveyer hypothesis of MT assembly explains the cause of stark distinctions in the disposition of MTs in different cells: in motile blood cells and in melanophores, the system is radial (Schliwa, 1975; Schliwa et al., 1978; Anderson et al., 1982; Schliwa et al., 1982); slowly moving cells (fibroblasts) form a network often without a clearly distinct center (Osborn and Weber, 1976);and in practically immotile epithelial cells, a MT convergence focus may not be detectable at all (Bershadsky et al., 1978a). According to the conveyer hypothesis, cells with a developing network of MTs would have an ever-increasing number of them farther away from the center. An increase in the number of MTs at some distance from the centrioles is indeed observed during radial spreading of fibroblasts on glass (Gudima et al., 1983a). The number of MTs radiating from the center should increase during a transition of the cell from immotility to motility, and this has also been observed in cultured hepatocytes and bibroblasts (de Brabander et al., 1978; Gudima et al., 1983b). Irrespective of a concrete mechanism for centrioles to operate as MTOCs, we should note that their role in the organization of cell MTs may change depending on different physiological conditions of the cells, and such changes have been demonstrated at least for two cases: for transition from interphase to mitosis and vice versa and for the aging of the cultured cells. As we have said above, during transition to mitosis, the interphasic forms of MTNC give way to the mitotic halo, and MTs are assembled into a spindle upon disassembly of the preceding interphase network. Obviously, other MTNCs-the chromosomal kinetochores specific for mitosis only-have a significant part to play in spindle formation. The injection of a fraction of interphasic centrioles isolated from neuroblastoma cells into Xenopus eggs revealed the ability of these centrioles to induce formation of cytasters only in interphase-activated eggs, while exogenous nuclei or chromosomes are needed for that in mitotic cells

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(with artifical delay of activation) (Karsenti et al., 1984a). According to the authors, conditions for assembly of MTs in mitotic cells are such that MTs cannot assemble even on the centrioles (and this explains disassembly of MTs at the end of the G , period); they reassemble only with the aid of additional chromatin-related factors. Two subpopulations have been detected in cultured Chinese hamster fibroblasts: normal fibroblasts and abnormally large ones, the polygonal cells. The latter are accumulated with the aging of the culture (in late passages). If, in the normal cells, nocodazole-induced depolymerization of MTs proceeded from the periphery to the center (the way it should be if a MT has its minus end anchored in the center), the pattern was reversed for the large cells: the MTs in the center were disassembled first, and thereupon, only those at the periphery (Raes et al., 1984).The mechanism of the latter process has received no satisfactory explanation as yet, but it is certainly connected with functional changes in MTOC. We may conclude that the role of the cell center in organizing a system of MTs needs further investigation and that the actual mechanism for its action may turn out to be more complex than hitherto believed. VIII. The Centrosome and the Cell

Microtubules are involved in sustaining a “spatially organized asymmetric form of the cell” (Solomon, 1980). Unlike actin filaments, which generate local forces bringing the cells or their parts into motion and which must have multiple organizing centers (Lazarides, 1976; Schliwa, 1982), the MTs organize the cell as a whole and usually have a single organizing center. As said above, the centrioles and the structures surrounding them operate as MTOCs in animal cells. The number of centrioles in the cells varies and so does the amount of MTNC substance. Nevertheless, experiments on MT growth initiation described in Sections VI and VII indicate that the number of MTOCs is as a rule one to two per cell (Brinkley et al., 1975; Osborn and Weber, 1976; Frankel, 1976; Watt and Harris, I 980). How does the cell regulate the number of MTOCs? First, by regulating the distribution of the MTNC substance. Usually it is present only in the centriolar region, and the number of MTOCs corresponds to the number of active centrioles. A diploid cell has two centrioles, of which only one is active (Dalcq, 1964; Schaffer, 1%9; Vorobjev and Chentsov, 1977, 1982). Additional MTOCs without centrioles are a rare exception (Schliwa et al., 1982, 1983; Sato et al., 1983). Such MTOCs consist of one or few aggregates of the MTNC substance. An increase in the number of such

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centriole-free MTOCs after 1000-rad irradiation is accompanied by a loss of their MT-polymerizing activity and the death of cells (Sato et al., 1983). Fewer MTs come to the centriole-free (containing only a halo fragment) pole of the pathological multipolar mitotic spindle than to the poles with centrioles (regardless of the number of these centrioles), and a contractile ring near the centriole-free pole takes a longer time to form (Keryer et al., 1984). Concentration of MTNC substance around the centrioles is still a great enigma. Since only the centrioles persists in the cell center, and MTNCs appear and disappear [for instance, in the cell cycle (Erlandson and de Harven, 1971; Vorobjev and Chentsov, 1982; Rieder and Borisy, 1982)], it seems probable that the centrioles act as a matrix for assembly or as a frame to which MTNC substance is attached in animal cells. The amount of MTNC depends on the physiological condition of the cells and may increase or decrease as, for instance, under dispersion and aggregation of granules in melanophores (Schliwa et al., 1979). Under polarization and motion of fibroblasts (Gudima et al., 1983a, b) or after lymphocyte stimulation (Schweitzer and Brown, 1984), the amount of MTNC substance increases. There is no ground for an assumption that the centrioles are directly involved in MTNC substance generation, for it would contradict the available data on the pathways of biosynthesis of proteins. Probably, a process similar to self-assembly of MTNCs and their accumulation in the cell center takes place, for instance, when centrioles are injected into eggs. Mitotic halo substance is accumulated on the centrioles, with cytasters being formed (Kuriyama and Kanatani, 1981; Hamaguchi and Kuriyama, 1982). Second, the number of centrioles and their distribution in the cell is regulated. As is known, diploid cells have only two centrioles. Since the centrioles double in tandem with DNA synthesis, the number of centrioles in polyploid cells should correlate with ploidy. Thus, tetraploid hepatocytes have four centrioles, and octaploid ones have eight (Onischenko, 1978). The same dependence of the number of centrioles on ploidy has also been observed for Allomyces (Borkhardt and Olson, 1979). At the same time, polyploid cells at the terminal stages of differentiation (megakaryocytes) and also cells (syncytia) formed via the fusion of dipolid cells (muscle fibers and giant cells appearing in inflammation processes) have fewer centrioles compared with their ploidy (Przybylski, 1971; Konishi et d., 1973; Sapp, 1976; Moskvin-Tarkhanov and Onischenko, 1978). One possible suggestion is that centriolar replication is inhibited or that resorption of some of the centrioles occurs (Mahovald et al., 1979). The reasons for the imbalance between ploidy and the number of centrioles deserves special investigation. It should be mentioned that only a deficit of centrioles

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is observed, but most probably, there exist no superfluous centrioles in the cells. Whether the cell can reconstitute the lost centrioles is still an open question. There is evidence that regenerating diploid karyoplasts from centrioles again, namely, two centrioles per cell (Zorn et al., 1979); but there are also opposing data that, during enucleation, some karyoplasts may retain centrioles, and only these karyoplasts are reconstituted into normal cells (Brown et al., 1980). Whatever the number of centrioles in a cell, they and the structures surrounding them are usually aggregated into a single cell center, and it is rarely that they are spaced far apart (Spiegelman et al., 1979b). Centrioles are found in a compact group even in giant syncytia, the homosynkaryons (Heidenhain, 1907; Matthews et al., 1967; Wang et al., 1979). Multiple centrioles are grouped at the spindle poles in heterokaryons, the neuroblastoma cells, and hepatocytes (Pera, 1975; Onischenko, 1978; Peterson and Berns, 1979; Ring e? al., 1982). Extra centrioles outside the mitotic spindle are rather an exception to the rule (Peterson and Berns, 1979; Brenner et al., 1977). As the cell changes its functional activity, they (the centrioles) may split. Centrioiar “splitting” takes place in neutrophils under the effect of chemoattractant or tumor promoter (Schliwa et al., 1982, 1983), and in cultured cells, under the effect of the growth factor and other mitogenetic agents (Sherline and Mascardo, 1982a, b). As a result of “splitting,” each centriole either active or inactive forms a MTOC of its own. Divergence of diplosomal centriolar pairs at the prophasic spindle poles is a process similar to “splitting.” This divergence is not synchronized with the condensation of chromosomes in the nucleus (Mole-Bajeret al., 1975; Aubin et al., 1980) and apparently does not depend on MTs (Snyder and McIntosh, 1975; Anderson et al., 1981; Cabral et al., 1983). Under prolonged incubation of cells in colcemid or in p-mercaptoethanol, the centrioles move apart and each may form its own MTOC (Watt and Harris, 1980; Watt et al., 1980), including a mitotic spindle pole (Went, 1977; Onischenko et al., 1979). Early embryogenesis, i.e., egg fertilization and division, may be a good model for studying cell MTOC regulation. Mature unfertilized eggs contain significant amounts of tubulin and MAPS, but very few MTs (Schatten et al., 1983; Balczon et al., 1983). Formation of MTs (asters and spindles) begins only after a spermatozoon penetrates the egg. It was believed earlier that the role of the spermatozoon is to bring the centrioles, absent in the egg, to initiate aster formation and division (Wilson, 1925). Indeed, after the spermatozoon enters the egg, its basal body loses its connection to the flagellum and becomes surrounded by osmiophilic material; MTs start growing from it, and a spermal aster is formed (Longo

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and Anderson, 1968). This picture was described with the aid of electron and light microscopy. Yet staining egg cells with tubulin antibodies revealed that no direct relationship exists between the sperm aster and the mitotic figure, and there is a period when, in the zygote, no MTs, emerging from any centers, are detected (Harris et al., 1980). It was also learned that some eggs may have a centriole (Zamboni, 1971; van Assel and Brachet, 1968). On the other hand, basal bodies (centrioles) are absent from many spermatozoa where they, in the course of spermiogenesis, are replaced by electron-dense material, which resembles a centriole by its contour only (Woolley and Fawcett, 1973). Finally, in mouse and rabbit fertilized eggs, though we see a classical combination of an egg without centrioles and a spermatozoon with basal bodies (Szollosi et al., 1972), no centrioles are present at the spindle poles during first divisions until a stage of 16 blastomeres. Instead, electrondense material (MTNC), organized into a single complex, is found (Burkholden e? al., 1972; Calarco-Gillam et al., 1983; Szollosi et al., 1972; Longo, 1974, 1976). Various treatments (hypertonic solution heating, microneedle prick, ammonia, heavy water, etc.) may induce parthenogenetic development of eggs in many animal species. Long-term effect of some of these agents results in the appearance of multiple asters, the cytasters, in the ooplasm. As shown in sea urchin and frog egg cells, such cytasters contain centrioles in the center (Koichi and Masao, 1971; Miki-Noumura, 1977; Dirksen, l%l). In sea urchin ooplasm, centrioles, close to a nuclear envelope or annulate lamellae, are frequently formed de novo (Kallenbach, 1982; Kallenbach and Mazia, 1982). Possibly, the latent centriolar precursors, found in significant amounts in the egg, are activated (Kallenbach, 1983). It is far more difficult to resolve the problem of the origin of multiple centrioles appearing upon injection of basal bodies or centrioles isolated from various objects. It was taken for granted that the basal bodies and centrioles thus injected underwent certain functional alterations and started functioning in the ooplasm as cytasters. In other words, they became surrounded by a halo from which MTs started growing (Heidemann and Kirschner, 1975, 1978; Maller et al., 1976; Heidemann et al., 1977; Kuriyama and Kanatani, 1981; Hamaguchi and Kuriyama, 1982; Karsenti et a l . , 1984a). Yet a thorough EM study or basal body labeling is needed, otherwise, it may not be excluded that the injected basal bodies operate only as promoters for the appearance of functioning centrioles in the ooplasm. It is known that only basal bodies or centrioles are capable of inducing cytaster formation (Hirano and Ishikawa, 1979; Heidemann and Kirschner, 1975). Their inducing activity is sensitive to proteases and RNase (Heidemann e? al., 1977).

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Egg cell cytasters, irrespective of their origin, divide synchronously with mitotic asters (Wilson, 1925). A mitotic aster can divide upon removal of the zygote nucleus, while the zygote is capable of regenerating the mitotic aster removed from the egg (Lorch, 1952). All these facts prove that egg centrioles, albeit not a fully autonomous structure, are sufficiently independent of the nucleus. It appears that an egg has a pool of centriolar and mitotic halo precursors, which are mobilized, due to various activating effects setting off cell division (Weisenberg and Rosenfeld, 1975). In the course of cell division, however, the extra cytasters are lost, and each cell of the embryo gains two centrioles (Wilson, 1925). Also, if the division sets in without centrioles at the mitotic poles, then centrioles appear at the morula stage two per cell (Szollosi et al., 1972). There is only one indication that father centrioles persist in the embryo, i.e., a giant centriole of the spermium Chrysopa carnea is detectable in one of the blastula cells (Friedlander, 1980).Thus, animal cells can “take count” of their centrioles and MTOCs. They lose this capacity during oocyte maturation and early division when the centrioles gain relative autonomy. It is desirable to look into a mechanisms of control which the cell establishes over the centrioles in the course of the organism’s ontogenesis.

IX. Localization and Orientation of Centrioles in Cells Centriolar location in cells was the object of many investigations in the past (reviews: Heidenhain, 1907; Wilson, 1925). The last few years have seen a renaissance in related studies, but at a higher level, with the aid of EM and immunofluorescent methods, and also, using cultured cells as an object. Since an EM shows the centriole as a differentiated structure and it has proved to be a polar structure (Vorobjev and Chentsov, 1980), one can now study not only the location of centrioles, but also their orientation in cells. It is hard to perceive some causal relationship between the facts of nonrandom disposition of centrioles described below and other intracellular processes. The presence of one MTOC in most animal cells accounts for cell disymmetry-the greater, the closer is the MTOC to some surface of the cell; conversely, a cell with a centrally located MTOC retains a spheric symmetry. As shown near the end of the last and early in this century, the centrioles in some cells may move away from the nucleus onto the surface intestine or trachea epithelial cells which are a case in point. In most instances, however, centrioles are located in the central region and are pushed aside

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by the nucleus only (Heidenhain, 1907). Two groups of cells may be singled out as the extreme types: those with perinuclear and those with periplasmalemmal disposition of centrioles. The first type is represented by motile cells (leukocytes and fibroblasts) and epithelial cells, still capable of division. The centrioles are located near the nucleus, often in its recess. They are frequently surrounded by Golgi complexes (Kupfer et al., 1982; Nemere et al., 1985). The centrioles of epithelial cells and fibroblasts may produce a primary cilium (Sorokin, 1962, etc.) (see Section 11; Allenspach and Roth, 1967), but they do not form specialized stereo- or kinocilia. The second type is represented by some differentiated immotile cells. Their centrioles lie far from the nucleus and close to the free surface, usually in the apical part (Heidenhain, 1907; Tucker, 1984). They may form a stereocilium [for instance, in sensory organs (Dahl, 1963; von Narnack, 1963; Tilney et al., 1980)] or serve as a matrix for basal bodies (Sorokin, 1968; Steinman, 1968; Dirksen, 1971). The centrioles moving toward the cell edge can participate in the formation of a specialized system of MTs, for example, in the growth of axon neurotubules (Spiegelman et al., 1979b), when they lie in the axon hill, or in forming an ulterior bundle of MTs in invertebrate nuclear erythrocytes (Cohen and Nemhauser, 1980). The centriolar shift to the erythrocyte periphery is genetically determined and controlled by one gene (Searle and Bloom, 1979). In secretory cells, the centrioles may be connected with secretory vesicle clusters (Zeligs, 1979). Tissue culture cells occupy an intermediate position between the two cell types described above: the distance between the nucleus and the plasmalemma is very small, and the centrioles, lying not far from the nucleus, may form an external cilium (Albrecht-Buehlerand Bushnell, 1979; Jensen et al., 1979; Rieder and Borisy, 1982). The nuclei and centrioles from cells of the first type and from cultured cells may be isolated together (Dales et al., 1973; Gould and Borisy, 1977), which suggests their mechanical cohesion (Bornens, 1977; Nadezhdina et al., 1979; Mar0 and Bornens, 1980; Nelson and Traub, 1982; Kuriyama and Borisy, 1981b). This cohesion is insensitive to detergents (Nadezhdina et al., 1979), i.e., the centrioles are attached not to the nuclear membrane, but to some submembrane structures of the nucleus. No concrete structures, responsible for the centriolenucleus association have been detected so far. Intermediate filaments are the putative candidates, even though centriole-nucleus complexes may be isolated in cells having no intermediate filaments (Nelson and Traub, 1982). The centriolar connection to the nucleus is weakened by cytochalasin B (Maro and Bornens, 1980), and therefore, when cells are enucleated with cytochalasin, the centriole remains in the cytoplast (Goldman et al., 1975). There is contradictory evidence concerning the effect of colcemid or nocodazole on the centriole-

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nucleus association. Aronson was the first to demonstrate a colcemiddependent centriolar connection to the prophasic nucleus in dividing sea urchin eggs (Aronson, 1971). But centrioles were also found to form MTindependent links to chromosomes (Bahr and Sugler, 1977). In tissue-culture cells, under the effect of colcemid or nocodazole, the centrioles may retreat to the cell periphery (de Brabander and Borgens, 1975), and it is more difficult to isolate them together with the nuclei (Maro and Bornens, 1980). During cell enucleation in the presence of nocodazole or cytochalasin B, some of the centrioles transfer into karyoplasts (Karsenti et al., 1984b),as the authors believe, because the MTs anchor the centrioles inside the cells. Centrifuged cells are an exciting example of centriole-nucleus association (Fais et al., 1984). If the cultured cells are centrifuged at 20,00040,OOO g, in such a way that the centrifugal force is parallel to the substrate plane, the nucleus is shifted in the centrifugal direction and so does the centriole in its wake: they push back other heavier organelles. In the presence of cytochalasin B, the centriole does not follows the nucleus, and the distance between them significantly increases. A single cell center causes cell asymmetry. It may be that there is some mechanism regulating the location of cell center in polarized cells. Motile cells, observed in v i m , are the most convenient model for studying this problem. It was shown that, in fibroblasts and endothelial cells polarized at the monolayer edge, the centrioles move to the front that becomes the leading edge of the cell (Gotlieb et al., 1981; Gudima et ul., 1983b) (Fig. 12). The same holds true for polarization of neutrophils in a chemoattractant gradient (Malech et al., 1977). The data on moving cells are more contradictory. Thus Gudima and coauthors write that, in moving fibroblasts polarized after attachment on the glass surface, the centrioles are always (in 100% of the cells) located between the nucleus and the leading edge of the cell (Gudima et al., 1983a,b). Albrecht-Buehler and Bushnell, contrary to what they had described in a summary of their work, observed lateral location of the centrioles (in 60% of the cells) in the direction of the fibroblast movement (Albrecht-Buehler and Bushnell, 1979). It may be that the authors traced the direction of cell movement by the phagokinetic track and not by the leading edge. In moving neutrophils, according to some data, the centrioles migrate toward the leading edge (Bessis and Breton-Gorius, 1967; Schliwa et al., 1982), and according to others, they remain in the center of the cell (Anderson et al., 1982; Gudima et al., 1984). No cogent explanation of these different results has been offered. In moving lymphocytes (T killers), the centrioles are located in the uropod (Gudima et al., 1984), i.e., in the part of the T killer by which it attaches to the target cell (Geiger et al., 1982).

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FIG.12. Centrioles in a moving fibroblast. A, Side view; B, top view: m. mother centriole: d. daughter centriole; c, cilia; N . nucleus: le, leading edge of the cell.

How are the centrioles located with respect to each other? During replication in animal cells, the daughter centriole is formed near the proximal end of the mother centriole and at 90”. In most (if not in all) cells investigated to date, the two centrioles after mitosis are not perpendicular to each other (Bessis, 1964; Murray et al., 1965; Robbins et al., 1968; Erlandson and de Harven, 1971; Schaffer, 1969; Fawcett, 1966; Vorobjev and Chentsov, 1977). Unfortunately, this fact is seldom mentioned in reviews and manuals. The mother and daughter centrioles lose their mutually perpendicular orientation at the end of mitosis or at the beginning of the interphase that follows it (Robbins et al., 1968; Erlandson and de Harven, 1971; Vorobjev and Chentsov, 1982). Thereby the centrioles can move far apart, to a distance of several micrometers (Vorobjev and Chentsov, 1982), and come together subsequently. A similar divergence of the centriolar pair was also described for centriolar replications in PtK, cells (Rieder and Borisy, 1982). Activation of neutrophils causes the centrioles to split rapidly as far as 1 Fm and more (up to 10 pm), whereas they are quite near to one another in the control cells (Schliwa et ul., 1982; Sherline and Mascardo, 1982a). It is believed that “centriolar splitting,” as the author calls it, is related to the work of actin microfilaments (Schliwa et al., 1982; Sherline

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and Mascardo, 1982b). The same explanation applies to centriolar splitting in the course of the transition from mitosis to interphase: as the cells spread, actin filaments are restructured in them. Since the centriole is a cylinder-like structure and a polar one at that, one may assume, proceeding from only geometrical concepts, that the centriolar cylinder can take a definite position in the cell-for instance, perpendicular or parallel to the substrate on which the cell lives, and in mitosis, a definite position with respect to the spindle axis is possible, etc. Nonrandom (predominantly perpendicular) disposition of the centrioles with respect to the substrate plane was described for PE cells early in interphase (Vorobjev and Chentsov, 1982), for normal mouse fibroblasts at the stage of radial spreading and during polarization (Gudima et al., 1983a),and for 3T3 fibroblasts polarizing at the monolayer edge (Gudima et al., 1983b). In spreading fibroblasts, centriolar orientation depends on the presence of MTs and does not change upon destruction of microfilaments (Gudima et al., 1983a). In mitosis the active centrioles are located mainly perpendicularly to the spindle axis at the stage of metaphase and anaphase [see Tables 1 and 111 in Vorobjev and Chentsov (1982)l. One should also point to the phenomenon of nonrandom disposition of centrioles in tissues, when they are found at one and the same level in a layer of epithelial cells (Heidenhain, 1907; Wilson, 1925; Tucker, 1984), in a malignant tumor strand (Schaffer, 1969), or are shifted within the cells in the course of embryogenesis (Foe and Alberts, 1983; Tucker, 1984).

X. Conclusion Since the publication of Fulton’s review (197I), we have learned much about the centrioles. Yet as it often happens, the new data have produced far more questions than answers. At present, we have a thorough knowledge of the three-dimensional centriolar structure (in mammals, at any rate). A large number of EM observations enable us to conceive an overall image of both centrioles and basal bodies. These organelles comprise, besides nine MT triplets, a large number of fine substructures. A detailed study into the genesis of centriolar cylinders and experiments on dissociation in vitro of isolated centrioles bring us to the conclusion that the most conspicuous centriolar cylinder component, the triplets, is not a structural basis of this cylinder; this role appears to be reserved for the centriolar matrix, a rim of amorphous material, surrounding the triplets and the axial hublike structures (the cartwheel and the pinwheel).

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Bernhard and de Harven (1960) showed that the centriole is a polar structure. Today we know that the polarity of the centriolar cylinder manifests itself, on the one hand, in the orderly organization of its substructures; and on the other, centriolar cylinder polarity is a sequel to the polarity of the MTs within the centrioles and basal bodies. The past few years, owing to the appearance of immunofluroescent methods for detection of cytoskeletal structure, have provided some fresh insights into the role of centrioles in organizing cell MTs. Although not all the MTs in interphase cells are connected to the centrosome, it has a virtual monopoly on forming new MTs. The MTs grow from the center toward the periphery; they have their minus ends in the cell center and the plus end at the periphery. It is the plus-ends that are elongated. Consequently, the cell center seems to regulate only the finding of MTs; other factors must account for their growth. The number of MTs growing from the cell center varies. It may vary depending on the cell cycle, the functional changes of the cell, and during its morphogenetic reactions. The number of MTs radiating from the cell center apparently does not depend how dense the MT network is in a cell, but on the renewal rate of that network. As indicated by immunofluorescent investigations, the MT system undergoes two drastic alterations in the cell cycle: as the cell enters mitosis, the cytoplasmic MTs disassemble and form a spindle; upon completion of mitosis, the spindle is destroyed and reconstitution of the cytoplasmic network takes place. Formation of MTs in interphase and in mitosis is related to different structures of the cell center, and so a regular succession of these structures (pericentriolar satellites in interphase and the halo in mitosis) in the cell cycle precedes a restructuring of the MT system and, most probable of all, is the cause of this restructuring. Back in the early 1960s, Dalcq (1964) pointed to the nonequivalence of the two centrioles in a cell. Detailed studies of centrioles in a cell cycle have made it possible to explain their nonequivalence, as well as the fact that at least two centrioles are present in a cell (if they are there at all!). The point is that the centriolar formation process, beginning in the S period of the cell cycle, embraces a cell cycle and a half and thus only one mature centriole, capable of performing all of its functions, gets into the cell after division, and one immature centriole, capable of replication only, gets into the cell after division. In the 1970s, centrioles were detected in fractions of isolated nuclei. Subsequent investigations have shown the centrioles to be structurally connected to the cell nucleus, and this association does really exist in a living cell.

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The nonrandom disposition of centrioles in a cell was described long ago at the light microscopic level (review: Wilson, 1925), and the latest EM observations enable us to speak about the nonrandom location of the centriolar cylinders in vitro. In certain events in the life of cultured cells (spreading and motion), the centriolar cylinders may be perpendicular to the substrate plane; and during cell division (in meta- and anaphase), the mother centrioles may be perpendicular to the spindle axis. The least results appear to have been achieved in the field of biochemical studies of centrioles and basal bodies. The only relevant fact is that the cell center and the basal bodies contain no DNA, but they contain RNA. Of late there has been a gradual resurgence of the concept of centrioles as the central structures in the life of an animal cell. Two independent approaches are applied: one focuses on the unique geometrical properties of the centrioles, their orderly structure with the ninth order central symmetry. The centrioles are attributed the role of an intracell gyroscope (Bornens, 1979); it is also presumed that they ensure a higher probability of the cell turning at 40” and not at any other angle (Albrecht-Buehler, 1981).

The other approach, in the footsteps of Pickett-Heaps (1974), includes MTOCs on the evolutionary standpoint. The centrioles are regarded as derived from the basal bodies of ancient Flagellata. According to this approach, the centrioles are no more than a structure for template-guided axonemal polymerization; they are put by in a cell for emergence, if the need arises to construct a cilium or flagellum. Furthermore, it is supposed (Onischenko, 1982) that, in the process of evolution in animal cells, a structural unification of centrioles and mitotic and cyloplasmic MTOCs into a single polyfunction system, the cell center, took place. Besides, having originated as a motility organ, the flagella and cilia, in the case of multicellular animals, acquired a function of reception. It is suggested therefore that centrioles, as derivatives from basal bodies, have retained the former receptory functions of the cilia and evolved into “intracellular receptors” (Chentsov, 1984). For all the attraction of the above-sketched suggestions, the only waterproof concept of centriolar functions is as follows: orderly initiation of the growth of the MT network. Concerning further trends in centriolar studies, we would like to stress these points: 1. Hitherto centriolar investigations have not involved mutant cell lines or mutant species, except for a few studies on Chlumydomonus basal bodies. We have found only one reference in the literature to the possibility

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of hereditary changes in the behavior of human centrioles (Afzelius, 1980). No such observations have been registered with respect to animals. So, the cytogenetic trend in centriolar apparatus studies is still an open area of research. 2. The centriole possesses considerable difficulties for biochemical analysis. Most of the functions ascribed to it are not accessible to such analysis at present and require preliminary phenomenological investigation. On the other hand, until recently, centriolar behavior could be described only with the aid of EM on serial sections. This is the most reliable, even though the most labor-consuming, and thus, inefficient method. Lately, autoimmune antibodies against centrioles have been found (Connoly and Kalnins, 1978; Maunory, 1979), which should certainly speed up investigations. However, obtaining autoimmune antibodies, unlike the conventional procedure for polyclonal antibodies, is more difficult and this may be the reason why antibodies against centrioles are not as widespread as antibodies against tubulin. Therefore isolation of a centriolar (or basal body) antigens is an urgent problem. 3. No good experimental models have been found as yet for studying centriolar functions (apart from the direct induction of MT polymerization). Only initial steps have been taken toward this end. Thus it has been shown that centriolar splitting occurs under nonspecific activation of granulocytes (Schliwa et al., 1982)and under stimulated proliferation of certain cultural cells (Sherline and Mascardo, 1982a, b); nonrandom orientation of the centrioles to the substrate in morphogenetic reactions of cells in vitro has been discovered (Gudima et al., 1983a, b). In all cases, the effect applied to a portion of the cells is from 15 to 50%. As the next step, it is essential to find some methods for comprehensive effects (i.e., in 100% of the cells) on the location and transition of centrioles, or on their activity in MT polymerization. 4. The most straightforward approach would be to obtain cells without centrioles. Such eosinophils have been obtained recently by microirradiation (Koonce et al., 1984), as well as cytoplasts, by enucleation in the presence of nocodazole and cytochalasin B (Karsenti et al., 1984b),and by a line of centriole-free cells (Debec et al., 1982). Comparing the life of the centriole-free cells, we may gain a direct understanding of what the centriole is needed for.

ACKNOWLEDGMENTS We thank Professor Yu. S. Chentsov for critical remarks and fruitful discussion. We also thank Dr. R. G. W. Anderson and Dr. 0.G . Borisy for their permission to reproduce their pictures and Dr. L. G . Bergen for a copy of his figure.

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REFERENCES Abel, J. H., Tokelainen, P. T., and Verhage, H. G. (1972). Cytobiologie 6, 468472. Afzelius, B. A. (1980). I n “International Cell Biology 1980-81” (H. G. Schweiger, ed.). pp. 440-447. Springer-Verlag, Berlin and New York. Aguas. A. P.. and Nickerson, P. A. (1981). Tissue & Cell 13, 491499. Aist, J. K., and Williams, P. H.(1972). J. Cell Biol. 55 368-389. Albrecht-Buehler. G. (1981). Cell Motil. 1, 237-245. Albrecht-Buehler, G., and Bushnell, A. (1979). Exp. Cell Res. 120, I 1 1-1 18. Albrecht-Buehler, G., and Bushnell, A. (1980). Exp. Cell Res. 126, 427437. Allen, C., and Borisy, G. G. (1974). J. Mol. Biol. 80, 381402. Allen, R. D. (1969). J. Cell Biol. 40, 716-733. Allenspach, A. L., and Roth, L. E. (1967). J . CellBiol. 33, 179-1%. Amano, S . (1957). Cytologia (Tokyo) 22, 193-212. Amos, W. B., Grimstone, A. V.. Rothschild, L. J., and Allen, R. D. (1979). J. Cell Sci. 34, 139-164. Anderson, D. C., Wible, L. J., Hughes, B. J., Smith, C. W., and Brinkley, B. R. (1982). Cell 31, 719-729. Anderson, L. C., Lehto, V.-P., Stenman, S.. Badley. R. A., and Virtanen, 1. (1981). Nature (London). 291, 247-248. Anderson, R. G . W. (1971). J. CellBiol. 50, 18, 22, 26. Anderson, R. G. W. (1972). J. Cell Biol. 54, 246265. Anderson, R. G. W. (1974). J. Cell Biol. 60, 393-404. Anderson, R. G. W. (1977). J. Cell Biol. 74, 547-561. Anderson, R. G. W., and Brenner, R. M. (1971). J . Cell Biol. 50, 10-34. Anderson, R. G. W., and Floyd, A. K. (1980). Biochemistry 19, 5625-5631. Archer, F.. and Wheatley, D. (1971). J. Anat. 109, 277-292. Aronson, J. F. (1971). J. Cell Biol. 51, 579-583. Ates, Y., and Sentein, P. (1977). Biol. Cell. 29, 31-36. Aubin. J. C., Osborn, M.. and Weber, K. (1980). J . Cell Sci. 43, 177-194. Bahr, G. F., and Sugler, W. F. (1977). Chromosoma 63, 291-303. Balczon, R., Schatten, H., and Schatten, G. (1983). J. Cell Biol. 97, (Pt.2). 245a. Barnes, B. G. (l%l). J. Ultrastruct. Res. 5,453461. Barrau M. D., Blackburn, G. R., and Dewey, W. C. (1978). Cancer Res. 38, 2290-2294. Ben-Zetv A., Farmer, S. R., Penman, S. (1979). Cell 17, 319-325. Bergen, L. G., and Borisy, G. G. (1980). J. Cell Biol. 84, 141-150. Bergen, L. G., Kuriyama, R.. and Borisy. G. G. (1980). J . CellBiol. 84, 151-159. Berkowitz, S. A., Katagiri, J., Binder, H. K., and Williams, J. R. C. (1977). Biochemistry 25, 5610-5617. Bernhard, W.. and de Harven, E. (1960). Electron Microsc. Proc. I n t . Congr. j t h , 1958 2, 2 17-227. Berns, M., and Richardson, S. M. (1977). J . Cell Biol. 75, 977-982. Berns, M. W., Leonardson, K., and Witter, M. (1976). J. Morphol. 149, 327-337. Berns, M. W., Rattner. J. B., Brenner, S., and Meredith, S . (1977). J . Cell Biol. 72, 351367. Bershadsky,A. D.,andGelfand, V. 1. (1981). Proc. Natl.Acad. Sci. U . S . A .78,3610-3613. Bershadsky, A. D., Tint, 1. S.,Gelfand, V. I., Rosenblat, V. A,, Vasiliev. Yu. M.,and Gelfand, 1. M. (1978a). Cell Biol. Int. Rep. 2, 345-351. Bershadsky, A. D., Gelfand, V. I.. Svitkina, T. M.,and Tint, I. S . (1978b). Cell Biol. In?. Rep. 2,425432.

THE CENTROSOME AND MICROTUBULE ORGANIZATION

285

Bershadsky. A. D., Gelfand, V. I., Svitkina, T. M., and Tint, I. S. (1979a). Cell Eiol. Int. Rep. 3, 12-20. Bershadsky, A. D., Tint, I. S. . Gelfand, V. I., Rosenblat, V. A., Vasiliev, Yu. M., and Gelfand, I. M. (1979b). Unfogenez 10, 231-235. Bessis. M. (1964). In “Electron Microscopic Anatomy” (S. M. Kurtz, ed.), pp. 149-181. Academic Press, New York. Bessis. M., and Breton-Gorius. J. (1957). NOUV. Rev. Fr. Hematol. 7, 601-620. Bessis, M.. Breton-Gorius, J., Theiry, J. P. (1958). Rev. Hematol. 13, 363. Binder, L.. Dentler. W., and Rosenbaum, J. L. (1975). Proc. Natl. Acad. Sri. U . S . A . 72, 1122-1 126. Black, M. M.. and Kurdyla, , J. T. (1983). J. Cell Eiol. 97, 1020-1028. Blackburn, G . R.. Barrau, M. D., and Dewey. W. C. (1978). Exp. Cell Res. 113, 183-187. Bloom, G. S., Luca, F. C.. and Vallee, R. B. (1984). J. Cell Eiol. 98, 331-368. Bordas, J . , Mandelkow, E.-M., and Mandelkow, E. (1983). J . Mol. Eiol. 164, 89-135. Borisy, G . G. (1978). J . Mol. B i d . 124, 565-570. Borisy, G. G . , and Could, R. R. (1978). Mitosis: Facts Quest Proc. Workshop, 78-87. Borkhardt, B., and Olson, L. W. (1979). Protoplasma 100, 323-344. Bornens, M. (1977). Nature (London) 270, 80-82. Bornens, M. (1979). B i d . Cell. 35, 115-132. Bouck, G. B., and Brown, D. L. (1973). J . Cell Eiol. 56, 340-359. Brenner, S. L., and Brnikley. B. R. (1982). Cold Spring Harbor Symp. Quanf. Eiol. 46, 24 1-254. Brenner, S.. Branch, A., Meredith, S., and Berns, M. W. (1977). J. CeNEiol. 72, 368-380. Brinkley, B. R., and Stubblefield, E. (1970). Adv. Cell Biol. 1, 119-185. Brinkley, B. R., Fuller. G. M., and Highfield, D. P. (1975). Proc. Natl. Acad. Sci. U.S.A. 72, 49814985. Brinkley. B. R., Fuller, G. M., and Highfield, D. P. (1976). Cold Spring Harbor Con$ Cell Proliferation 3, (Book A), 435453. Brinkley, B. R., Fistel, S. M.. Marcum, J. M., and Pardue, R. L. (1980). Int. Rev. Cytol. 63, 59-95. Brinkley, B. R., Cox, S. M., Pepper, D. A., Wible, L., Brenner, S. L., and Pardue, R. L. (1981). J . Cell Eiol. 90, 554-562. Brown, D. L., Massalski. A., and Patenaude. P. (1976). J. Cell Eiol. 69, 106-125. Brown, D. L., Steams, M. E., and Macrae. T. H. (1983). In “The Cytoskeleton in Plant Growth and Development” (C. Lloyd, ed.), pp. 55-84. Academic Press, London. Brown, R. L., Wible, L. J., and Brinkley, B. R. (1980). Cell Eiol. Int. Rep. 4, 453458. Burgos, M. N., and Fawcett, D. W. (1956). J . Eiophys. Eiochem. Cytol. 2, 223-240. Burkholden. J . D., Okada, 1. A., and Conings, D. E. (1972). Exp. Cell. Res. 75, 497-511. Burnasheva. S. A., and Solovjeva, G. A. (1980). Dokl. Akad. Nauk SSSR 254,487489. Byers, B., Shriver, K., and Goetsch, L. (1978). J. Cell Sci. 30, 331-352. Cabral, F., Wible, L.. Brenner, S., and Brinkley, B. R. (1983). J. Cell Eiol. 97, 30-39. Calarco-Gillam, P. D., Siebert, M.C., Hubble, R.. Mitchison, T., and Kirschner, M. (1983). Cell 35, 621-629. Carlier, M.-F., Hill, T. L., and Chen, J. (1984). Proc. Natl. Acad. Sci. U.S.A. 81,771-775. Chalfie, M. (1982). Cold Spring Harbor Symp. Quant. Eiol. 46,255-262. Chalfie, M., and Thomson, J. N. (1979). J . Cell Eiol. 82, 278-289. Chentsov, Yu. S. (1984). “Obschaya Cytologya.” Moskovsky Universitet, Moscow. Chesin. E. M . (1967). Tsitologiya 9, 1411-1413. Child, F. M.. and Mazia, D. (1956). Experientia 12, 161-162. Chung, J . W., and Keefer, D. A. (1976). Cell Tissue Res. 175, 421.

286

1. A. VOROBJEV AND E. S. NADEZHDINA

Cleveland, D. W., Hwo, S. -J., and Kirschner, M. W. (1977). J . Mol. Biol. 116, 207-226. Cleveland, D. W., Lopata, M. A., Sherline. P., and Kirschner, M. W. (1981). Cell 25,537546. Cleveland, D. W., Pittenger, M. F., and Feramisco, J. R. (1983).Nature (London)305,738741. Clevenger. C. V., and Epstein, A. L. (1984). Exp. Cell Res. 151, 194-207. Cohen, W. D., and Nemhauser. J. (1980). J . Cell Biol. 86, 286-291. Connoly. J. A., and Kalnins. V. I. (1978). J. Cell Biol. 79, 526-532. Dahl, H. A. (1963). Z . Zellforsch. Mikrosk. Anat. 60, 369-386. Dalcq, A. M.(1964). Bull. CI. Sci. Acad. R . Belg. 50, 1408-1449. Dales, S., Hsu, K. C.. and Nagayama, A. (1973). J. Cell Biol. 59, 643-660. Danylova. L. V. (1982). “Sovremennye Problemy Spermatogeneza,” Nauka, Moscow. Dave, A. J., and Godward, M. B. E. (1982). J. Cell Sci. 58, 345-362. David-Ferreira, 3. F. (1962). I n “Electron Microscopy” (S. S. Breese. ed.), Vol. 11, pp. XX-4. Academic press, New York. Debec, A.. Szollosi, A., and Szollosi, D. (1982) Biol. Cell. 44, 133-138. de Brabander. M. (1982). Cell Biol. Int. Rep. 6, 901-916. de Brabander, M., and Borgens, M. (1975). J. Cell Sci. 19, 331-340. de Brabander, M., Wanson. J.-C., Mosselmans, R., Genes, G . . and Drochmans, P. (1978). Biol. Cell. 31, 127-140. de Brabander, M., Geuens. G.. Nuydens, R., Willebrords, R., and De Mey, J. (1980). In “Microtubules and Microtubule Inhibitors” (M. de Brabander and J. De Mey, eds.), pp. 255-265. Elsevier Amsterdam. de Brabander, M.. Geuens, G., Nuydens, R., Willebrords, R., and De Mey, J. (1982). Cold Spring Harbor Symp. Quant. Biol. 46,227-240. Deery, W. J., and Brinkley, B. R. (1982). I n “Gene Regulation” pp. 327-341. Academic Press, New York. Deery, W. J.. and Brinkley, B. R. (1983). J . Cell Biol. 96, 1631-1641. Deery, W. J., Means, A. R., and Brinkley, B. R. (1984). J. Cell Biol. 98, 904-910. De Foor, P. H., and Stubblefield. E. (1974). Exp. Cell Res. 85, 136-142. de Harven. E. (1968). In “The Nucleus” (A. J. Dalton and F. Haguenau, eds.), Vol. 3, pp. 197-227. Academic Press, New York. de Harven. E., and Bernhard, W. (1956). Z . Zellforsch. Mikrosk. Anat. 45, 375-398. Dentler, W. L. (1977). Tissue & Cell 9, 209-222. Dentler, W.. Granet. S . , Witman, G . B., Rosenbaum, J. L. (1974). Proc. Natl. Acad. Sci. U.S.A. 71, 1710-1714. Dentler, W. L., Granet, S., and Rosenbaum, J. L. (1975). J. Cell B i d . 55, 237-241. de The, G. (1964). J. Cell Biol. 23, 265-275. Deutch, A. H., and Shumway, L. K. (1973). Protoplasma 76, 3 8 7 4 2 . Dingemans. K. P. (1969). J. Cell Biol. 43, 361. Dingle, A. D., and Fulton, C. (1966). J. Cell Biol. 31,43-54. Dingle, A. D., and Larson, D. E. (1981). BioSystems 14, 345-358. Dippell, R. V. (1968). Proc. Natl. Acad. Sci. U.S.A. 61, 461468. Dippell, R. V. (1976). J . Cell Biol. 69, 622-637. Dirksen, E. R. (1961). J. Biophys. Biochem. Cytol. 11, 244-247. Dirksen, E. R. (1971). J. Cell Biol. 51, 286-302. Dirksen, E. R.. and Crocker, T. T. (1966). J. Micros. (Paris) 5 , 629-644. Doolin. P. F., and Birge, W. F. (1966). J. Cell Biol. 29, 333-345. Dustin, P. (1980). “Microtubules” Elsevier, Amsterdam. Dustin, P. (1983). Blood Cells 9, 449-453. Erlandson, R. A., and de Harven, E. (1971). J. Cell Sci. 8, 353-397.

THE CENTROSOME AND MICROTUBULE ORGANIZATION

287

Esponda, P.. and A h a , P. (1983). Eur. J . Cell Biol. 30, 313-315. Fais, D., Nadezhdina, E. S., Chentsov, Yu. S. (1984). Eur. J . Cell Biol. 33, 190-1%. Fawcett, D. W. (1966). In “The Cell, Its Organelles and Inclusions,” pp. 49-62. Saunders, Philidelphia, Pennsylvania. Fawcett, D. W.. and Porter, , K. P. (1954). J . Morphol. 94, 221-264. Flavell, R. A., and Jones, 1. G. (1971). J . Cell Sci. 9, 719-726. Flock, A., and Duvall, A. F. (1%5). J. Cell Biol. 25, 1-8. Foe, V. E., and Alberts, B. M. (1983). J. Cell Sci. 61, 31-70. Fonte, V. G., Searls, R. L.. and Hiffer, S. R. (1971). J . Cell B i d . 49,226-229. Franke, W. W., and Reau, P. (1973). Arch. Mikrobiol. 90, 121-129. Frankel, F. R. (1976). Proc. Nufl. Acud. Sci. U.S.A. 73, 2798-2802. Friedlander, M. (1980). J . Cell Sci. 42, 221-226. Friedlander, M., and Wahrman, J. (1970). J. Cell Sci. 7, 65-89. Fuller, G. M., Brinkley, B. R., and Boughter, J. M. (1975). Science 187, 948-950. Fulton. C. (1971).I n “Origin and Continuity of Cell Organelles” (J. Reinert and H. Urspring, eds.). pp. 170-221. Springer-Verlag. Berlin and New York. Cachet. J., and Thiery, J.-P. (1964). J. Microsc. 3, 253-268. Gall, J. G. (1961). J . Biophys. Biochem. Cyfol. 10, 163-193. Gavin, R. H. (1977). J . Morphol. 151, 239. Gavin, R. H. (1980). J . Cell Sci. 44, 317-333. Gavin, R. H. (1984). J . CellSci. 66, 147-154. Geiger, B., Rosen, D., and Borke, G. (1982). J . Cell Biol. 95, 137-143. Gelfand. V. I., Ivanova, 0. Yu., Mittelman, L. A., and Pletyushkina, 0. Yu. (1984). Bull. Exp. Biol. Med. (Engl. Trunsl.) 97, 491494. Gibbons, Y . R. (1961). J . Biophys. Biochem. Cyfol. 11, 170-205. Gifford, E., and Larson, S.(1980). A m . J . B o f . 67, 119-124. Gillot, M. A., and Triemer, R. E. (1978). J. Cell Sci. 31, 25-35. Gilula, N. B., and Satir, P. (1972). J . Cell Biol. 53, 494-509. Goldman, R. D., Pollack, R., Chang, C. M., and Bushnell, A. (1975). Exp. Cell Res. 93, 175-183. Gordon, R. E., Lane. B. P., and Miller, F. (1977). J . Cell Biol. 75, 586-595. Gordon, R. E., Williams, K. B., and Puszkin, S. (1982). J . CellBiol. 95, 57-64. Gotlieb, A. I., May, M. L., Subrahmanyan, L., and Kalnins, V. I. (1981). J. Cell Biol. 91, 589-594. Gould, R. R. (1975). J . Cell Biol. 65, 65-74. Gould, R. R., and Borisy, G. G. (1977). J. Cell Biol. 73, 601-615. Gudima, G. O., Vorobjev, I. A.. and Chentsov, Yu. S. (1983a). Biol. Nuuki (Moscow) N7, 45-50. Gudima, G. 0.. Vorobjev, I. A., and Chentsov, Yu. S. (1983b). Tsifologiyu 24, 883-888. Gudirna, G. 0.. Vorobjev, I . A.. and Chentsov, Yu. S. (1984). Tsifologiya 26, 1002-1007. Haimo, L. T., Telzer, B. R., and Rosenbaum, J. L. (1979). Proc. Natl. Acud. Sci. U.S.A. 76, 5759-5763 Hamaguchi. Y.,and Kuriyama, R. (1982). Exp. Cell Res. 141, 450-454. Harris, P., Osborn, M., and Weber, K. (1980). J. Cell Biol. 84, 668-679. Hartman, H., Puma, J. P., and Gurney, T. (1974). J . Cell Sci. 16, 241-259. Heath, J. B. (1974). Mycologiu 66, 354-359. Heath, J. B. (1980). Inr. Rev. Cyfol. 64, 1-80. Heath, J. B.. and Greenwood, A. D. (1970). J. Gen. Microbiol. 62, 139-148. Heidemann, S. R., and Kirschner, M. W. (1975). J. Cell Biol. 67, 105-1 17. Heidemann, S. R., and Kirschner, M. W. (1978). J. Exp. Zool. 206, 431-444. Heidemann, S. R.. and Mclntosh, J. R. (1980). Nature (London) 286, 517-519.

288

1.

A. VOROBJEV AND E. S. NADEZHDINA

Heidemann, S. R., Sander, G., and Kirschner. M. W. (1977). Cell 10, 337-350. Heidenhain, M. (1907). “Plasma und Zelle,” Fischer Jena. Henneguy, L. F. (1898). Arch. Anal. Microsc. Morphol. Exp. I , 481. Herzog. W., and Weber, K. (1977). Proc. Nafl. Acad. Sci. U . S . A . 74, 1860-1864. Herzog, W., and Weber, K. (1978). Eur. J. Biochem. 91, 249-254. Hill, T. L., and Chen, J. (1984). Proc. Nafl. Acad. Sci. U.S.A. 81, 5772-5776. Himes, R. H., Burton, P. R., and Gaito, J. M. (1977). J. Biol. Chem. 252, 6222-6228. Hirano. K.-J., and Ishikawa, M. (1979). Dev. Growth Differ. 21, 473-481. Hoffman, E. J. (1%5). J . CellBiol. 25, 217-228. Hufnagel, L . A. (1969). J. Cell Sci. 5, 561-573. Hyams, J . S. (1984). Nature (London) 308,604-605. Hyams, J. S., and Borisy, G. G. (1978). J . Cell B i d . 78, 401-414. Hyams, J. S., and Chasey, D. (1974). Exp. Cell Res. 84, 381-387. Ishikawa, M., Ohta, T., and Kato. K. H. (1979). Acru Embryo/. Exp. 1, 21-32. Jameson, L., and Caplow. M. (1981). f r o c . Narl. Acad. Sci. U.S.A. 78, 3413-3417. Jensen. C., Jensen, L., and Rieder, C. L. (1979). Exp. Cell Res. 123, 444-449. Jirsova, Z., Kraus, R., and Martinek, J. (1974). In “Biogenesis of Cell Organelles” (M. Dvorak, ed.), pp. 249-251. J . E. Purkynd University, Bmo. Kaganovskaya, E. A. (1976). Tsitologiya 18, 171-177. Kallenbach. R. J. (1982). Biosci. Rep. 2, 959-966. Kallenbach, R. J. (1983). Eur. J . Cell Biol. 30, 159-166. Kallenbach, R. J., and Mazia, a, D. (1982). Eur. J. Cell Biol. 29, 68-76. Kalnins, V. I., and Porter, K. R. (1969). Z . Zellforsch. 100, 1-30. Karsenti, E., Newport. J., Hubble, R., and Kirschner, M. (1984a). J. Cell Biol. 98, 17301745. Karsenti, E., Kobayashi, S., Mitchison, T., and Kirschner, M. (1984b). J . Cell B i d . 98, 1763-1776. Kato. K. H., and Sugiyama, M. (1971). Dev. Growth Differ. 13, 350-366. Keryer, G., Ris, H., and Borisy, G. G. (1984). J . Cell Biol. 98, 2222-2229. Kiefer, B. J. (1970). J. Cell Sci. 6, 177. Kierhart, D. P. (1981). J . Cell Biol. 88, 604-617. Kirschner, M. W. (1980). J. Cell Biol. 86, 330-334. Koichi, K. H., and Masao, S. (1971). Dev. Growth Differ. 13, 359-366. Konishi, A., Beacham, W. S., and Hunt, C. C. (1973). J. Cell Biol. 59, 749-755. Koonce, M. P., Cloney, R. A., and Berns, M. W. (1984). J. Cell Biol. 98, 1999-2010. Krishan, A.. and Buck, R. C. (1965). J. Cell Biol. 2 4 , 4 3 3 4 . Kupfer, A.. Louvard, D., and Singer. S. J. (1982). f r o c . Natl. Acad. Sci. U.S.A. 79,26032607. Kuriyama, R. (1984). J . Cell Sci. 66, 277-295. Kuriyama, R., and Borisy, G. G. (1981a). J . Cell Biol. 91, 814-821. Kuriyama, R., and Borisy G. G. (1981b). J . Cell Biol. 91, 822-826. Kuriyama, R., and Kanstani, H. (1981). J. Cell Sci. 49, 33-49. Kuznetsov, S. A., Gelfand. V. 1.. Rodionov, V. I., Rosenblat, V. A., and Gulayeva, J. G. (1978). FEBS L e f f .95, 343-346. Kuznetsov, S. A., Rodionov, V. I., Gelfand, V. I., and Rosenblat, V. A. (1980). Cell Biol. I n f . Rep. 4. 1017-1023. Larson, D. E., and Dingle, A. D. (1981). J . Cell Biol. 89, 424-432. Lauweryns, J. M., and Boussauw, L. (1973). J. Ulfrasfruct.Res. 42, 25-28. Lazarides, E. (1976). Cold Spring Harbor Conf. Cell Proliferation 3, 347-360. Lenhossek, M. (1898). Verh. Anaf. Ges. 12, 106. Lockwood, A., and Pendergast. M. (1980). Eur. J . Cell B i d . 22, 308.

THE CENTROSOME AND MICROTUBULE ORGANIZATION

289

Longo. F. J. ( 1974).J . Cell B i d . 63, 199. Longo. F. J . (1976). J. C ~ ~Blild . 69, 539-547. Longo. F. J.. and Anderson. E. (1968). J. CeIlBiol.'39, 339-368. Lorch, 1. J . (1952). Q . J . Microsc. Sci. 93, 475-486. Lutz, D. A.. and Huebner, E. (1980). Tissue & Cell 12, 773-794. McDonald. K.. Pickett-Heaps, J. D., Mclntosh, 1. R.. and Tippit, D. H. (1977).J . CellBiol. 74, 377-388. McGill, M.. and Brinkley, B. R. (1975). J. Cell B i d . 67, 189-199. McGill, M., Highfield, D. P., Monshan, T. M., and Brinkley, B. R. (1976). J . Ultrmtruct. Res. 57, 43-53. Mclntosh. J. R. (1983). I n "Modern Cell Biology" ( J . R. Mclntosh, ed.), Vol. 2, pp. I 15142. Liss. New York. McNitt. R. (1974). frofc~plirsmii80, 91-108. McNiven. M. A,. Wang, M., and Porter, K. R. (1984). Cell 37, 753-765. Mahovald, A. P.. Caulton. J. H., Edwards, M. K., and Floyd, A. D. (1979). Exp. Cell Res. 118, 404-410. Malawista, S. E.. De Boidfleury-Chevance, A., Maunory, R., and Bessis, M. (1983). Blood Cells 9, 443-448. Malech. H. L., Root, R. K., and Gallin, J. I. (1977). J . Cell Biol. 75, 666-693. Maller, J.. Poccia, D., Nishioka, D., Kidd, P., Gerhart, J.. and Hartman, H. (1976). Exp. Cell Res. 99, 285-294. Manton. J.. Clarke, B., and Greenwood, A. D. (1956). J . Exp. Bor. 6, 126-128. Margolis. R. L., and Wilson, L. (1978). Cell 12, 1-8. Markham, R.. Frey, S.. and Hills, G. (1963). Virology 20, 88-102. Maro, B., and Bornens, M. (1980). Biol. Cell. 39, 287-290. Matera, E. M., and Davis, W. J. (1982). Cell Tissue Res. 222, 25-40. Matsusaka. T. (1967). J . Cell B i d . 33, 203-208. Matthews, J . L.. Martin, J. H., and Race, G. L. (1967). Science 155, 1423-1424. Maunory, R. (1979). B i d . Cell. 36, 91-94. Melkonian, M. (1980). BioSysfems 12, 85-104. Meza. I . , Bryan, J., and Nagle. B. W. (1975). I n "Microtubule and Microtubule Inhibitors" (M. de Brabander and J . DeMey. eds.). pp. 91-101. Elsevier, Amsterdam. Miki-Noumura, T. (1977). J . Cell Sci. 24, 203-216. Millecchia, L. L., and Rudzinska. M. A. (1970). J. Cell Biol. 46, 553-563. Mitchison, T., and Kirschner, M. (1984a). Nature (London) 312, 232-237. Mitchison, T.. and Kirschner, M. (1984b). Nature (London) 312, 237-242. Mizukami, 1.. and Gall, I. (1966). J . Cell Biol. 29, 97-1 11. Mohri, H. (1968). Nature (London) 217, 1053-1054. Mole-Bajer. J . . Bajer. A.. and Owczarzk, A. (1975). Cyfobios 13, 45-65. Moser. J . M., and Kreitner, G. L. (1970). J. Cell Biol. 44, 454459. Moskvin-Tarkhanov, M . I . , and Onischenko, G. E. (1978). T.sitologiya 20, 1436-1438. Munn. E. A. (1970). Tissue & Cell 2, 499-512. Murphy, D. B., and Borisy, G. G. (1975). Proc. Nut/. Acud. Sci. U.S.A. 72, 2696-2700. Murray, R. G., and Murray, A. S., and Pizzo. A. (1965). J . Cell Biol. 26, 601-619. Nadezhdina, E. S., Fais, D.. and Chentsov, Yu. S. (1978). CellBiol. Int. Rep. 2, 601-606. Nadezhdina, E. S., Fais, D., and Chentsov, Yu. S. (1979). Eur. J . CeIlBiol. 19, 109-115. Nadezhdina, E. S ., Fais, D., and Chentsov, Yu. S. (1980). Macromol. Funct. Cell, Sov. Itrrl. Svmp. 2nd 1980, pt. 2, 163. Nayak, R. K. (1972). J. Hisrochem. Cytochern. 20, 841-842. Nelson, W. J., and Traub, P. (1982). Cell Biol. In!. Rep. 6, 215-223. Nemere, J . , Kupfer. A., Singer. S. J. (1985). Cell Motil. 5, 17-29.

290

I. A. VOROBJEV AND E. S. NADEZHDINA

Nemhauser, I . , Joseph-Silverstein, J.. and Cohen, W. D. (1983). J . Cell Biol. 96, 979-989. Ohta, T., and Ishikawa, M. (1979). Dobursugaku Zasshi 88, 269-279. Oliver, J. M., Osborne, W. R. A., Pfeiffer, J. R., Child, F. M., and Berlin, R. D. (1981). J . Cell Biol. 91, 837-847. Olmsted, J. B., Marcum, J. M., Johnson, K. A.. Allen, C., and Borisy, G. G. (1974). J . Supramol. Strucr. 2, 429-450. Olson, G. E., and Rattner, J. B. (1975). J. Ultrastruct. Res. 51, 409417. Onischenko, G. E. (1978). Tsitologiya 20, 395-399. Onischenko. G. E. (1982). Usp. Sovrem. Biol. 94, 360-375. Onischenko, G. E., Bystrevskaya, V. B., and Chentsov, Yu. S. (1979). Dokl. Acad. Nuuk SSSR 248, 1443-1446. Osborn, M., and Weber, K. (1976). Proc. Natl. Acad. Sci. U.S.A. 73, 867-871. Osborn, M., and Weber, K. (1977). Cell 12,561-571. Pardue, R. L., Perry, G. W.,Brady, R. C., and Dedman, J. R. (1983). J. Cell Biol. 96, 11491154. Patzelt, C., Singh, A., Le Marchand, Y., and Jeanrenaud, B. (1975). Microtubules Microtubule Lnhib. Proc. In?. Symp. 1975. Pepper, D. A., and Brinkley, B. R. (1977). Chromosoma 60,223-235. Pepper, D. A., and Brinkley, B. R. (1979). J. Cell Biol. 82, 585-591. Pepper, D. A., and Brinkley, B. R. (1980). Cell Moril. 1, 1-15. Pera, F. (1975). Exp. Cell Res. 92, 419427. Perkins, F. P. (1970). J. Cell Sci.6, 629-653. Perkins, F. O., and Amon, J. P. (1969). J. Prorozool. 16, 235-257. Peterson, S. P., and Berns, M. W. (1978). J. Cell Sci. 34, 289-301. Peterson, S. P., and Berns, M. W. (1979). Exp. Cell Res. 120, 223-236. Peterson, S. P., and Berns, M. W. (1980). In?. Rev. Cytol. 64, 81-108. Phillips, D. M. (1967). J . Cell Biol. 33, 73-92. Phillips, D. M. (1970). J . Cell Biol. 44, 243-277. Phillips, S. G . , and Rattner, J. B. (1976). J. Cell Biol. 70, 9-19. Pickett-Heaps, J. D. (1%9). Cyrobios 1, 257-280. Pickett-Heaps, J. D. (1974). BioSysterns 6, 37-48. Pitelka, D. R. (1974). In “Cilia and Flagella” (M. A. Sleigh, ed.), pp. 437-469. Academic Press, New York. Przybylski, R. P. (1971). J. Cell Biol. 49, 214-221. Punin, M. Yu. (1981). Tsitologiya 23, 1109-1 116. Raes. M., de Brabander, M., and Remade, J. (1984). Eur. J. Cell Biol. 35, 70-80. Randall, J., and Disbrey, C. (1965). Proc. R . SOC. Ser. B. 162,473491. Rash, J. E., Shay, J. W., and Biesele, J. J. (1%9). J. Ulrrastruct. Res. 29, 470. Rattner, J. B., and Phillips, S. G. (1973). J . Cell Biol. 57, 359-372. Raykov, 1. B. (1978). I n “Yadro Prosteyshikh. Morphologya i Evolutsiya.” Nauka, Leningrad. Reese, T. S. (1965). J . Cell Biol. 25, 209. Reichle, R. E. (1%9). Mycologia 61, 30-51. Renaud, F. L., and Shift, H. (1964). J. Cell Biol. 23, 339. Rhein, L., Cagan, R., Orkand, P., and Dolack, M. (1981). Tissue & Cell 13, 577-587. Rieder, C. L. (1979). J. Cell Biol. 80, 1-9. Rieder, C. L., and Borisy, G. G. (1981). Chromosoma 82, 693-716. Rieder, C. L., and Borisy, G. G. (1982). Biol. Cell 44, 117-132. Rieder, C. L., Cynthia, G., Jensen, L., and Jensen, C. W. (1979). J. Ulrrastrucr. Res. 68, 173-185. Ring, D., Hubble, R., Caput, D., and Kirschner, M. W. (1980). In “Microtubules and Mi-

THE CENTROSOME AND MICROTUBULE ORGANIZATION

29 1

crotubule Inhibitors” (M. de Brabander and J. De Mey, eds.). pp. 297-310. Elsevier. Amsterdam. Ring, D., Hubble, R.. and Kirschner, M. W. (1982). J. Cell Biol. 94, 549-556. Ringo. D. L. (1967). J. Ullrastruct. Res. 17, 266-277. Robbins, E., and Gonatas, N. K. (1964). J . Cell Biol. 21, 4 2 9 4 2 . Robbins, E., and Jentzsch, G. (1969). J . Cell Biol. 40, 678-692. Robbins, E., Jentzsch, G., and Micah, A. (1968). J . Cell B i d . 36, 329-339. Rodionov. V. 1.. Gelfand. V. I., and Rosenblat, V. A. (1978). Dokl. Acad. Nauk SSSR 239, 23 1-233. Roobol, A., Havercraft, J. C., and Gull, K. (1982). J . Cell Sci. 55, 365-382. Roos, U.-P. (1973). Chromosoma 41, 43-82. Ross, A. (1968). J. Ulrrasrrucr. Res. 23, 537-539. Rubin, R. W.. and Cunningham, W. P. (1973). J . Cell Biol. 57, 601-612. Runge, M., Detrich, H. W., and Williams, R. C. (1979). Biochemisrry 18, 1689-1697. Sabatini, D. D., Bensch, K., and Barnett, R. J. (1963). J. Cell Biol. 17, 19-31. Sakaguchi, H. (1965). J. Ultrastruct. Res. 12, 13-21. Salisbury. J. L. (1982). J . Cell Sci. 58, 4 3 3 4 3 . Salmon, E. D., Leslie, R. J., Saxton, W. M., Karow. M. L., and Mclntosh. J. R. (1984). J . Cell Biol. 99, 2165-2174. Sapp. J. P. (1976). Lab. Invest. 34, 109-114. Satir, P., and Satir, B. (1964). J. Theor. Biol. 7, 123-128. Sato, C., Kuriyama, R., and Nishizawa. K. (1983). J. Cell Biol. 96, 776-782. Sauron. M. E., Marty, M. C., Maunory, R., Couvalin, J. C., Maro, B., and Bornens, M. (1984). J. Submicrosc. Cylol. 16, 133-135. Saxton, W. M., Stemple, D. L., Leslie, R. J., Salmon, E. D., Zavortink, M., and Mclntosh, J. R. (1984). J . Cell Biol. 99, 2175-2186. Schaffer, P. W. (1969). Science 164, 1300. Schatten, G., Simerly, C., Cline, C., and Schatten, H. (1983). J. Cell Biol. 97, 27a. Schiff, P. B., and Horwitz, S. 9. (1980). Proc. Natl. Acad. Sci. U.S.A. 77, 1561. Schliwa, M. (1975). Microtubules and Microrubule Inhib. Proc. Int. Symp. p.p. 215-228. Schliwa. M. (1982). J. Cell Biol. 92, 79-91. Schliwa, M., Osborn, M., and Weber, K. (1978). J . Cell Biol. 76, 229-236. Schliwa, M., Euteneuer, U., Herzog, W., and Weber, K. (1979). J. Cell Biol. 83,623-632. Schliwa, M., Pryzwansky, K. B.. and Euteneuer, U. (1982). Cell 31, 705-717. Schliwa, M., Pryzwansky, K. B., and Borisy, G. G. (1983). Eur. J . Cell Biol. 32, 32, 7585. Schmidt, K. (1969). Z. Zellforsch, 99, 357-388. Schweitzer, J., and Brown, D. L. (1984). Biol. Cell. 52, 147-160. Searle, B. M., and Bloom, S. E. (1979). J. Hered. 70, 155-160. Sharp, G., Osborn, M., and Weber, K. (1981). J. Cell Sci. 48, 1-24. Sherline, P., and Mascardo, R. N. (1982a). J . Cell Biol. 93, 507-511. Sherline, P., and Mascardo, R. N. (1982b). J . Cell Biol. 95, 316-322. Silver, R. B.. Cole, R. D., and Cande, W. Z. (1980). Cell 19, 505-516. Simpson, P. A., and Dingle, A. D. (1971). J . Cell Biol. 51, 323-328. Sloboda, R. D.. and Rosenbaum, J. L. (1978). Biochemistry, 18, 48-55. Smith-Sonneborn, J., and Plaut, W. (1967). J. Cell Sci. 2, 225-234. Smith-Sonnebom, J . , and Plaut, W. (1969). J . Cell Sci. 5, 365-372. Snell, W. J., Dentler, W. L., Haimo, L. T., Binder, L. I., and Rosenbaum, J. L. (1974). Science 185, 357-360. Snyder, J. A. (1980). Cell Biol. I n t . Rep. 4, 859-866. Snyder, J. A., and Mclntosh, J. R. (1975). J. Cell B i d . 67, 744-760.

292

I. A. VOROBJEV AND E. S. NADEZHDlNA

Solomon, F. (1980). Cell 22, 331-332. Sorokin, S. (1962). J. Cell Biol. 15, 363-377. Sorokin, S. P. (I%@. J . Cell Sci. 3, 207-230. Spiegelman, B. M., Lopata, M. A., and Kirschner, M. W. (1979a). Cell 16, 239-252. Spiegelman, B. M., Lopata, M. A., and Kirschner, M. W. (1979b). Cell 16, 252-263. Starosvetskaya, N. A. (1969). Tsitologia 21, 1228-1233. Starosvetskaya, N. A., and Kazanjev, V. V. (1977). Tsitologia 29, 275-280. Steams, M. W., and Brown, D. L. (1979). Proc. Narl. Acad. Sci. U.S.A. 76,5745-5749. Steams, M. W., and Brown, D. L. (1981). J . Ultrasrruct. Res. 77, 366-378. Steams, M. W., Connoly, J. A., and Brown, D. L. (1976). Science 191, 188-191. Steinman. R. M. (1%8). Am. J . Anat. 122, 19-56. Steinman, R. M. (1970). J. Ultrastruct. Res. 30, 423. Stephens, R. E. (1975). J . Cell Biol. 64, 408-415. Stephens, R. E. (1982). J . Cell Biol. 94, 263-270. Stockinger, L., and Cireli, E. (1965). Z . Zellforsch. 68, 733-740. Stubblefield, E. (1968). In “The Proliferation and Spread of Neoplastic Cells” (M. D. Anderson, ed.), pp. 175-189. Williams and Wilkins, Baltimore, Maryland. Stubblefield, E., and Brinkley, B. R. (1967). In “Formation and Fate of Cell Organelles” (K. B. Warren, ed.), Vol. 6, pp. 175-218. Academic Press, New York. Swale, E. M. F. (1969). Arch. Microbiol. 67, 71-90. Szollosi, D. (1964). J . Cell Biol. 21, 465-479. Szollosi, D., Calarco, P., and Donahue, R. P. (1972). J . CeIl Sci. 11, 521-541. Tamm, S. L. (1972). J . Cell Biol. 54, 39-55. Telzer, B. R., and Rosenbaum, J. L. (1979). J. Cell Biol. 81, 484-497. Thompson, W. C., Asai, D. J.. Dresdent. C., Punch, D. L., and Wilson, L. (1983). J. Cell Biol. 97, 201a. Thornhill, R. A. (1967). J . Cell Sci. 2, 591-602. Tilney, L. G., and Goddard, J. (1970). J . Cell Biol. 46, 564-575. Tilney, L. G . , Derosier, D. J., and Mulroy, M. J. (1980). J. Cell Biol. 86, 246259. Tsukita, S., and Ishikawa, H. (1980). Eur. J. Cell Biol. 22, 348-356. Tucker, J. B. (1982). In “Microtubules in Microorganisms” (P.Cappuccinelli and N. R. Moms, eds.), pp. 351-437. Dekker, New York. Tucker, J. B. (1984). J. Cell Biol. 99, 5 5 ~ 4 2 s . Tucker, R. W. (1983). Cell Muscle Motil. 3, 259-295. Tucker, R. W., Scher, C. D., and Stilles, C. D. (1979a). Cell 16, 1065-1072. Tucker, R. W., Fardee, A. B., and Fujiwara, K. (1979b). Cell 17, 527-535. Tuffanelli, D. L., McKeon, F., Kleinsmith, D., Burnham, T. K., and Kirschner, M. (1983). Arch. Dermatol. 119, 560-566. Turksen, K., Aubin, J. E., and Kalnins, V. I. (1982). Nature (London) 298, 763-765. Vallee, R. B., and Bloom, G. S. (1983). J. Cell Biol. %, 1523-1529. van Assel, R. S., and Brachet, J. (1968). J. Embryo/. Exp. Morphol. 19, 261-272. Vandre, D. D., Davis, F. M., Rao, P. N., and Borisy, G. G. (1984a). Proc. Natl. Acad. Sci. U.S.A. 81, 4439-4443. Vandre, D. D., Kronebush, P., and Borisy, G. G. (1984b). In “Molecular Biology of the Cytaskeleton” (G. G. Borisy, D. W. Cleveland and D. B. Murphy, eds.), pp. 3-16. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. von Narnack, V. M. (1%3). Z . Zellforsch. 60, 432-451. Vorobjev, I. A,, and Chentsov, Yu. S. (1977). Tsirologiya 19, 598-603. Vorobjev, 1. A., and Chentsov, Yu. S. (1980). Cell Biol. Int. Rep. 4, 1037-1044. Vorobjev, I. A., and Chentsov, Yu. S. (1982). J. Cell Biol. 98, 938-949. Vorobjev, I. A., and Chentsov, Yu. S. (1983). Eur. J . Cell Biol. 30, 149-153.

T H E CENTROSOME A N D MICROTUBULE ORGANIZATION

293

Vorobjev, 1. A,, and Chentsov, Yu. S . (1985). Tsirologiyu 27, 1101-1 105. Wang, E., Connoly, J.. Kalnins, V. I., and Choppin. P. W. (1979). Proc. Nurl. Acud. Sci. U . S . A . 76, 5719-5723. Wachmann, E., and Hennig, A. (1974). Zeitschrit fur morphologie der Tiere 77, 337-344. Waterhouse, P. D., Anderson, P. J., and Brown, D. L. (1983). Exp. Cell Res. 144, 367-376. Watt, F. M.. and Harris, H. (1980). J. CellSci. 44, 103-121. Watt, F. M., Sidebottom. E., and Harris, H. (1980). J. Cell Sci. 44, 123-133. Weber. K., and Osborn. M. (1981). In “Cytoskeletal Elements and Plasmomembrane Organization” ( G . Poste and G. L. Nicolson, eds.), pp. 1-55. North-Holland Publ., Arnst e rdam . Weber. K.. Pollack, R., and Bibling, T. (1975). Proc. Nurl. Acud. Sci. U . S . A . 72,459-463. Weisenberg, R. C. (1972). J. Cell Biol. 54, 266-278. Weisenberg, R. C., and Rosenfeld, A. C. (1975). J . CeNBiol. 64, 146-158. Welsh, M. G . , Dedman, G. R., Brinkley, B. R., and Means, A. B. (1978). Proc. Nut/. Acud. Sci. U.S.A. 75, 1867-1871. Went, H. A. (1977). Exp. Cell Res. 108, 63-73. Wheatley, D. N. (1968). Experientiu 24, 1157. Wheatley. D. N. (1969). J . Anut. 105, 351-362. Wheatley, D. N. (1974). J . Gen. Virol. 24, 395-399. Wheatley, D. N. (1982). I n “The Centriole: A Central Enigma of Cell Biology,” pp. 1-232. Elsevier, Amsterdam. Williams, N. E., Vaudaux, P. E., and Skriver, L. (1979). Exp. Cell Res. 123, 31 1-320. Wilson, E. B. (1925). “The Cell in Development and Heredity.” Macmillan, New York. Witt, L.. and Borisy, G. G. (1980). Chromosomu 81,483-505. Wolfe. J. (1970). J . Cell Sci. 6, 679-700. Wolfe, J. (1972). Adv. Cell Mol. Biol. 2, 151-192. Wolff. H. G . (1969). 2. Zellforsch. 100, 251-270. Woolley, D. M., and Fawcett, D. W. (1973). Anut. Rec. 177, 289-301. Wright, M., Moisand, A., and Mir, L. (1979). Protoplusmu 100, 231-250. Wright, R. L., Chojnacki. B., and Jarvik, J. (1983). J . Cell Biol. 96, 1697-1707. Yamasa, E. (1956). Proc. 1st. Conf. Asiu Oceania Tokyo, 247-250. Younger, K. B., Banejee, S., Kelleher, J. K., Winston, M. D., and Margulis, L. (1972). J . Cell Sci. 11, 621-637. Zackroff, R. V.. Rosenfeld, A. C., and Weisenberg, R. C. (1976). J. Suprumol. Struct. 5 , 577-589. Zamboni, L. (1971). “Fine Morphology of Mammalian Fertilisation.” Harper, New York. Zamboni, L. (1975). A m . J . Anur. 144, 521-531. Zavarzin, A. A., Punin, M. Yu.. and Tsvetkov, A. G. (1984). Tsitologiya 26, 52. Zeligs. J . D. (1979). Cell Tiss. Res. 201, 11-21. Zickler, D. (1970). Chromosomu 30, 287-304. Zorn, G. A., Lucas. J. J., and Kates. J. R. (1979). Cell 18, 659-672.