Mitosis: Dissociability of Its Events

Mitosis: Dissociability of Its Events Sibdas Ghosh*vt and Neidhard Paweletzt ‘Centre of Advanced Study in Botany, University of Calcutta, Calcutta 700019, India tResearch Program IV, German Cancer Research Center, D-6900 Heidelberg, Germany

1. Introduction Although the regulation of cell division is one of the most important basic questions in the context of development and differentiation, it still remains one of the least understood processes in cell biology. An understanding of the control mechanism of mitosis is essential not only to understand the basic control of the cell cycle but also to obtain a better insight in the basis for malignant growth. One approach to understanding this process is to analyze events associated with this process and to see how far these are interlinked. At its simplest, mitosis can be conceived as a linear sequence of events, each of which is required to take place before the next can occur. All these events may be induced by a common signal and they proceed in a cascade. On the other hand, the events may be only casually but not causally linked. In such case, omission or blocking of one or two events may not inhibit the occurrence of other events. Moreover, in such cases, the induction of these events would demand separate initiation factors or activators. In this review, we analyze some events associated with mitosis, such as condensation of chromosomes, breakdown of the nuclear envelope, microtubule rearrangement, development of trilaminar kinetochore, centrosome-kinetochore interaction, chromatid separation, chromosome movement, and nuclear reformation. Our main intention is to see how far these events are dissociable, independent, and inducible. As such, we have concentrated mainly on this particular aspect. A large number of reviews on mitosis have appeared in recent years. Some of them are referred to in this discussion. Apart from them, the readers’ attention is drawn to two special issues of journals (Science 1989, 246, 537-724; J . Cell Sci. 1989, Suppl. 12) in which a number of fine articles on the cell cycle and mitosis appeared. Experimental induction of mitotic events was first achieved by fusing mitotic cells with cells in interphase by Rao and Johnson (1970) using the cell-fusion 217

Copyright 0 1993 hy Academic Press. Inc. All rights of reproduction in any form reserved.

218

SIBDAS GHOSH AND NEIDHARD PAWELETZ

technique. Components responsible for the induction of some of the mitotic events such as chromosome condensation and nuclear envelope breakdown were first identified in Xenopus oocytes and were termed maturation promoting factor (MPF) (Masui and Markert, 1971; Wu and Gerhart, 1980). A similar activity has been subsequently reported from Xenopus embryos, yeast, sea cucumber, and mammalian cells (Sunkara et d., 1976; Weintraub et d . , 1982; Kishimoto et al., 1982; Gerhart et al., 1984). It has been suggested that MPF triggers entry of cells into mitosis by initiating a cascade of protein phosphorylation reactions leading to the expression of a series of mitotic events (MiakeLye and Kirschner, 1985; Burke and Gerace, 1986). If true, this hypothesis would mean that a blockage of one early event should result in the disruption of later events. In that case, these events would not be dissociable. The last few years have witnessed a spurt of activities in understanding the mechanism of mitosis through concerted approaches by geneticists, cell biologists, and biochemists. At this juncture, we consider that a critical analysis of the events associated with this process would help toward a better understanding of the mechanism of governing the fundamental process of cell divisionmitosis.

II. Mitotic Events A. Historical Background According to Mazia (1961), Schneider described nearly all the stages of mitosis without giving exact interpretations in 1873. By 1878-1879, several workers including Strassburger and Flemming had arrived at the definitive picture of mitosis (Paweletz, 1974). The early studies were mainly descriptive (Wilson, 1925). Experimental investigations on cell reproduction began around 1950. In his classical review on mitosis (1961) Mazia presented a list of events that were regarded at that time to be associated with the process of mitosis. Even at that time, he noted that these events could run parallel, as well as sequentially. He also described continued condensation of chromosomes even when the breakdown of the nuclear envelope was inhibited by treating sea urchin eggs exposed to 0.75 M mercaptoethanol 25 min before prophase. Evidently, it was assumed that changes of the nuclear membrane were not directly geared to the condensation of the chromosomes. In the same year, Lettr6 (1961) proposed two extreme possibilities for the regulation of cell division. These are (a) that the different mitotic events are all causally connected with each other, so that one step can take place only when the previous one has been successful (cascade hypothesis) and (b) that mitotic events are only chronologically associated with each other so that the failure of one step would not necessarily prevent the next one from

MITOTIC EVENTS

219

occurring. From a host of experimental data and earlier observations, he concluded that (a) several mitotic steps are independent of each other, (h) some events are causally linked to each other, and (c) others are only chronologically connected. Unfortunately, these studies did not receive much attention from later workers. Only recently has the regulation of mitotic events again become a matter of great interest.

6. Standard Type of Mitosis To examine the dissociability of mitotic events, we make a survey of the variations that can be observed in the mitotic processes in different organisms, particularly in lower eukaryotes. While talking of variations, one should expect a standard type of cell division to which all deviations can be compared. Although it will be difficult to define a standard type, since no standard type naturally exists, the general mitotic process that is encountered in the higher eukaryotes including mammalian system may be taken as a standard. A standard mitosis (as seen in mammalian cells; Paweletz, 1987) may be described as follows. The first visible sign of prophase is the condensation of chromatin. The fine or coarse network of chromatin strands develop into chromosomes. This process starts within the nuclear boundaries. In mammalian cells, the nucleus enlarges and becomes light. The centrosomes that have replicated in the preceding S phase develop an aster of microtubules and begin to separate. The nuclear envelope forms a funnel-shaped depression that transforms into a zone of deep folds and indentations, and the nucleolus disintegrates. In the cytoplasm, the membranes of the Golgi apparatus and of the endoplasmic reticulum rearrange and the Golgi apparatus disintegrates into vesicles. Prophase turns into prometaphase; the condensation of chromatin continues. In tissue culture, some type of cells lose contact with the neighbors and round up. Cytoskeletal microtubules disappear and the mitotic spindle is formed. The nuclear envelope opens at the base of the crypts and microtubules penetrate into the nuclear area; the nuclear envelope then breaks down. The trilaminar structure of kinetochores becomes identifiable, and kinetochore and microtubule attachment occur. The two centrosomes continue their migration to the prospective poles, thereby creating the two half-spindles to form the bipolar mitotic apparatus. At the end of prometaphase, the chromosomes become arranged in the equatorial plate of the spherical cell. In metaphase, the bipolar mitotic apparatus exhibits its typical spindle-shaped form and condensation of chromosomes reaches its maximum. The chromosomes are found oscillating around the equatorial plate. The membranous vesicles of the former Golgi apparatus are distributed all over the spindle area while the majority of the cisternae of the endoplasmic reticulum-nuclear envelope complex encase the mitotic apparatus.

220

SIBDAS GHDSH AND NEIDHARD PAWELETZ

Anaphase starts with the separation of sister chromatids and the chromatids are translocated toward the poles. Chromatin begins to decondense. A new part of the spindle, the midbody, is formed during the separation of the chromosome groups; the osmiophilic streak that is the zone of overlapping of the half-spindle becomes visible. The spherical shape of the cell transforms into an ellipsoid, which continues to elongate. Cytokinesis begins with a ring-shaped shallow cleavage furrow. The cytoplasmic membranous system undergoes further rearrangements. Telophase is characterized by the continuation of cytokinesis. The chromosomes have reached their destinations. The nuclear envelope reforms around the chromosomes and surrounds the chromosome masses to form the daughter nuclei. The midbody regresses, and the Flemming body is formed in the middle of the cytoplasmic bridge between the two daughter cells. Nucleologenesis begins, the chromatin decondenses, and endoplasmic reticulum and Golgi apparatus reappear. The cell flattens, the cytoplasmic bridge breaks between the two daughter cells, and the Flemming body is pinched off. The mitotic cycle comes to an end (Paweletz, 1987). Unlike animal cells, cleavage does not take place in plant cells. Cytokinesis in plant cells involves the construction of new plasma membranes and a new cell wall that bisects the cell. The process depends on vesicle transport and fusion directed by a microtubule complex termed the phragmoplast. The phragmoplast appears at the equator of the mitotic apparatus at late anaphase or telophase. A cell plate is formed; the cell plate and the phragmoplast grow toward the surface to make the cytokinesis complete (see Bajer and MoE-Bajer, 1972; Inout, 1981). All these events may not be directly associated with the process of mitosis; for example, nucleolar dispersion, which was earlier considered to be an essential mitotic event (Das, 1962; Gimenez-Martin et al., 1971, 1977), takes place as a consequence of mitosis (Ghosh, 1987). Even in 1961, Mazia regarded the breakdown of the nucleolus not to be an obligatory event of mitosis. For the sake of simplicity in this review, we concentrate mainly on some events that are directly associated with the process of mitosis, e.g., chromosome condensation, nuclear envelope breakdown, centrosome activation, formation of kinetochoremicrotubule attachment, splitting of centromeres, translocation of chromatids toward poles, chromosome decondensation, nuclear envelope reformation, and cytokinesis. These events can be classified in three different series: (a) the processes that take place in the cytoplasm, (b) the events linked with the formation of the mitotic apparatus, and (c) the events taking place within the nucleus and associated with the chromosomes.

C. Variations in Lower Eukaryotes Mitotic division is a multistep process by which the genetic material is equally distributed to two daughter cells, which, in general, do not differ from their

MITOTIC EVENTS

221

mother. This can be realized in a number of different ways. If during this process one or the other step is lacking or an additional step is included, but the result of the process remains almost the same, this can be regarded as a variation of the standard type. Such variations point out that the sequence of events is not strictly obligatory for the successful completion of this process. Although the variation of mitotic events is limited in higher plant and animal cells, there is much variation in lower plants and animals. An extensive and comprehensive review of variant mitoses in lower eukaryotes is presented by Heath (1980), who describes the differences of the mitotic process in algae, fungi, and protists in detail and presents them in tables. Interested readers are also referred to Kubai’s review (1975) in which the differences are discussed in relation to the evolution of the mitotic spindle. It is not the purpose of this brief survey to enumerate all major and minor variations of mitotic events in all cell types investigated so far. However, we will point to some important alterations that indicate the dissociability of mitotic events. Let us first consider the behavior of the nuclear envelope. Whereas in higher plants and animals nuclear envelope breakdown is a major event, in many lower eukaryotes the envelope persists during mitosis. Two major forms of persistent nuclear envelopes are known: In some species, particularly in Zoomastigina and Ciliophora, the nuclear envelope remains completely intact during the entire course of mitosis (Franke, 1974; Kubai, 1975; Heath, 1980). During separation of chromatids in anaphase of these cells the nucleus greatly elongates and is cleaved to give rise to two daughter nuclei; cytokinesis then follows. This type of mitosis is termed “closed cell division” or “closed mitosis.” There is a gradual transition from closed to open mitosis (our standard mitosis-as described above). In some fungal species, belonging to Heterobasidiomycetes (McCully and Robinow, 1971a,b), only a few holes are formed, which reseal around the microtubules. In some species of Rhodophyceae and fungi (see Heath, 1980), gaps in the nuclear envelope forming “polar fenestrae” develop through which the spindle is formed. A few further special features of the closed mitosis will be mentioned here. In micronuclei of some ciliates, the nuclear envelope of the daughter nuclei develops within the sheath of the mother envelope, indicating that reformation of the new envelope is independent of the existence of an “old” envelope (Franke, 1974; Heath, 1980). A very rare case occurs in Stylocephalus, in which the nuclear envelope disperses but each chromosome becomes enwrapped within a layer of double membranes (nuclear envelope) and these micronuclei are then incorporated into the spindle. In some species, the nuclear envelope remains completely intact at the beginning of mitosis, but opens in postmetaphase stage (see Heath, 1980). A very interesting case can be observed in Physarum. Aldrich (1969) described the persistence of the nuclear envelope in cenocytic plasmodia1 mitoses,

222

SIBDAS GHOSH AND NEIDHARD PAWELETZ

whereas the nuclear envelope breaks down at prophase in myxamebae of the same species. In this context, the existence of cytoplasmic control (factor) for either persistence or breakdown is discussed (Ross, 1968). We can assume that this factor is quite independent of other mitotic factors and neither the occurrence nor the lack of nuclear envelope breakdown is essential for the process of cell division, and the other mitotic events in this organism are likely to be regulated independently. In cell types with open mitosis, the complete disintegration of the nuclear envelope into smaller cisternae and patches seems to be compensated by a layer of cisternae of the endoplasmic reticulum-nuclear envelope complex around the major parts of the mitotic apparatus (Paweletz, 1981). In some algae and a few other species, a system of the endoplasmic reticulum is formed around the mitotic nuclei, resembling the formation of two concentric nuclear envelopes around the mitotic chromosomes as in Stylocephalus (Heath, 1980). A large number of vesicles are found within the closed or around the open mitosis in many species of fungi or algae, as in higher organisms. One of the most conspicuous events of mitosis is the condensationdecondensation cycle of chromatin. In higher eukaryotes, the condensation of chromatin starts at the end of the S phase and culminates at metaphase; decondensation starts in anaphase, continues in telophase, and culminates in the G , phase. However, there are some differences between the condensation-decondensation cycle of the hetero- and euchromatin of the same nucleus (Frenster, 1974). In lower eukaryotes, we can find two extremes. One is that there is no condensation of chromosomes, which are very small and show no differentiation into kinetochores, centromeres, or nucleoli-organizing regions, as in Saprolegnia (Heath, 1978) and Saccharornyces (Peterson and Ris, 1976). It is not only the size of the chromosomes that determines the condensation process, since cells with an equally small amount of DNA per chromosome as in Coprinus clearly show chromatin condensation. The other extreme is no decondensation at the completion of mitosis and the chromosomes also remain in their condensed state during interphase (e.g., in some dinoflagellates such as Euglena) (Grell, 1964; Ris and Kubai, 1974). If we assume the existence of a factor controlling the process of condensation and decondensation of chromatin, it then follows that this factor is quite independent of the signals controlling other events of mitosis. Another interesting feature can be observed in Cryptophytes and Diatoms. The condensation process leads to an apparently unorganized accumulation of chromatin around the central spindle (Pickett-Heaps and Tippit, 1978), but the chromatin does not condense into individual chromosomes. There is also a wide variation in the spindle structure and function in lower eukaryotes compared to higher organisms. In higher eukaryotes, the structure and function of the mitotic apparatus are in principle the same and the spindle is always involved in the arrangement and distribution of chromosomes. In general, two basic components can be observed: a framework of microtubules

MITOTIC EVENTS

223

(MTs) that either continuously or by overlapping runs from pole to pole (PMTs) and microtubules that connect the chromosomes (kinetochores) to the poles (KMTs). The latter are mainly involved in the movement of the genophores. In many lower eukaryotes, however, this is not the case. Excellent reviews on this subject have been presented by Kubai (1975), Heath (1980), and Fuge (1982). Functionally, the microtubules in some lower eukaryotes are quite different, which may indicate that microtubule-oriented chromosome movement in higher eukaryotes originated quite independently of other mitotic events. In some dinoflagellates, as in Crypfhecodinium cohnii, it can be seen that microtubules are present in the cytoplasm during mitosis but do not participate in the movement of chromosomes inside the nucleus (Soyer, 1969; Kubai and Ris, 1969; Ris and Kubai, 1974; Kubai, 1975). The chromosomes are transported along the inner nuclear membrane to polar regions in the elongated nucleus; a cleavage then follows, developing two daughter nuclei. There the microtubules act as cytoskeletal elements. In some other dinoflagellates, as in Trichonympha agilis and Hypermastigina, microtubules attach to the nuclear envelope to which the chromosomes are fixed at the inner side (Kubai, 1973). Here, cooperation between the microtubules and the nuclear envelope is necessary to move chromosomes. In this case, special parts of chromosomes are attached to the nuclear envelope for contact with the microtubules outside the nucleus. In fact, special chromosomal regions (kinetochores) can be found at the inner nuclear membrane (as in C . cohnii) in some pockets of the nuclear envelope, which is attached to the microtubules (as in T. agilis), or in nuclear pores or holes in direct contact with the microtubules (as in Syndinium sp.; Kubai, 1973). Kinetochores have been considered part of the nuclear envelope (Franke, 1974; Pickett-Heaps, 1974), but perhaps they are distinct chromosomal regions that developed contacts with the nuclear envelope in some lower eukaryotes as a means of transportation to the poles. It is now well established that in higher eukaryotes the kinetochores are intranuclear without any attachment to the nuclear envelope (Ghosh and Paweletz, 1987a; Paweletz and Lang, 1988). In lower eukaryotes, the morphology of the kinetochores ranges from very small funnel- or disc-like structures, as in C . cohnii, Haplozoon axiothellae (Siebert and West, 1974), and Amphidinium sp. (Oakley and Dodge, 1974), which are almost indistinguishable from the nucleoplasm, to large multilayered complexes, as in Oedogonium (Coss and Pickett-Heaps, 1973, 1974). In most lower eukaryotes, the kinetochores are without typical trilaminar differentiation (in higher plants the kinetochores are ill-defined, less-electron-dense ball-like structures), but are quite able to fulfill their role in chromosome distribution in mitosis either by attachment to the nuclear envelope or the microtubules directly or by means of the nuclear envelope. In species in which mitosis is closed, the presence of intranuclear kinetochores is doubtful (see Heath, 1980). In the micronucleus of Paracineta limbata intranuclear MTs are formed at mitosis. These MTs are parallel and no definite attachments to either the nuclear envelope or the poorly defined chromosomes is

224

SIBDAS GHOSH AND NEIDHARD PAWELETZ

apparent (Hauser, 1972). In Trypanosoma rhodiense the MTs radiate from an intranuclear spindle pole body and elongate until the pole bodies reach the nuclear envelope. Microtubules lengthen and the polar bodies are pushed. Chromatins become distributed along the inner nuclear membrane, well-separated from the central microtubular mass. Here also, there are no definite connections between the MT and the chromosome (Vickerman and Preston, 1970). The nuclear envelope possibly plays a role in genophore distribution. In general, cell division comprises karyokinesis and cytokinesis. Under normal conditions, karyokinesis is followed by cytokinesis. In many lower eukaryotes, e.g., in Myxomycetes Physurum, the plasmodium is a syncytium in which intranuclear divisions take place synchronously without subsequent cytokinesis. Upon a special signal, however, cell walls are formed and cytokinesis proceeds to develop myxamebae (Lloyd et al., 1982). A large number of fungal, algal, sporozoal, and cilophora species present cenocytic conditions, in which karyokinesis is not followed by cytokinesis (Heath, 1980). There is a wide range of variation in the structure of the spindle pole in lower eukaryotes. As Heath (1980) states, “there is perhaps no other feature of mitosis that exhibits such a range of variation as the structures that lie at the poles of the mitotic spindle.” On the basis of the variation in the structure, they have different names such as spindle plaque, centrosomal plaque, nucleus-associated body, nucleus-associated organelle, spindle pole body, and centrosome (Heath, 1980), but the function of these polar structures (whether intra- or extranuclear) remains the same: to embody the mitotic pole. The presence of centriole is not always essential in the polar body (to be discussed later). It seems to be an accessory structure. The presence or absence of centrioles can be demonstrated in the same organism depending on its physiological state: in Myxomycetes (Nuegleria) and in few other lower organisms (Heath, 1980), centrioles are not always present during vegetative mitosis, but instead they synthesize centrioles de novo when flagellum production is necessary. Kubai (1975) and Heath (1980) have presented excellent accounts of mitotic variation in lower eukaryotes. They have tried to establish evolutionary sequences from very primitive types of cell division to a standard type such as that found in higher eukaryotes. This review does not focus on evolutionary sequence of mitosis. However, it is clear that many of the major mitotic events did not evolve simultaneously, as evidenced in lower eukaryotes. The events evolving independently are also likely to have independent control.

D. Variations in Higher Eukaryotes There have been consistent reports of different types of variations of the mitotic process in a few forms of higher eukaryotes, some of which later proved to be misinterpretations. There has been some consistent discussion on the possible

MITOTIC EVENTS

225

persistence of the nuclear envelope in plant cells as a boundary around the mitotic apparatus (Wada, 1955, 1957; cited in Mazia, 1961). However, these were light-microscope observations. It is now known that layers of cisternae of the endoplasmic reticulum-nuclear envelope complex can be present around the major parts of the mitotic apparatus (Paweletz, 1981). In light microscopy, this may give an impression of a persistent nuclear envelope. In many plant cell types, a DNA-replication cycle can be observed within the nuclear envelope and without spindle formation. These are endomitotic and endoreduplication cycles (Nagl, 1981). Both cycles lead to endopolyploidy. Endomitosis (Geitler, 1939) and endoreduplication (Levan and Hauschka, 1953) differ from each other in that structural changes comparable to those seen in mitosis occur in the former event, whereas no mitosis-like process can be seen during an endoreduplication cycle. Endomitosis as such can be regarded as an intraenvelope variant of mitosis, leading to a high degree of polyploidy in different plant tissues. Endomitosis leading to polyploidization can also be occasionally met in animal cells. Mammalian bone marrow megakaryocytes reach an octaploid state (Paulus, 1968). Such endopolyploidy is also noted in silk gland cells of Bombyx mori, trophocytes of insect ovaries, etc. (Nagl, 1981). Another interesting deviation from endomitosis is the nonseparation of sister chromatids in endoreduplication so that in the next mitosis each chromosome exhibits four (two pairs) chromatids (Takanari and Izutsu, 1981; Goyanes and Svartzman, 1981). In repetitive endoreduplication (Levan and Hauschka, 1953; Rizzoni and Palitti, 1973), chromosomes show four pairs of chromatids (quadruplochromosomes). The chromatids fail to separate, since the G, cells are believed to escape mitosis (to be discussed later). Apart from polyploidization due to endoreduplication and endomitosis, other types of polyploid cells, as caused by the action of antimitotic drugs are often formed in differentiated regions of plants (Nagl, 1981) and different animal tissues, as in liver and other glands, megakaryocytes, and vegetative ganglia (Brodsky and Uryvaeva, 1977). In specific tissues of mammals and also of some other animals, polyploid cells are formed as a result of aberration in the mitotic process during the later phases after the separation of chromosomes. Such a mode of formation of polyploid nuclei is termed “mitotic,” in contrast to endomitotic polyploidization. In Drosophila, fragmentation of the nuclear envelope occurs at the spindle pole only (Strafstrom and Staehelin, 1984), resembling the semiopen mitosis found in many lower eukaryotes. The most prominent type of “abnormal” mitosis found in higher eukaryotes (especially in plants) is the delayed cytokinesis and often cytokinesis not following karyokinesis. The most common occurrence can be found in nuclear endosperms. Free nuclear conditions may persist throughout as in Floerkea, Limanthes, and Oxyspora or the wall formation may take place later as in Helianthus, Triticum, and Haemanthus (Bhatnagar and Sawhney, 1981).

226

SIBDAS GHOSH AND NEIDHARD PAWELETZ

Such phenomena can also be encountered in animal cells. During the embryonic development of Drosophila, only nuclear divisions take place at the beginning and a syncytium is formed from the fertilized egg. The nuclei move to the cortex. From the fourteenth division on, however, the embryo becomes cellularized and all nuclear divisions are now always followed by division of the cytoplasm (Zalokar and Erk, 1976; Foe and Alberts, 1983). In following sections, experimental evidence will be put forward to show that karyokinesis is a process independent of cytokinesis, but that karyokinesis and cytokinesis have been interlinked to ensure smooth cellular division.

111. Dissociation of Mitotic Events A. Genetic Evidence The problem of the dissociability of mitotic events has been successfully approached by the study of cell cycle mutants and temperature-sensitive (ts) mutants. Three main cell types or organisms have been used for this purpose. Lower prokaryotes (especially fission and budding yeasts) have been studied extensively and they have contributed much to the understanding of cell division. Drosophila, another main object investigated by geneticists, has been shown to develop mutants that are defective in the regulation of individual mitotic steps. Mammalian cells cultivated in vitro represent the third group of cell types in which a number of mutants blocking or altering mitotic events have been observed. 1. Lower Eukaryotes In an excellent review, Hartwell (1978) has enumerated lower organisms in which cell or division cycle mutants have been found, such as S. cerevisiae, Schizosaccharomyces pombe, Aspergillus nidulans, Tetrahymena pyriformis, Ustilago maydis, Physarum polycephalum, and Chlamydomonas reinhardtii. However we will mainly concentrate on the results obtained from yeast. A cautious estimation of the number of genes involved in regulating the cell division cycle assumes as many as 400 genes; at present about 50 cell division cycle (cdc) genes have been isolated and identified (Meeks-Wagner et al., 1986). Since mitosis in yeast takes place within the nucleus, and cells and nuclei of yeast are very small, it is difficult to distinguish individual mitotic events. According to Hartwell (1978), the cell cycle in S. pombe can be subdivided into DNA synthesis, nuclear division, early plate formation, late cell plate formation, and cell separation stages. In S. cerevisiae spindle pole body (SPB) duplication, SPB separation, initiation of DNA synthesis, bud emergence, nuclear migration,

MITOTIC EVENTS

227

early nuclear division, medial nuclear division, late nuclear division, cytokinesis, and cell separation can be recognized. There are cell cycle mutants affecting all cell cycle events, but we discuss here only those mutants concerned with the mitotic events. All mutants detected so far are temperature-sensitive cells that traverse the cell cycle undisturbed at their permissive temperature. However, when they are transferred to the nonpermissive (restrictive) temperature they become arrested at a respective “landmark.” Seven mutated genes can be found in cells that show defects in nuclear divisions (Culotti and Hartwell, 1971). It is likely that defects could be correlated with normal mitotic events, as seen in mammalian cells; the authors have, however, used different terminology suited to yeast mitosis such as mitotic arrest at an early stage or a medial stage or a late stage of nuclear division. Four other genes are responsible for defects in cytokinesis (Hartwell, 1971). At the restrictive temperature cells of these mutants undergo several rounds of DNA synthesis and nuclear division, but cytokinesis is blocked, which leads to the development of multinucleate cells. Methylbenzimidazole-2-yl carbamate (MBC) inhibits the division cycle between DNA synthesis and the completion of nuclear divisions (Quinlan et al., 1980). Using MBC as a tool, Wood and Hartwell (1982) tried to analyze the MBC-sensitive step by means of cdc mutants. The completion of DNA replication was found to be independent of the execution of the MBC-sensitive steps. Uemura and Yanagida (1986) found mutations of the topoisomerase I1 locus (top 2). At the nonpermissive temperature, such mutants show an uncoordinated mitosis. Normally the spindle is formed and starts to pull, but because the chromosomes are not condensed, they behave abnormally. The spindles show normal kinetochore function and spindle elongation. Here the independence of the process of chromosome condensation from all other mitotic events is evident. The authors furthermore show that topoisomerase I1 is intimately involved in the condensation-decondensation cycle of chromatin. Hiraoka ef al. ( 1984) have isolated a ts mutant that is defective in the production of P-tubulin. At the restrictive temperature, this mutant lacks a spindle and cytokinesis does not take place, even though the chromosomes condense normally. Toda et al. (1984) also report a mutant defective in a-tubulin. Two genes [nim 1, (new inducer of mitosis) and cdc 251 are responsible for the initiation of mitosis, which may be induced at a reduced cell size (compared to the wild type) when cdc 25 is lacking and is compensated by an increased expression of nim 1 (Russel and Nurse, 1987a). Mutants of the Wee 1 locus start cell division at a smaller size than the wild type. If the mitotic inducer of cdc 25 genes is overproduced, the activity of the Wee 1 locus is necessary to prevent a lethal premature mitosis. Cell division is delayed until the cells have grown to a larger size as soon as the Wee 1 expression is increased (Russel and Nurse, 1987b). The product of Wee 1 is obviously an inhibitor of mitosis. These products of the mitosis-regulating genes have been shown to be protein kinase

228

SIBDAS GHOSH AND NEIDHARD PAWELETZ

homologs. Although these data have no bearing on the dissociability of mitotic events, they clearly show that the initiation of mitosis is regulated independently of the control of cell size, supporting the cell bi-cycle theory of Mazia (1974). Hartwell et al. (1974) proposed a scheme of the cell cycle of S . cerevisae derived from mutant phenotypes. According to this scheme, cdc mutants 9, 13, 16, 17, 20, and 23 block medial nuclear division; cdc 5, 14, and 15 block late nuclear divisions; and cdc 3, 10, l l , and 13 inhibit cytokinesis. Nurse et al. (1976) have proposed a scheme of the cell cycle of S . pomhe, showing diagnostic landmarks of the mutant types. According to this scheme, mutants 1, 2, 5 , and 6 block nuclear division; cdc 7, l l , 14, and 15 block early cell plate formation, and cdc 3, 4, 8, and 12 inhibit late cell plate formation. Thus, a large number of genes that control different events are involved in mitosis in both budding and fission yeasts. Naturally, these gene products may be considered independent factors. Frankel and colleagues have analyzed temperature-sensitive mutants in T. thermophila. In this organism, division is accompanied by the formation of a second oral apparatus and the development of a fission zone that precedes the cleavage furrow. In this ciliate, a macro- and a micronucleus are present, both of which under normal conditions go into mitosis before cleavage occurs. Frankel et al. (1980) isolated mutants in which micronuclear division takes place normally, whereas macronuclear division is totally suppressed. The formation of the fission zone is also prevented. In some other mutants, the fission zone is fully developed, but complete constitution is inhibited. Another type of mutant shows a somewhat altered development of the oral apparatus but cannot enter normal cell division (Frankel et al., 1980). Cleffmann and Frankel (1978) obtained mutants in which macronuclear division and cell divisions are blocked at the restrictive temperature, whereas the first stages of micronuclear division can take place undisturbed. DNA replication is also not affected. Frankel er al. (1980) described a series of cell division arrest (cda) mutants that shows different types of blocks in macro- and micronuclear division, development of the oral apparatus, and formation of the fission zone or cleavage at the restrictive temperature. Although the results obtained from Tetrahymena are not as conclusive as those obtained from yeasts, these mutants show that the typical stages of cell division in this species appear to be rather independent of each other. Similar ts mutants have also been isolated from Paramaecium tetraurelia (Jones and Berger, 1982). As in Tetrahymena the formation of a fission zone always precedes the cleavage furrow in normal division. In one mutant, only defective fission zones (dfz mutant) are formed, whereas karyokinesis and cytokinesis occur normally. In the defective constriction (dc) mutant nuclear division takes place undisturbed and the fission zone is formed, but constriction is only attempted and not completed. Thus, some of the main events of cell division can also be dissociated in Paramaecium. In A. nidulans, Morris (1976a) described a temperature-sensitive mutant that is blocked in nuclear division. This UV ts 706 mutant accumulates mitotic spindles

MITOTIC EVENTS

229

and condensed chromosomes at an elevated temperature. A normal course of mitosis is resumed as soon as the temperature is downshifted from 40 to 32°C. Moms therefore concludes that the block occurs at anaphase. In this stage, the chromosomes remain condensed, the spindle does not regress, and the nucleus cannot divide. This shows that despite the normal beginning of mitosis, the block can occur at anaphase. The effects of the UV ts 706 mutation can be compared to the action of a number of antimitotic drugs, but the block is likely to be explained here by the assumption of a factor(s) controlling the postmetaphase mitotic stage. Thus, the old idea that once mitosis has started normally it must continue can no longer be maintained. The role of an anti-MPF factor (Adlakha et al., 1983; Gerhart et al., 1984) and the dilution of MPF activity (Miake-Lye and Kirschner, 1985) in the progression of postmetaphase mitotic events will be discussed later. A heat-sensitive P-tubulin mutation, benA33 of A. nidulans, blocks nuclear division and nuclear movements at the restrictive temperature (Oakley and Morris, 1981). Shifting of benA33 to the nonpermissive temperature results in the inhibition of chromosome movement to the poles (anaphase movement). However, the formation of the spindle is not blocked and the mitotic apparatus appears normal. The product of benA33 hyperstabilizes the spindle microtubules, preventing disassembly and thus causing arrest of chromosome movement. These effects can be compared to the action of D,O or taxol on spindle movement (Burgess and Northcote, 1969; Schiff et al., 1979). Although products of benA33 increase the stability of mitotic microtubules, thereby blocking nuclear division at anaphase, tubA1 and tubA4 mutations (Gambino et al., 1984) destabilize spindle and cytoplasmic microtubules and thus also block mitosis. Weil et al. (1986) isolated mutants of A. nidulans (microtubule-interacting protein, mip) that can suppress the block of benA33 at restrictive temperature and allow normal nuclear division at that temperature. The mip mutations are cold-sensitive. In a detailed comprehensive study on mitotic mutants in A. nidulans, Morris (1976b) reported 45 temperature-sensitive mutants that are defective in nuclear division, septation, and distribution of nuclei within the mycelium. The ts blocked in mitosis (bim) E7 mutant overrides normal control systems that prevent mitosis from prematurely occurring during S or G, (Osmani et al., 1988). In the never in mitosis (nim) group, the cells are blocked just before mitosis and cell division cannot take place. The bim mutants enter mitosis but cannot complete it. In some of these mutants, chromosome condensation does take place normally and the intranuclear spindle is formed, but the spindle is much smaller than that in the wild type. Because of its small size, it cannot move the chromosomes. Moreover, normal chromosome condensation and spindle regression do not take place. Here again the regulation of mitosis does not follow a cascade, but is actually a coordination of several events. The dissociability of cytokinesis can be documented in the septation (sep) mutations in which nuclear division is completely normal, but septation is

230

SIBDAS GHOSH AND NEIDHARD PAWELETZ

inhibited. In the bimB strain (Boothroyd and Moms, 1986), giant nuclei are formed with a large number of small nucleoli. Here, chromosomes condense after reduplication, a spindle is not formed, and nuclear cleavage is prevented. After a definite time at the restrictive temperature, some of the giant nuclei cleave into smaller nuclei, generally with only one nucleolus, demonstrating that nuclear cleavage can be greatly delayed. Again, nuclear cleavage is independent of spindle formation. This situation is somewhat similar to the delayed cytokinesis in endosperm of certain plant species (Bhatnagar and Swahney, 1981). 2. Drosophila

The dissociability of the mitotic events has been extensively studied in mutants of Drosophila. Gelbart (1974) found a mutant (mitotic loss inducer, mit) in which definite chromosomes were lost in the course of mitosis. In these mutants, which appear to be very similar to some ts mutants of S. cerevisiae (Meeks-Wagner et al., 1986), neither karyokinesis nor cytokinesis is disturbed or inhibited. The failure of some chromosomes to be incorporated into the spindle may be genetically controlled and perhaps its regulation is independent of the system of regulations of all other mitotic events. A number of mutations in Drosophila were found by Smith et a/. (1985) and Gatti er al. (1983). They isolated mutants defective in condensation of chromatin. The functions of some mutant genes were necessary for the condensation of heterochromatin (mus 101) but not euchromatin. Other mutants affect the condensation of both types of chromatin (Gatti et al., 1983). Still other mutants, such as 1( 1) ZW. - 10, produce a large number of mitotic nondisjunctions, presumably due to premature centromere (kinetochore) separation (Smith er a/., 1985). The progeny of such cells exhibit an aneuploid genome. It has been shown (Smith et al., 1985) that mitosis is not inhibited or severely disturbed in these mutants, although some of the mitotic events are disturbed. Ripoll et al. (1985) describe a cell division mutant of Drosophila with an abnormally functioning spindle (asp). In this mutant, the spindle is altered. Cells are arrested in metaphase for a definite period of time and then the nuclear envelope is reformed, resulting in highly polyploid cells. Not only the mitotic but also the meiotic spindle is affected, resulting in e.g., diplo or nullo gametes. The light-microscopical images greatly resemble those of cells that are arrested in metaphase by colchicine-like spindle poisons. However, the authors demonstrated that a spindle was present in both mitotic and meiotic cells. In this respect, they resemble the benA33 mutant of A. nidulans. Thus, chromosome decondensation and nuclear envelope reformation are events that are independent of the presence and/or the function of the spindle. Freeman et al. (1986) describe a recessive maternal effect lethal mutant that they term giant nucleus (gnu). This mutant makes it evident that nuclear events of mitosis are uncoupled from many of the cytoplasmic ones. In gnu embryos,

MITOTIC EVENTS

231

the centrosomes replicate and separate independently of all other mitotic events. These centrosomes are responsible for the nucleation of the mitotic asters that can be found in the periphery of the embryo. As already described, in the wild-type embryo the first 13 nuclear divisions take place without cytokinesis and the nuclei are found in a syncytium. During the fourteenth nuclear generation, the syncytium forms cellular membranes and about 5000 cells can be found. In the gnu mutant, DNA synthesis continues despite the lack of nuclear divisions. This finally results in a few giant nuclei, from which the name for this mutant is derived. Although nuclear divisions do not take place at the beginning, the centrosomes continue to replicate and separate. They then migrate into the cortex of the syncytium and nucleate a few giant asters. The nuclei break down and some spindle-like structures are formed. From this mutant, it is evident that the centrosome cycles are independent of the nuclear cycle. A large number of cell cycle mutants of Drosophilu have been reported so far. An excellent review of these studies has been presented by Glover (1989). Some of these mutants, such as mit(3)R2, mit(3)R72, and mit(3)R135, mimick the effect of colchicine. Mutants l(l)d.deg3 and l(l)d.deglO display overcondensed chromosomes and split chromatids, with no anaphase, which thus leads to polyploid cells (Gatti and Baker, 1988; cited in Glover, 1989). Endoreduplication has also been noted by these authors in mutant 1(3)13m281. A mutant polo shows abnormal spindle poles (Sunkel and Glover, 1988). Another mutant, merry-go-round (mgr), shows functional monopolar spindles (Gonzalez et al., 1988), which possibly arise due to a failure of centrosome division or due to the failure of centrosome pairs to separate. Another mutant, string (stg), blocks G, of interphase 14 of Drosophila embryo, which is the first zygotically controlled mitosis (Edgar and O’Farrell, 1989). String protein has been shown to be homologous to cdc 25+ from S . pomhe, an activator of cdc 2+, a constituent of MPF (Edgar and O’Farrel, 1990; Jimenez et ul., 1990). There are more mutants in Drosophila (Freeman et al., 1986; Glover, 1989). Many of these mutants support the assumption that the process of cell division is not a cascade but rather a sequence of events at least some of which may be regulated independently in a temporal order. 3. Mammalian Cell Lines Mammalian cells are of particular interest for the study of the regulation of mitosis, including cytokinesis. The elaboration of new techniques in tissue culture of mammalian cells enabled scientists to look for mutants that could provide some information about the genetic control of cell division. In 1969, Naha was successful in isolating ts mutants from a monkey cell line that had been treated with a mutagen. Since then, a large number of ts mutants showing blocks and deficiencies in the course of the cell cycle have been selected (Wissinger and

232

SIBDAS GHOSH AND NEIDHARD PAWELETZ

Wang, 1983). Smith and Wigglesworth (1972) obtained a ts mutant that grew and divided normally at 31"C, but that developed binucleate cells when transferred to 39°C. Obviously, cytokinesis was inhibited, whereas karyokinesis could proceed undisturbed. Hatzfeld and Buttin (1975) isolated a ts mutant of a Chinese hamster cell line that was defective in cytokinesis at the restrictive temperature. However, along with rnultinucleate cells, cells with one giant nucleus with up to 100 chromosomes were also formed. Here, some unknown disturbance to karyokinesis also occurred in association with blockage of cytokinesis. Another ts mutant of Chinese hamster ovary (CHO) cells was isolated by Thompson and Lindl (1976). At the restrictive temperature, cytokinesis was inhibited. Both polyploid and multinucleate cells were produced. Ultrastructural studies revealed the failure of the midbody to develop, the part of the spindle between separating groups of chromosomes during anaphase and telophase. This is unique and has not been found elsewhere. There is no drug known that can mimick this effect. On the other hand, it is well known that the different parts of the microtubular spindle (aster, half-spindle, midbody, Flemming body) exhibit different sensitivity to cold or microtubular poisons, resulting in deficiency of only one part of the spindle at a threshold dose. This CHO ts mutant seems to be defective in production of compounds determining the differential sensitivity of the microtubules. Wang (1974) reported a ts mutant, ts-655, that grew and divided normally at the permissive temperature of 33°C. A shift to 39°C blocked many of the mitotic cells in metaphase. The light microscope showed that chromosome condensation and nuclear envelope breakdown took place normally but in metaphase the chromosomes accumulated in the central part of the cell. In this ts mutant, Wang et al. (1974) reported normal spindle formation at metaphase, which indicates that the blockage is due to some other reasons but not due to abnormal spindle function, unlike the asp mutant of Drosophila or BenA33 mutant of A. nidulans. It rather resembles the ts bim G mutant of Aspergillus (Doonan and Morris, 1989) and cold-sensitive disjoining-defective (cs-dis) mutants of fission yeast (Ohkura et al., 1988). Another interesting mutant was isolated by Wang (1976), which when transferred to 39°C showed defects in prophase progression. Chromatin condensed into dense clumps and no typical chromosomes were formed. Although the presence of the nuclear envelope was clearly evident at the beginning, the nuclear boundary could no longer be identified in later stages, indicating that the nuclear envelope had broken down in the normal sequence, the clumping of chromatin continued, and nuclear reformation could not be recognized. At the restrictive temperature, cells were arrested in prophase. The data show nuclear envelope breakdown without being followed by other mitotic events. In another mutant, ts 546 (Wang and Yin, 1976), the cells continued their cell cycle and proceeded until metaphase when switched to the nonpermissive temperature. Prophase and prometaphase took place undisturbed but then the chromosomes clumped and co-

MITOTIC EVENTS

233

alesced into aggregates. The nuclear envelope reformed around these clumps of chromatin and then the chromatin decondensed, but cytokinesis did not follow. A ts mutant of murine leukemic cells described by Shiomi and Sat0 (1976) was found to be blocked in karyokinesis (probably during metaphase) and cytokinesis did not take place. Chromatin condensation, breakdown of the nuclear envelope, arrangement of chromosomes into metaphase-like configuration at the beginning of the shift to the nonpermissive temperature, and the typical rounding-up process occurred undisturbed, indicating not only that these events can be dissociated from the late mitotic events, but also that the late mitotic events are independent of spindle formation and its function. Another interesting ts mutant of Syrian hamster cells has been reported by Wang el al. (1983). It shows some disturbances in mitosis at the nonpermissive temperature. Cells that have reached prometaphase or metaphase at the shift from 33 to 39°C pass through undisturbed and result in two normal daughter cells. After 15 min of exposure to the nonpermissive temperature, the spindle formation is altered. A bipolar spindle cannot be formed. Instead, the chromosomes are arranged in a shell at the cell periphery; microtubules are present and connect the chromosomes to the four closely associated centrioles, which obviously form a monopolar mitotic apparatus near the center of the cell. This spherical monopolar mitotic apparatus soon transforms into a conical half-spindle in which the chromosomes become arranged in a metaphase-like configuration. Chromosomes move within this half-spindle in an ordered way, but chromatids do not separate. The chromosomes decondense, the nuclear envelope is reformed, and all chromosomes remain in one nucleus. Cytokinesis is attempted, but fails. Such cells can go into several subsequent rounds for up to 5 days, resulting in cells with hundreds of chromosomes. Here the types of microtubules responsible for the separation of the centrosomes and the erection of a bipolar spindle are, perhaps, lacking. It is obvious that only one type of microtubule is defective, whereas the rest are functional. We now have enough evidence from mutants of lower eukaryotes, Drosophila, and ts mutants of animal cell lines that cell division is not a cascade event, but at least some of these events can be dissociated from others without endangering the whole process or affecting the later events. Other experimental evidences have also confirmed these observations.

6 . Other Experimental Evidence

1. Cytokinesis Is Independent of Other Mitotic Events In general, mitosis comprises karyokinesis followed by cytokinesis. It has already been mentioned that in a large number of lower eukaryotes karyokinesis

234

SIBDAS GHOSH AND NEIDHARD PAWELETZ

is not followed by cytokinesis resulting in the formation of syncytia or cenocytic cells. Such abnormality is also encountered in endosperm of higher plants and in the embryonic development of Drosophila. In yeast, a number of mutants, cdc 3, 10, l l , and 13, inhibit only cytokinesis, allowing other nuclear events to proceed. Cytokinesis can also be experimentally inhibited or delayed from the events of karyokinesis. In a classical experiment, Kihlman and Levan (1949) showed that caffeine could inhibit cytokinesis in plant cells, resulting in the formation of binucleate cells. Cytokinesis in plants itself comprises overlapping phases such as ( a ) production of Golgi vesicles, (h) vesicle accumulation, (c) vesicle arrangement, and (d)vesicle coalescence (Lopez-Saez et al., 1982). Caffeine inhibits vesicle fusion, thus inhibiting cytokinesis (Paul and Gaff, 1973). Other chemicals such as methylxanthine and 2,6-dichlorobenzonitrile are also known to cause inhibition of cytokinesis in plants (Gonzales-Reyes et al., 1986). In animal cells, cytochalasin B, a mold metabolite from Helminthosporium demafioideum, causes multinucleate cell formation by suppressing daughter cell separation following an otherwise normal nuclear division (Carter, 1967;Smith et al., 1967). In time lapse cinematographic observations, the treated mitotic cells round up, chromosomes segregate, and a normal cleavage furrow is seen to develop. The resulting daughter cells move away from each other, but remain connected by a bridge showing the midbody. This connecting bridge fails to break and the daughter cells reunite and form a large binucleate cell (Krishan and RayChaudhuri, 1969). Binucleate cells can also be induced by metabolic inhibitors such as sodium vanadate (Navas et al., 1986). This is possibly due to the inhibition of ATPase by this metabolic inhibitor (Cande and Wolniak, 1978).When the drug is withdrawn from the medium, binucleate cells are found to undergo cytokinesis and convert to the mononucleate state (own observations). This phenomenon may indicate that the factor for cytokinesis was present but could not act due to energy depletion. We have seen that karyokinesis may occur without being followed by cytokinesis. However, no cell type is known in which cytokinesis takes place under normal conditions without prior karyokinesis. Under definite experimental conditions (Lettrk, 1961),cleavage can be observed after which one “cell” is anucleate, whereas the second daughter cell contains all the genetic material. In erythroblasts, the nucleus is extruded under normal conditions, giving rise to anucleate erythrocytes. This process may be compared to the one referred to above. Hiramoto (1956, 1965) removed the mitotic apparatus in fertilized sea urchin eggs by micromanipulation or by injection of sucrose solution. He could also displace the mitotic apparatus by injecting paraffin, oil, or seawater. In all cases, cleavage took place almost normally despite the absence of the mitotic apparatus or its displacement. In the myxomycete Physarum, the plasmodium is a syncytium in which karyokinesis is not followed by cytokinesis. However, upon a special signal cell wall formation is initiated and karyoki-

MITOTIC EVENTS

235

nesis is followed by cytokinesis to develop myxamebae. Similarly, cytokinesis in Drosophila embryonic development follows karyokinesis only from the fourteenth division onward. Such late wall formation is also reported in the free nuclear endosperm of certain higher plants. In the developing endosperm of Citrullus fistulosus (Chopra, 1955; cited in Bhatnagar and Swahney, 198l), the haustorium becomes cellular by segmentation into multinucleate chambers. These chambers finally subdivide to give rise to uninucleate cells. Here, the process of cytokinesis in delayed cellularization is not immediately preceded by karyokinesis. These observations indicate that cytokinesis requires a special signal (factor), but in normal mitosis it is being coordinated with karyokinesis.

2. Dissociability of Mitotic Apparatus Associated Events from Other Mitotic Events About half a century back it was noted by Blackeslee (1937) that in plant cells the chromosome number could be doubled by treatment with colchicine. Later colchicine was found to be the most specific poison acting on nearly all types of cells of animal and plant kingdom, combining effectively with tubulin (Eigsti and Dustin, 1955; Deysson, 1975). Colchicine depolymerizes the microtubules and inhibits polymerization of tubulin, but allows the other mitotic events to proceed. The chromosomes condense and the nuclear envelope breaks down, but the events associated with the mitotic apparatus are inhibited. The chromosomes remain arrested in metaphase for a considerable length of time and fail to segregate, but the chromatids separate, indicating splitting of centromeres. Then, the decondensation of chromatin starts and a new nuclear envelope is formed (Fig. 1). Cytokinesis is inhibited, resulting in the formation of a polyploid cell or a cell with a number of nuclei formed from single or a group of chromosomes (Ghosh and Paweletz, 1984a). The effect of this chemical clearly shows that the events associated with the mitotic apparatus are quite independent of other mitotic events. Moreover, cytokinesis is also a dispensable event of mitosis. It also indicates that separation of sister chromatids is not a direct function of the microtubules (Lambert, 1980). After the discovery of colchicine several other substances were found to have an action similar to that of colchicine, the best known of which are podophyllotoxins, the vinca alkaloids such as vinblastine and vincristine (Kelly and Hartwell, 1954; LettrC, 1965). Hexachlorocyclohexane or gammexane was found to have a similar effect in plant cells (D’Amato, 1969). A large number of other substances have been reported to be microtubular poisons in specific cases (see Table 5.3, Dustin, 1978). It is of interest to note that an antibiotic, griseofulvin, obtained from Penicillum griseofulvum induces polyploidy in the myxomycete P. polycephalum, which has the closed type of mitosis (Gull and Trinci, 1974). A recent addition to the list of these inhibitors is nocodazole, which shows an effect almost identical to that of colchicine (DeBrabander et a/., 1986).

236

SIBDAS GHOSH AND NEIDHARD PAWELETZ

FIG. 1 Electron micrograph of a colcemid-treated rat kangaroo cell showing chromosome decondensation and nuclear envelope reformation around metaphase chromosomes, which failed to show anaphase movement. Bar, 1 pm. (From Ghosh and Paweletz, 1984a. with permission.)

Heavy water and taxol, a diterpenoid isolated from Taxus brevifolia, have effects opposite to those of colchicine and promote microtubule assembly (Burgess and Northcote, 1979; Schiff et al., 1979). Taxol also stabilizes microtubules against cold and microtubule inhibitors (DeBrabander et al., 1986). But the net effect of taxol on mitosis seems to be similar to that of microtubule inhibitors. Chromosome condensation proceeds, the nuclear envelope breaks down, and cells round up, but the chromosomes remain dispersed and immotile in an abortive metaphase stage followed by formation of restitution nuclei and readhesion of the cell. The inactivation of the mitotic chromosome movement is caused by the additional and abnormal assembly of microtubules (DeBrabander et a / . , 1986). Here again, we find that mitotic apparatus (MA)-associated events are quite dissociable from the other mitotic events. Spontaneously arising polyploid cells are occasionally met in differentiating plant tissues (Nagl, 1981) and in some animals, including mammalian cells (Brodsky and Uryvaeva, 1977). These polyploid cells are believed to be formed as a result of aberrations in the mitotic process itself. The mode of formation of such polyploid nuclei is termed mitotic (Brodsky and Uryvaeva, 1977). It seems

MITOTIC EVENTS

237

that antimitotic drugs mimick these naturally occurring polyploids and they result from a mitotic cycle in which some of the events are missing. That the events associated with the mitotic apparatus are independent of the chromosome cycle in mitosis is evident from some other experiments using metabolic inhibitors, such as 2,4-dinitrophenol and sodium azide, which cause inhibition of anaphase chromosome movement (Hepler and Palevitz, 1985; Spurck et al., 1986; Ghosh el al., 1989). These arrested chromosome groups are able to form restitution nuclei. These results are somewhat comparable to the effect of taxol. Even the anaphase type of chromosome movement is not sequential to other mitotic events. McNeill and Bems (198 1) irradiated one of a pair of kinetochores of chromosomes arranged in the equatorial plane with a laser microbeam. Immediately, all the chromosomes moved to the pole to which the other chromatid was attached. Thus anaphase-type movement including the shortening of KMT fibers may occur even during metaphase. With respect to that particular chromosome, the MA is monopolar. Monopolar mitotic apparatus has been reported in a ts mutant of Syrian hamster cells (Wang et al., 1983), as already discussed. The Drosophila cell line mutant mgr is a!so functionally monopolar (Gonzalez et al., 1988). Such monopolar MA was experimentally induced in fertilized sea urchin eggs after treatment with mercaptoethanol (Mazia et al., 1981). In such cells chromosomes condense, the nuclear envelope breaks down, and chromosomes are aligned in a metaphase-like configuration and are then transported to one pole in an anaphase-like movement. The second halfspindle does not develop. A nucleus is reconstructed, complete cleavage fails, but attempts are made. Similar results are described in newt lung cells (Bajer et al., 1980), where a monopolar mitotic apparatus is spontaneously formed but as a rare event. In sea urchin, Harris (1983) noted a caffeine-induced monoaster cycling in fertilized eggs. In cold-sensitive lethal mutant ndc-1-1 of yeast, chromosomes remain attached to one pole and thus delivered to one daughter cell only (Thomas and Bottstein, 1986). It is a mutant of a cell cycle gene required for attachment of chromosomes to the spindle pole. However, normally a monopolar mitotic apparatus is formed due to the failure of the centrosomes to migrate to opposite poles and the chromosome-to-pole connections are made only by kinetochores that face centrosomes (Mazia et al., 1981). The centrosome embodies the spindle pole and its presence is universal in eukaryotic cells (Mazia, 1987), which has been fully established after the demonstration of the presence of centrosomes in higher plants using anticentrosomal antibodies (Wick, 1985). There can be accessory structures, which can be temporarily or permanently associated with centrosomes. Centrioles represent such structures and often serve as indicators of centrosomes in different cell types. However, centrioles are absent in typical barrel-shaped spindles in plant cells (Bajer and MoE-Bajer, 1972). As such, centrioles are not regarded necessary for mitosis. In multipolar spindles of higher animal cells, some poles can lack centrioles (Keryer et al., 1984). Mitosis proceeds undisturbed after selective laser

238

SIBDAS GHOSH AND NEIDHARD PAWELETZ

microbeam destruction of the centriolar region in PtK2 cells in prophase (Berns and Richardson, 1977). In cells of higher animals, centrosomes undergo a cyclic development (Paweletz et al., 1984; Mazia, 1984, 1987). Although in general the centrosome cycle is coordinated to the mitotic cycle, it can be uncoupled by experimental means. In cells with arrested chromosome cycle with DNA synthesis inhibited by arabinosyl cytosine, the centrioles divided independently (Rattner and Phillips, 1973). Paweletz et al. (1984) demonstrated that the centrosomal cycle of fertilized sea urchin eggs can proceed whereas the chromosomal cycle is arrested by mercaptoethanol. In some insects (e.g., the gall midge; Wolf, 1980) asters can undergo up to four divisions, although nuclear divisions do not take place. When PtK1 cells are arrested in metaphase by means of colcemid for a long period, the number of centrosomes increases steadily by duplication, although nuclear division does not take place (Dey et al., 1989). The independence of the chromosome cycle from the centrosome cycle has been precisely recorded by Sluder et al. (1986). They showed that centrosomes in an enucleated cytoplasm in sea urchin eggs replicated with precise periodicity, indicating cytoplasmic control of the centrosome cycle. Naturally, it is expected that the mitotic events associated with the chromosomes and nuclei would be independent of events associated with the centrosomes. This has also been demonstrated in multinucleate cells obtained by cell fusion. Using a peroxidase-antiperoxidase method for the detection of polymerized tubulin in fused multinucleate cells, we (Armas-Portela et al., 1988) demonstrated that the transition of the microtubular cytoskeleton from interphase to mitosis is independent of the factor(s) responsible for chromatin condensation and nuclear envelope breakdown. Mitotic asters can be induced to form even around interphase nuclei (Fig. 2). In an earlier publication, we (Ghosh and Paweletz, 1987b) showed that S phase prematurely condensed chromosomes (PCCs) fail to interact with microtubules, although they contained fully formed kinetochores (Fig. 3). Consequently, they very often fail to segregate. Okadaic acid-induced PCCs also fail to organize metaphase spindle. It is likely that the premature centrosomes fail to respond to the factor( s) that induces nuclear envelope breakdown and premature chromosome condensation in the corresponding nuclei. Recently we have shown (Ghosh et al., 1992) that okadaic acid (0A)-induced PCCs in HeLa cells not only fail to organize a metaphase spindle, but also fail to develop trilaminar kinetochores.

3. Dissociability of Chromosomal and Nuclear Events in Mitosis The most conspicuous events in mitosis are the changes associated with chromosomes. The chromosomal events include condensation of chromatin in distinct chromosomes containing two chromatids, appearance of trilaminar

MITOTIC EVENTS

239

FIG. 2 Asynchrony in the rearrangement of microtubules in a fused multinucleate HeLa cell. (a) Bright-field microscopy reveals the presence of mitotic asters near the prometaphase chromosome groups, but interphase microtubule in the other part. Note one of the mitotic asters in front of an interphase nucleus (arrow). (b) Fluorescence microscopy with Hoechst 222358. Bar, 20 pm. (From h a s - P o n e l a ef al., 1988, with permission.)

kinetochores, and separation of centromeres along with the chromatids followed by decondensation of chromatin, whereas the nuclear events consist of breakdown of the nuclear envelope and disintegration of the nuclear lamina and their reorganization in telophase. Like the disintegration of the nucleolus, the disorganization of the nuclear matrix network (Ghosh el al., 1978; Ghosh and Dey, 1986) may also be regarded as a nuclear event. But these are only consequences and are not directly involved in the process of mitosis per se. The first indication of the independence of the chromosome-related events came from observations on endomitotic cycles (Nagl, 1981). Endomitosis is regarded as an intraenvelope variant of mitosis, leading to high degrees of polyploidy in different plant tissues and mammalian cells, already described. Endoploidy has also been induced by various chemicals such as azaguanine (Nuti-Ronchi et al., 1965), hydroxylamine sulfate (Lin and Walden, 1974), and 3-deoxyadenosine (Gimenez-Martin et a!., 197I).

240

SIBDAS GHOSH AND NEIDHARD PAWELETZ

FIG. 3 (a) S-phase PCC with very low degree of chromosome condensation but with fully developed kinetochore plates (arrow). (b) exhibits PCC kinetochores without microtubule attachment. Bar, 1 pm. (From Ghosh and Paweletz, 1987b. with permission.)

In this type of mitosis, the chromosomes condense, the kinetochores split, and the chromatids separate all within the nuclear envelope and without the formation of a mitotic spindle. It indicates that the chromosome cycle is independent of other mitotic events, although in normal cells they are highly coordinated. Moreover, it can be inferred that centromere splitting is a chromosomal event, which is also apparent from colchicine-treated cells (Lambert, 1980). Endoreduplication also leads to endopolyploidy (Levan and Hauschka, 1953), but here the chromosomes do not show structural change and the centromeres fail to separate. Endoreduplication has been observed to arise spontaneously in a number of cell types, as already mentioned. A larger incidence of this phenomenon can be induced by colcemid (Herreros and Gianelli, 1967; Rizzoni and Palitti, 1973), mitomycin C (Takanari and Izutsu, 1983), hydrazine (Speit ef al., 1984), and a number of other chemicals and also in plant cells by 8azaguanine (Nuti Ronchi et al., 1965) and hexyl mercury bromide (Levan, 1971). However, data on endoreduplication are scarce. In this case, we must presume that the G , chromatin in the absence of mitotic division must enter the second cell cycle to undergo another round of DNA replication to give rise to diplochromosomes in the following mitosis (Schwarzacher and Schnedl, 1965). However, it is known that G, nuclei are unable to synthesize DNA in fused multinucleate cells even in the presence of S-phase nuclei (Rao and Johnson,

MITOTIC EVENTS

241

1970). The chromosomes have a continuous coiling cycle and at the end of G, they are maximally decondensed when S phase and DNA replication can begin (Mazia, 1987; Pederson, 1972). The G, chromosomes, although not so condensed as the mitotic chromosomes, must undergo decondensation before the initiation of the next DNA replication phase. As such, in endoreduplication the G2 cells are presumed to enter the next G , phase without entering the M phase. The trilaminar kinetochores appear in prometaphase (Rieder, 1982). Their development could be observed even on prematurely condensed chromosomes (Ghosh and Paweletz, 1987b). It can be assumed that in the endoreduplicated cells, the kinetochores do not develop in the absence of the mitotic factors, and centromeres fail to split and the chromatids fail to separate in absence of mitosis. Endoreduplication represents a cell cycle that omits the mitotic phase, whereas in endopolyploidy mitosis takes place without involving the nuclear and the centrosome-associated mitotic events (intraenvelope). Other evidence indicating the dissociability of chromosomal and nuclear events of mitosis can be considered in two broad categories: (a) the early mitotic events, including chromosome condensation, nuclear envelope and nuclear lamina breakdown, development of trilaminar kinetochores, and splitting of chromatids; (b) postmetaphase mitotic events, such as reformation of the nuclear envelope and nuclear lamina and decondensation of chromatin.

a. Early Mitotic Events Mitotic chromosome condensation is part of the chromosome-coiling cycle (Mazia, 1987) and the chromosomes are already well condensed when the nuclear envelope breaks down at the onset of prometaphase. As such, these two events are unlikely to be controlled by a common factor. Mazia (1961) noted continued condensation of chromosomes even when the nuclear envelope breakdown was inhibited in mercaptoethanol-treated sea urchin eggs. In fused multinucleate cells, we (Ghosh and Paweletz, 1984b) observed that the nuclear envelope breakdown was often delayed in certain nuclei but the chromosomes could reach a fully condensed state (Fig. 4). Obviously, these events are not likely controlled by a single factor and should be regarded as dissociable. This is also supported by the observations of Wagenaar (1983a). He noted that sea urchin embryos did not show chromosome condensation and mitosis when the protein synthesis was inhibited 25 min after fertilization. When the protein synthesis was inhibited 30 min after fertilization, the chromosomes condensed but the nuclear envelope failed to break down and the cells were arrested at prophase. The results indicate that chromosome condensation and nuclear envelope breakdown are controlled by two separate factors. When mitotic cells are fused with interphase cells, the chromatin of interphase cells shows premature chromosome condensation (Rao et al., 1977). The PCCs lack the nuclear envelope and show different degrees of chromosome condensation. In PCCs chromosome condensation initiates only after breakdown of the nuclear envelope (Peterson and Berns, 1979; Ghosh and Paweletz, 1984b). This

242

SIBDAS GHOSH AND NEIDHARD PAWELETZ

FIG. 4 Electron micrograph of a fused multinucleate HeLa cell, showing one well-advanced nucleus in close proximity to two less-advanced nuclei. The chromosomes are highly condensed but are still enclosed with the nuclear envelope. Bar, 1 fim. (From Ghosh and Paweletz, 1&7a, with permission.)

sequence of events is quite different from that found in normal mitosis. This phenomenon actually represents premature nuclear envelope breakdown with incomplete chromosome condensation. Premature chromosome condensation is induced by the MPF synthesized or activated by another nucleus residing in the same cytoplasm. As such, premature chromosome condensation cannot be observed in mononucleate cells. A temperature-sensitive mutant, BN 2 of the BHK cell line (Syrian hamster fibroblast), may undergo premature chromosome condensation and other early mitotic events at the restrictive temperature (Nishimoto et al., 1978, 1985).

MITOTIC EVENTS

243

The block to M-phase initiation can be overcome by treatment with caffeine, which induces premature chromosome condensation, nuclear envelope breakdown, and mitosis-specific phosphoprotein synthesis in synchronized BHK cells arrested in early S phase (Schlegel and Pardee, 1986). The dependence of mitosis on completion of DNA synthesis is lost in Wee mutants in fission yeast (Enoch and Nurse, 1990) (discussed later in relation to the checkpoint hypothesis). The ts bim E7 in Aspergillus also overrides the normal control system that prevents mitosis from occumng prematurely during S or G, (Osmani et al., 1988). Okadaic acid at a concentration specifically inhibiting phosphatase 1 can also induce PCCs (Yamashita et al., 1990). A possible molecular mechanism of this phenomenon has been discussed by Enoch and Nurse (1991). Mitosis in the absence of chromosome replication is disastrous, so cells had to develop a control maintaining dependence of mitosis on chromosome replication. However, even the initiation of mitosis is not nondissociably coupled with the completion of S phase. From their biochemical experiments using an in vitro system, Newport and Spann (1987) conclude that chromosome condensation occurs independently of nuclear envelope breakdown and lamina depolymerization. Chromosome condensation can be specifically inhibited by competition for a putative binding protein, whereas lamina depolymerization remains unaffected. Chromosome condensation is also blocked by inhibitors of topoisomerase I1 (Wright and Schatten, 1990). Topoisomerase I1 has been identified as a major component of protein fractions derived from mitotic chromosomes (Eamshaw et al., 1985; Eamshaw and Heck, 1985). The development of the trilaminar structure of kinetochores can be seen only after nuclear envelope breakdown (Rieder, 1982). This could indicate that kinetochore plate formation might depend on nuclear envelope breakdown, being triggered by cytoplasmic factors diffusing into the rupturing nucleus (Roos, 1973). However, we (Ghosh and Paweletz, 1987a) were able to demonstrate the presence of fully developed kinetochores on chromosomes still enclosed within the nuclear envelope (Fig. 5). This could indicate that the same factor is not responsible for the breakdown of the nuclear envelope and the development of the kinetochore plates. Fully developed kinetochores have been observed on PCCs belonging to G I , S, and G, phases (Szollosi et al., 1986; Ghosh and Paweletz, 1987b). It is possible that the same factor that controls chromosome condensation also controls the development of kinetochores. However, the top 2 mutant of yeast shows formation of normal kinetochore with normal function on abnormal chromosomes that fail to condense (Uemura and Yanagida, 1986). This shows that kinetochore plate formation is an event independent of even chromosome condensation. The PCCs belonging to the G I or S phase often fail to be connected to the spindle fibers (Ghosh and Paweletz, 1987b), due to unsynchronized chromosomal and centrosomal cycles, as has already been discussed. However, kinetochore development itself seems to be a chromosomal event.

244

SIBDAS GHOSH AND NEIDHARD PAWELETZ

FIG. 5 Electron micrograph of a fused multinucleate HeLa cell (part), showing condensed chromosomes still enclosed within the nuclear envelope. Fully formed trilaminar kinetochores are visible on two chromosomes (arrow). Bar, 1 pm. (From Ghosh et al., 1987a. with permission.)

Balczon and Brinkley (1987) reported the presence of a specific protein complex on metaphase chromosomes that is contiguous with kinetochore-bound tubulin and may be involved in microtubule-kinetochore interactions during mitosis. In that case, kinetochore-microtubule interaction may depend on the synthesis of a special type of protein. Cells entering into mitosis when treated with protein synthesis inhibitors often show irregularities in microtubule kinetochore attachment (Wagenaar, 1983b), which may indicate that the factor controlling the microtubule-kinetochore interaction may be synthesized during G,, before the cells enter mitosis. Recently the role of a group of proteins (inner centromeric proteins, INCENPs) isolated from the mitotic chromosome scaffold of MSB 1 cells (chicken) in sister chromatid pairing and separation has been claimed (Cooke et al., 1987, 1990). In metaphase chromosomes, these proteins have been located all along between two chromatids. In colcemid-blocked diplochromatids they appear restricted to the centromere. Another type of protein, chromatid linkage protein (CLiP) has been isolated from sera from a CREST (calcinosis, Raynauds phenomenon, esophageal dismotility, sclerodactyly, and telangiecta-

MITOTIC EVENTS

245

sia) patient, which has been shown to have identical chromosomal locations (Rattner et a!., 1988). Although these two groups of proteins seem to have identical functions, they differ structurally. It has long been claimed that chromatid separation is a chromosomal function and that chromatids can separate even in the absence of microtubule attachment to the kinetochores, as in colchicinetreated cells (Mol&-Bajer, 1958; Lambert, 1980). Even in acentric chromosomal fragments, sister chromatids are seen to separate simultaneously with their centric partners (Carlson, 1938). The assumed role of INCENPs or CLiPs in chromatid separation could explain this behavior (Earnshaw and Rattner, 1989). It is possible that some modifications of INCENPs or CLiPs occur at metaphase stage, which change their chromatid linking property, and the chromatids fall apart. This modification would then be governed by some definite mitotic factor. Alterations of such factors by mutations may also explain the behavior of ts bimG mutants of Aspergillus and cs-dis mutants of fission yeasts. It is interesting to note that both of these mutants fail to encode phosphoprotein phosphatase 1 (Doonan and Moms, 1989; Ohkura et al., 1989). On the contrary, the high frequency of aneuploid nuclei in Drosophila (1) ZW-I0 mutants appears to be the consequence of premature separation of sister chromatids at prophase-metaphase and their subsequent independent regulation at anaphase (Smith et al., 1985). This mutant may lack the CLiP or INCENP necessary for the cohesion of sister chromatids. Recently, however, it was suggested that the INCENPs represent a new class of proteins, chromosomal passenger proteins, that are canied to the spindle equator by the chromosomes and subsequently perform a cytoskeletal role following their release from the chromosomes at the metaphase-anaphase transition (Earnshaw and Cooke, 1991). Recently we demonstrated (Ghosh and Paweletz, 1992) that phosphatase 1 inhibition at metaphase by okadaic acid induces failure of sister chromatid separation even in mammalian cells. The visible nuclear event during initiation of mitosis is the breakdown of the nuclear envelope. Disassembly of the nuclear envelope begins at prophase when the pore complexes disappear and the nuclear membranes are fragmented, forming small vesicles that disperse throughout the cytoplasm and become indistinguishable from membranes of the endoplasmic reticulum (Roos, 1973). The nuclear lamina which is a supramolecular protein assembly associated with the nucleoplasmic surface of the inner nuclear membrane is depolymerized in coincidence with the disassembly of the nuclear envelope (Gerace and Blobel, 1980). Phosphorylation of lamin may lead to the disassembly of the nuclear lamina which may in turn trigger nuclear envelope breakdown. However, there is ample evidence to indicate that the nuclear envelope breakdown and lamina disassembly are dissociable events. The lamina polypeptides appear in the cytoplasm long before the nuclear envelope has disappeared (Jost and Johnson, 1981). Using a cell-free system, it has been observed that structural proteins of the nuclear lamina are hyperphosphorylated within 15 min after addition of MPF, followed by gradual depolymerization of the nuclear lamina

246

SIBDAS GHOSH AND NEIDHARD PAWELETZ

until the nuclear envelope breaks down 30 min later (Miake-Lye and Kirschner, 1985). Even the disassembly of the nuclear lamina itself may not be responsible for the breakdown of the nuclear envelope. In oocytes, the nuclear lamina disappears during zygotene, but then reappears in diplotene (Stick, 1987) within an intact nuclear envelope.

b. Postmetaphase Mitotic Events Apart from anaphase A and anaphase B movements, other postmetaphase mitotic events are chromosome decondensation, nuclear envelope reformation, and reassembly of lamina. These are chromosomal and nuclear events. That these events are not dependent on successful completion of anaphase movement is evident from the effect of a large number of drugs that lead to the arrest of anaphase movement. Examples of these drugs are colchicine, nocodazole, taxol, and dinitrophenol, whose effect on microtubules has already been discussed. In fused multinucleate cells, we (Ghosh and Paweletz, 1987c; Ghosh et al., 1988) observed that neither decondensation of the chromatin nor dissolution of the spindle was associated with the induction of nuclear envelope reformation (Fig. 6 ) . Similarly, in colcemid-induced multinucleate cells nuclear reformation was found to be induced around metaphase chromosomes by diffusible factors from nearby telophase groups. In 2,4-dinitrophenol-treatedcells, nuclear envelope reformation could be observed (Ghosh et al., 1988, 1989) around condensed anaphase chromosomes with distinct trilaminar kinetochores and microtubular attachment (Fig. 7). It seems that ATP depletion does not inhibit nuclear envelope reformation per se. On the other hand, a striking chromatin condensation results from ATP depletion (Newmayer et al., 1986). It is very likely that these two events are dissociable. The assembly of the nuclear lamina is concurrent with the reformation of the nuclear envelope (Gerace and Blobel, 1980). Burke and Gerace (1986) noted a telophase-like reconstruction of the nuclear envelope around endogenous mitotic chromosomes in a cell-free system involving total homogenates from CHO metaphase cells along with dephosphorylation and assembly of the lamina around metaphase chromosomes in that array. Neither of these processes require free ATP. However, nuclear envelope reformation can take place without the formation of a lamina, as has been demonstrated by Benavente and Krohne (1986) after microinjection of lamina antibodies. Although these events are dissociable, they may be triggered by a common factor and both processes are regulated by protein dephosphorylation (Burke and Gerace, 1986). Fusion between mitotic and interphase cells demonstrates that cells in mitosis contain cytoplasmically transmissable factors that are able to induce both breakdown of the nuclear envelope and condensation of chromatin in interphase cells (Rao and Johnson, 1970). Subsequently, this cytoplasmic factor has been isolated and has been purified to a great extent (Sunkara et al., 1976; Wu and Gerhart, 1980; Adlakha et al., 1985; Lokha et al., 1988).

MITOTIC EVENTS

247

FIG. 6 Electron micrograph of part of a fused multinucleate HeLa cell exhibiting meta- and/or anaphase chromosomes with microtubule attachment to kinetochores (arrow).Nuclear envelope formation around these chromosomes is almost complete. Bar, 2 pm. (From Ghosh and Paweletz, 1 9 8 7 ~with . permission.)

Originally, the activity of the MPF-inducing germinal vesicle breakdown (GVBD) and chromosome condensation was demonstrated in amphibian oocytes leading to meiotic maturation. Mitotic maturation has also been reported to be induced by MPF (Kishimoto et al., 1982; Halleck et al., 1984). Maturation promoting factor activity has been observed even in starfish, in sea cucumber (Kishimoto et al., 1982), and in yeast cells (Weintraub et al., 1982); MPF has been isolated from metaphase chromosomes, also (Adlakha er al., 1982). Even in a cell-free system MPF has been found to induce early mitotic events such as chromosome condensation, nuclear envelope breakdown, lamina disassembly including hyperphosphorylation of the lamina, and formation of the spindle (Lokha and Maller, 1985; Miake-Lye and Kirschner, 1985; Suprynowicz and Gerace, 1986). Spindle formation obviously indicates the development of trilaminar kinetochores on condensed chromatin through induction by MPF. It has been suggested that MPF triggers entry of cells into prophase by initiating a cascade of protein phosphorylation reactions leading to chromosome condensation, nuclear envelope breakdown, and other early mitotic events (Miake-Lye and Kirschner, 1985; Burke and Gerace, 1986). The activity of the MPF can be preserved by a cytostatic factor (CSF), which is thought to maintain metaphase arrest by stabilization of MPF (Masui er al., 1980; Newport and Kirschner, 1984).

248

SIBDAS GHOSH AND NEIDHARD PAWELETZ

FIG. 7 Electron micrograph of part of an “anaphase” cell treated with 2.4-dinitrophenol showing reconstruction of nuclear envelope around chromosomes with distint trilaminar kinetochores and microtubule attachment. Bar, 1 pm. (From Ghosh et al., 1988, with permission.)

As MPF can induce mitotic events that appear one after another, it is believed that MPF induces a cascade mechanism that ultimately induces all the nuclear events (Miake-Lye and Kirschner, 1985; Burke and Gerace, 1986). In the cascade mechanism there is a substrate-product relationship. As such, the completion of earlier events is necessary for the initiation of later events. in this survey work we have presented many examples where later mitotic events run almost unhampered even though earlier events failed to occur. As an alternative to the cascade hypothesis Hartwell and Weinert ( 1989) have proposed a checkpoint hypothesis. According to this hypothesis the completion of an earlier event acts as a checkpoint for the later events. This hypothesis is mainly based on observations of some yeast ts mutants defective in DNA replication functions. Apart from the budding yeast mutants, several other mutants of fission yeast (Enoch and Nurse, 1990) and A. niduluns (Osmani et ul., 1988) and BN, mutants of a BHK cell line (Nishimoto et al. 1978) are known in which M phase does not depend on the completion of the S phase. This dependency is also abolished in

MITOTIC EVENTS

249

hamster cells treated with caffeine (Schlegel and Pardee, 1986) or mammalian cells treated with okadaic acid (Yamashita er al., 1990; Ghosh er al., 1992). In fission yeast activation of the cdc 2 kinase at M phase requires the product of the cdc 25 gene. Again, the cdc 25 activity is countered by two inhibitors, Wee 1 and mik 1, which together maintain the cdc 2 in an inactive state (Enoch and Nurse, 1991). At the molecular level the entry into M phase requires tyrosine/phosphorylation of p34 and dephosphorylation is blocked when replication is inhibited. Uncoupling of M phase from S phase may be induced by premature dephosphorylation of p34, through loss of inhibitors or by overexpression of activators or by chemical treatments. Thus the dependency is not a block in the cascade (substrate-product relationship), but is due to an intrinsic control, which is a coordination of several molecular events. Hartwell and Weinert (1989) have also cited a dependence of anaphase on metaphase, and the delay in the transition when metaphase is hampered, as evidence of a checkpoint. The best example of such delay can be observed in colchicine-treated cells. Here, the arrest in metaphase is accompanied by delayed degradation of cyclin B (Minshull et al., 1989; Lewin, 1990). Anaphase transition is also delayed in mutants deficient in phosphatase 1 activity (Doonan and Moms, 1989; Ohkura et al., 1989). These are, as such, intrinsic molecular controls. Actually, the induction of mitosis by MPF alone does not preclude the possibility of the dissociability and independence of different mitotic events. As chromosomes condense at mitosis, histone H, and H, become highly phosphorylated (Gurley ef al., 1978). Similarly the nuclear lamina becomes phosphorylated before disassembly (Gerace and Blobel, 1980). Sahasrabuddhe er al. ( 1984) noted phosphorylation of eight major nonhistone proteins (NHPs) before the initiation of mitosis. These NHPs were rapidly dephosphorylated during M-G, transition. Naturally, it may be surmised that MPF in turn activates a number of protein kinases to induce mitotic maturation. Actually, Murray and Kirschner (1989) have proposed a model in which MPF has been suggested to induce nuclear envelope breakdown, chromosome condensation, spindle assembly, etc., independently from each other (possibly acting on different substrates). Indeed, it has been shown by Newport and Spann (1987) that MPF is not the immediate effector of mitosis. The MPF can be depleted of activities required to promote nuclear envelope breakdown by preadsorption to DNase-treated nuclei, which implies that the primary interactions between MPF and nuclei involve proteins rather than DNA. Again, preincubation of MPF is not the immediate effector of mitotic breakdown. In another elegant experiment, Lokha and Maller (1985) have shown that when sperm chromatin or somatic cell nuclei were incubated with isolated MPF, they did not show mitotic changes. However, the same supernatant containing the MPF could induce nuclear envelope breakdown, chromosome condensation, and spindle assembly when added to extracts in which particulate components were abundant. These results suggest that particulate components are also required for the nuclear changes and that the MPF

250

SIBDAS GHOSH AND NEIDHARD PAWELETZ

cannot act directly on the chromatin. Obviously, the particulate material in this experiment contained inter alia nonchromosomal bodies such as centrosomes. Moreover, there is a possibility that MPF itself may contain more than a single factor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the partially purified mitotic factor indicated the presence of several polypeptides with a major band of 50 kDa (Adlakha et al., 1985). Even in a highly purified MPF preparation from Xenopus, Lokha et al. (1988) noted the presence of several proteins in minor amounts but two proteins of 45 and 32 kDa (34 kDa) were consistently present. The last few years have witnessed a confluence of genetical and biochemical approaches to the understanding of the cell cycle control. It has been established that S. pombe cdc 2+ and its homolog in S. cerevisiae cdc 28+ encode a protein kinase of 34 kDa, p34 (Hindley and Phear, 1984; Reed et al., 1985; Simanis and Nurse, 1986; Russel and Nurse, 1987a). Further, the Xenopus homolog of p34 cdc 2/28 has been shown to be a component of MPF (Dunphy et al., 1988; Gautier et al., 1988). This kinase probably acts in a complex with several other polypeptides to phosphorylate specific proteins to bring about changes associated with initiation of mitosis. The p34 level in the cells is remarkably stable throughout the cell cycle (Wittenberg and Reed, 1988; Draetta et al., 1989). However, in HeLa cells its enzymatic activity increases at least 70-fold as the cells move from G, to mitosis. It becomes inactivated during metaphase. This inactivation is associated with loss of a 62-kDa subunit from the protein kinase complex (Draetta and Beach, 1988). In G, cells, p34 becomes associated with p62 and is phosphorylated and maximally active as a protein kinase. On the contrary, another group of cell cycle proteins, the cyclins, accumulate during interphase but undergo rapid degradation at the end of each mitosis. The oscillation of cyclin levels is regulated by selective proteolysis which occurs at the metaphase/anaphase transition (Evans et al., 1983; Swenson et al., 1986; Standart et al., 1987). Earlier, it was proposed that cyclin A may have a role as an activator of MPF (Swenson et al., 1986). However, recently obtained evidence indicates that sea urchin and clam cyclins, which are a S. pombe pl3+ homolog, may be the second MPF (p45) component (Pines and Hunt, 1987; Booher and Beach, 1988; Solomon et al., 1988). It has been further shown that clam p34 is found in association with both cyclin A and cyclin B, probably not in a lrimolecular association, but as separate p34/cyclin A, p34/cyclin B complexes (Draetta et al., 1989). These authors have proposed a model depicting the relationship between p34 and cyclins in the activation-inactivation cycle of MPF. They have proposed that active MPF is created by post-translation modification of cyclin A and B/cdc 2+ complexes and is responsible for driving the cells into mitosis. It is possible that divergent regions of cyclin A and B sequences might differentially regulate the properties of the protein kinases. Indeed, the complex containing cyclin B has much more histone 1 kinase activity than that containing cyclin A (Draetta et al., 1989), although both cyclin A

MITOTIC EVENTS

251

and B are equally capable of causing nuclear envelope breakdown (Lewin, 1990). The association of cyclin A with p34 begins at S phase, but that of cyclin B during G, (Pines; cited in Lewin, 1990). Some potential functional differences between the forms of the kinase assembled with either of these cyclins are suggested by the differences in the timing of their maximum activity. A new class of cyclin, PRAD 1 (Motokura et al., 1991), has been recently reported. It can also bind and activate p34. At present at least four G,-specific cyclins are known (Surana et al., 1991). All these facts point to the possibility that different types of MPF activities may be needed for the initiation of mitosis. Sloboda (cited in Lewin, 1990) reported that phosphorylation of a 62-kDa protein of the mitotic apparatus correlates with the solubilization of microtubules of the mitotic apparatus. The kinase that undertakes this activity appears to be a calcium/ calmodulin-dependent enzyme present in the mitotic apparatus. Okadaic acid, which induces a rapid activation of MPF in S-phase cells, fails to organize metaphase spindles. In S. pombe, cdc 13 may interact with microtubules, as in cdc 13-1 17 mutants cytoplasmic interphase microtubules appear cytologically normal but the mitotic spindles fail to form (Hagan and Hayams, 1988). In S. cerevisiae cdc 28-1N cells arrested with fully formed mitotic spindle indicate that they are defective in executing certain other aspects of mitosis (Surana et al., 1991). These authors have further shown that clb 2 and cdc 28-1N mutants have high levels of kinase activity and yet are delayed or completely defective, respectively, in executing mitosis. These observations strongly suggest that cdc 28 kinase (cdc 2 kinase = p34 kinase) activity per se is not sufficient for mitosis. Similarly, Osmani reports (cited in North, 1991) the requirement of a second protein kinase, encoded by the nim A gene along with the cdc gene products for the initiation of mitosis in A. nidulans. Similarly, Obara et al. (1975) noted a requirement for continuous protein synthesis in the interphase cells before fusion to induce nuclear reformation (antiMPF). It is now known that MPF inactivation requires cyclin degradation (Woodgett, 1991), which is likely mediated by phosphatases. In yeast, a number of mutants are reported which show blockage of late nuclear events. Likewise, UV ts 706 mutant of A. nidulans (Morris, 1976a) cannot proceed to anaphase at elevated temperature. Another mutant of this species, ts bim G, fails to complete anaphase (Doonan and Moms, 1989). Schizosaccharomyces pombe cs-dis mutants are defective in sister chromatid disjoining (Ohkura et al., 1988). All of them indicate that gene products control these mitotic events. Since protein phosphorylation is important for the G2/M transition, it is likely that dephosphorylation could be required for the transition from mitosis to G1 (Lewin, 1990). Both A . nidiilans bim G+ and S. pombe dis 2+ encode phosphoprotein phosphatase 1, which is highly homologous to mammalian protein phosphatase 1 (Doonan and Morris, 1989; Ohkura et d., 1989). However, only four types of serinetthreonine-specific protein phosphatases have been found in mammalian cells (Ingebritsen and Cohen, 1983). There may be more

252

SIBDAS GHOSH AND NEIDHARD PAWELETZ

awaiting discovery or the restricted number of protein phosphatases may have a wide variety of substrate specificities to control different late mitotic events.

IV. Conclusion A critical survey of mitotic events in lower and higher eukaryotes shows that all the events that are associated with this process are not causally coupled. The evolutionary sequence of mitosis in lower eukaryotes also indicates the dissociability of these events. Genetic studies in cell cycle mutants in lower eukaryotes, especially in yeasts and Aspergillus, in Drosophilu, and in mammalian cell lines showing blockage at particular mitotic stages at restrictive temperatures demonstrate that particular gene functions may be required at those stages for completion of the process. In other words, different mitotic events may be governed by different gene products. Other evidence provided by experiments using the cell fusion technique, chemicals affecting different events of mitosis, antibodies and inhibitors, and cell-free systems strongly supports the dissociability of many of the mitotic events. Finally, recent biochemical studies including those on the cell cycle mutants indicate the possibility of the presence of multiple forms of MPF and protein kinases that are likely to be responsible for mitotic induction and initiation of different mitotic events. The roles of protein kinases for the transition of G,/M and phosphatases for M/G, also indicate that separate activities initiate early and late mitotic events. We conclude that several mitotic events are dissociable and run parallel and are perhaps governed by independent factors. However, we do not exclude the possibility that some mitotic events may be interdependent and may be controlled by common causal factors and that all these events are strongly coupled in normal mitotic division in a temporal order.

Acknowledgments This work is part of an Indo-German Science Collaboration Project between the Indian Council of Medical Research, New Delhi, and the Gesellschaft fur Strahlenforschung, Munich. S.G. is grateful to the German Cancer Research Center for financial assistance. We also thank Dr. D. Schroeter for help in preparing this manuscript and Ms. C. Kamp, Ms. E. Gundel, and Mrs. A. Wohlfahrt for careful secretarial work.

References Adlakha, R. C., Sahasrabuddhe, C. G.. Wright, D. A., Lindsey, W. F., and Rao, F. N. (1982). J . Cell Sci. 54, 193-206.

MITOTIC EVENTS

253

Adlakha, R. C., Sahasrabuddhe. C. G., Wright. D. A,, Sahasrabuddhe, H.. Bigo, H., and Rao, P. N. (1983). J. Cell B i d . 97, 1707-1713. Adlakha. R. C.. Wright. D. A.. Sahasrabuddhe, C. G., Davis, F. M.. Prashad, N., Bigo, H., and Rao, P. N. (1985). E.rp. Cell Res. 160, 471-482. Aldrich, H. C. (1969). Am. J . Bot. 56, 290-299. Armas-Portela, R., Paweletz, N., Zimmermann, H.-P., and Ghosh. S . (1988). Cell Moril. Cytoskel. 9, 254-263. Bajer, A. S., DeBrabander, M., Moll.-Bajer, J., DeMey, J., Paulaitis, S., and Guvens. G. (1980). In “Microtubules and Microtubule Inhibitors” (M. DeBrabander and J. DeMey, eds.), pp. 399-425. Elsevier/North-Holland, New York. Bajer, A. S.. and Moll.-Bajer. J. (1972). In “International Review of Cytology” ( G . Bourne et al., eds.), suppl. 3, pp. 1-272. Academic Press, San Diego. Balczon. R. D.. and Brinkley, B. R. (1987). J . Cell B i d . 105, 855-8562. Benavente, R . , and Krohne. G. (1986). J. Cell Biol. 103. 855-862. Berns, M. W., and Richardson, S. M. ( 1 977). J . Cell Biol. 75, 977-988. Bhatnagar, S. P., and Swahney, V. (1981). In “International Review of Cytology” (G. Bourne e t a / . . eds.), Vol. 74, pp. 55-102. Academic Press, San Diego. Blackeslee, A. (1937). C. R. Acad. Sci. (Paris) 205, 476-496. Booher. R., and Beach, D. (1988). EMBO J. 7, 2321-2327. Boothroyd, E. R., and Moms, N. R. (1986). J. Cell Biol. 103, 186a. Brodsky. W. Ya., and Uryvaeva, I. V. (1977). In “International Review of Cytology” (G. Bourne et a/.. eds.), Vol. 50, pp. 275-332. Academic Press, San Diego. Burgess, J., and Northcote, D. H. (1979). Nature (London) 277, 665-667. Burke, B., and Gerace, L. (1986). Cell (Cambridge, Mass.) 44, 639-644. Cande, W. Z., and Wolniak, S. M. (1978). J. Cell B i d . 79, 573-580. Carlson, J. G. (1938). Proc. Natl. Acad. Sci. U.S.A. 24. 500-507. Carter, S . B. (1967). Nature (London) 213,261-264. Cleffmann, G., and Frankel, J. (1978). ESP. Cell. Res. 117, 191-194. Cooke, C. A., Bernat, R. L., and Eamshaw, W. C. (1990). J. Cell Biol. 110, 1475-1488. Cooke, C. A.. Heck, M. M. S . , and Eamshaw, W. C. (1987). J. Cell Biol. 105, 2053-2067. Coss, R. A,, and Pickett-Heaps. J. D. (1973). Protoplasma 78, 21-39. Coss. R. A,, and Pickett-Heaps, J. D. (1974). J , Cell B i d . 63, 84-98. Culotti. J., and Hartwell, L. H. (1971). Exp. Cell Res. 67, 389-401. D’Amato, F. (1969). Caryologia 13, 339-35 1 . Das, N. K. (1962). J. CellBiol. 15. 121-130. DeBrabander, M., Guvens. G., Nuydens, R., Willebrords, R., Aerts, F., and De Mey. J. (1986). In “International Review of Cytology” (G. Bourne et a/., eds.), Vol. 101. pp. 215-238. Academic Press, San Diego. Dey, R. Paweletz, N., and Ghosh, S. (1989). Eur. J . Cell B i d . 48,227-238. Deysson, G. (1975). In “Microtubule Inhibitors” (M. Borgers and M. DeBrabander, eds.), pp. 427-45 1. Elsevier, New York. Doonan, J. H., and Moms, R. (1989). Cell (Cambridge. Mass.) 57, 987-996. Draetta, G., and Beach, D. (1988). Cell (Carnhridge. Mass.) 54, 17-26. Draetta, G.. Luca, F., Westendorf, J., Brizuela, L., Ruderman, J., and Beach, D. (1989). Cell (Cambridge, Mass.) 56, 829-839. Dunphy, W. G., Brizuela, L., Beach, D., and Newpon, K. J. (1988). Cell (Cambridge. Mass.) 54, 423-43 I . Dustin, P. (1978). “Microtubules.” Springer, Berlin. Eamshaw, W. C.. and Cooke, C. A. (1991). J . Cell Sci. 98,443-461. Eamshaw, W. C., Halligan, B., Cooker, C. A., Heck, M. M. S., and Liu. L. F. (1985). J . Cell Biol. 100, 1706-1715.

254

SIBDAS GHOSH AND NEIDHARD PAWELETZ

Earnshaw, W. C., and Heck, M. M. S. (1985). J. CellBiol. 100, 1716-1725. Eamshdw, W. C., and Rattner, J . B. (1989). In “Mechanism of Chromosome Distribution and Aneuploidy” (M. A. Resnick and B. K. Vig, eds.), pp. 33-42. Alan R. Liss, New York. Edgar, B. A,, and O’Farrel, P. H. (1989). Cell (Cambridge, Muss.) 57, 177-187. Edgar, B. A,, and O’Farrel, P. H. (1990). Cell (Cambridge, Muss.) 62, 469-480. Eigsti, 0. J., and Dustin. P., Jr. (1955). “Colchicine in Agriculture, Medicine, Biology and Chemistry.’’ Iowa State Coll. Press, Ames, Iowa. Enoch, T., and Nurse, P. (1990). Cell (Cambridge, Mass.) 60, 665-673. Enoch. T., and Nurse, P. (1991). Cell (Cambridge, Muss.) 64, 921-923. Evans, T., Rosenthal, E. T., Youngbloom, J., Distel, D., and Hunt, T. (1983). Cell (Cumbridge, MUSS.)33, 389-396. Foe, V. E., and Alberts, B. M. (1983). J . Cell Sci. 61.31-70. Franke, W. W. (1974). In “International Review of Cytology” (G. Bourne et al., eds.), Suppl. 4, pp. 72-236. Academic Press, San Diego. Frankel, J., Mohler, J., and Frankel, A. K. (1980). J. Cell Sci. 43, 59-74. Freeman, M., Niisslein-Volhard, C., and Glover, D. M. (1986). Cell (Camhridge,Mass.) 46, 457-580. Frenster, J. H. (1974). In “The Cell Nucleus’’ (H. Busch ed.), Vol. I , pp. 565-580. Academic Press, New Yorknondon. Fuge, H. (1982). Biol. unserer Zeir 12, 161-167. Gambino, J., Bergen, L. G., and Morris, N. R. (1984). J. Cell Biol. 99, 830-839. Gatti, M., Smith, D. A.. and Baker. B. S. (1983). Science 221, 83-85. Gautier, J., Norbury, C., Lokha, M., Nurse, P., and Maller, J. (1988). Cell (Cambridge, Mass.) 54, 433-439. Geitler, L. (1939). Chromosomu 1, 1-22. Gelbart, W. M. (1974). Genetics 76, 57-63. Gerace, L., and Blobel, G. (1980). Cell (Camhridge, Mass.) 19, 277-287. Gerhart, J., Wu, M., Cyert, M., and Kirschner, M. (1985). Cytobios 43, 335-347. Gerhart, J., Wu, M., and Kirschner, M. (1984). J. Cell Biol. 98, 1247-1255. Ghosh, S. (1987). In “International Review of Cytology,” Suppl. 17, pp. 573-597. Academic Press, San Diego. Ghosh, S., and Dey, R. (1986). Chromosoma 93,429-434. Ghosh, S., and Paweletz, N. (l984a). Chromosomu 89, 197-200. Ghosh, S., and Paweletz, N. (1984b). Chromosoma 90, 57-67. Ghosh, S . . and Paweletz, N. (1987a). Cell Biol. Int. Rep. 11, 165-169. Ghosh, S., and Paweletz, N. (3987b). Chromosoma 95, 136-143. Ghosh, S., and Paweletz, N. (1987~).Exp. Cell Res. 171, 243-249. Ghosh, S., and Paweletz, N. (1992). Exp. Cell Res. 200, 215-217. Ghosh, S., Paweletz, N., and Armas-Portela, R. (1988). Cell Biol. Inr. Rep. 12, 443-447. Ghosh, S., Paweletz, N., and Armas-Portela, R. (1989). Indian J. Exp. Biol. 27, 317-324. Ghosh, S., Paweletz, N., and Ghosh, 1. (1978). E.rp. Cell Res. 111, 363-371. Ghosh, S., Paweletz, N., and Schroeter, D. (1992). Exp. Cell Res. 201, 535-540. Gimenez-Martin, G., De La Torre, C., Lopez-Saez, J. F., and Esponda, P. (1977). Cyrobiologie 14, 426-462. Gimenez-Martin, G., Gonzalez Fernandez, A., de La Torre C., and Femandez Gomez, M. E. (1971). Chromosomu 33, 361-371. Glover, D. M. (1989). J . Cell Sci. 92, 137-146. Gonzalez, C., Casal, J., and Ripoll, P. (1988). J. Cell Sci. 89, 39-47. Gonzalez-Reyes, J . A., Navas, P., and Garcia-Herdugo, G. (1986). Protoplasma 132, 172-176. Goyanes, V. J., and Svartzman, J. B. (1981). Chromosoma 83,93-102. Grell, K. G. (1964). In “The Cell” (J. Brachet and A. E. Mirsky, eds.), Vol. 6, pp. 1-79. Academic Press, New York.

MITOTIC EVENTS

255

Gull, K., and Trinci, A. P. J. (1974). Proroplasma 81, 37-48. Gurley, R.. D’Anna, J-A., Barham, S.S., Deavan, L. L., and Tobey, R. A. (1978). Eur. J. Eiochem. 84, 1-15.

Hagan, I. M., and Hayams, T. S. (1988). J. Cell Sci. 89, 343-357. Halleck, M. S., Reed, J. A., Lumley-Sapansky, K., and Schlegel, R. A. (1984). Exp. Cell Res. 153, 561-569. Harris, P. (1983). Dev. Eiol. 96, 277-284. Hartwell, L. H. (1971). Exp. Cell Res. 69, 265-276. Hartwell, L. H. (1978). J. CellEiol. 77, 627-637. Hartwell, L. H., Culotti, J., Pringle, J. R., and Reid, B. J. (1974). Science 183,46-51. Hartwell, L. H., and Weinen, T. A. (1989). Science 246, 629-634. Hatzfeld, J.. and Buttin, G. (1975). Cell (Cambridge. Mass.) 5, 123-129. Hauser, M. (1972). Chromosoma 36, 159-175. Heath, I. B. (1978). In “Nuclear Division in the Fungi” (I. B. Heath, ed.), pp. 89-176. Academic Press, New YorkLondon. Heath, I. B. (1980). In “International Review of Cytology” (G. Bourne et a/.. eds.), Vol. 64, pp. 1-80, Academic Press, San Diego. Hepler, P. K., and Palevitz, B. A. (1985). J . Cell B i d . 102, 1995-2005. Herreros, B., and Gianelli, F. (1967). Nature (London) 216, 286-288. Hindley, J., Phear, G. A,, Stein, M., and Beach, D. (1987). Mol. Cell Eiol. 7 , 504-511. Hiramot0.Y. (1956). E.rp. Cell Res. 11, 630-636. Hiramoto, Y. (1965). J. Cell B i d 25, 161-167. Hiraoka. Y., Toda, T., and Yanagida, M. (1984). Cell (Cambridge, Mass.) 39, 349-358. Ingebritsen, T. S., and Cohen, P. (1983). Science 221, 331-338. Inoue, S. (1981). J. Cell B i d . 91, 131s-147s. Jimenez, J., Alphey, L., Nurse. P., and Glover, D. M. (1990). EMEO J . 9, 3565-3571, Jones, D., and Berger, J. D. (1982). Can. J. Zool. 60, 2296-2308. Jost, E., and Johnson. R. T. (1981). J . Cell Sci. 47, 25-53. Kelly, M. G., and Hartwell, J. L. (1954). J . Narl. Cancer Insr. 14, 967-1010. Keryer, G.. Ris, H., and Borisy, C. G. (1984). J. Cell Eiol. 978. 2222-2229. Kihlman, B. A., and Levan, A. (1949). Hereditas 35, 109-11 I . Kishimoto, T., Kuriyama, R., Kondo, H., and Kanatoni, H. (1982). Exp. Cell Res. 137, 121-126. Krishan, A., and RayChaudhuri, R. (1969). J. Cell B i d . 43, 618-621, Kubai, D. F. (1973). J. CellSci. 13, 511-552. Kubai, D. F. In “International Review of Cytology” (G. Bourne e t a / . . eds.), Vol. 43, pp. 167-227. Academic Press, San Diego. Kubai, D. F., and Ris, H. (1969). J . Cell Eiol 40, 508-528. Lambert, A. (1980). Chromosoma 76, 295-308. Lettre, H. ( 1961). Forschungen Fortschritte 35, 39-44. Lettre, H. (1965). E.werta Med. Int. Congr. Ser. 106, 43-49. Levan, A. ( I 97 I ). J . Indian Eot. Soc. Golden Jubilee A 50, 340-349. Levan, A,. and Hauschka, T. S. (1953). J . Natl. Cancer Inst. 14, 1-43. Lewin, B. (1990). Cell (Cambridge, Mass.) 61, 743-752. Lin, M. S., Walden, D. B. (1974). Exp. Cell Res. 86, 47-52. Lloyd, D., Poole, R. K., and Edwards, S. W. (1982). “The Cell Division Cycle.” Academic Press, New YorkLondon. Lokha, M. J., Hayes, M. K., and Maller. J. L. (1988). Proc. Nail. Acad. Sci. U.S.A.85, 3009-3013. Lokha, M. J., and Maller. J. L. (1985). J. Cell Eiol. 101, 518-523. Lopez-Saez, J. F.. Mingo, R.. and Gonzalez Fernandez. A. (1982). Eur. J. Cell Eiol. 27, 185-190. Masui, Y., and Markert, C. (1971). J. Exp. Zool. 177. 129-146. Masui. Y.. Mayerlof, P. G., and Miller, M. A. (1980). Svmp. Soc. Dev. Eiol. 38,235-258.

256

SIBDAS GHOSH AND NEIDHARD PAWELETZ

Mazia, D. (1961). In “The Cell” (J. Brachet and A. A. Mirsky, eds.), Vol. 3. Academic Press, New York. Mazia, D. (1974). In “Cell Cycle Control” (G. N. Padilla, T. L. Cameron, and A. Zimmermann, eds.), pp. 265-272. Academic Press, New York. Mazia, D. (1984). Exp. Cell Res. 153, 1-15. Mazia. D. (1987). In “International Review of Cytology” (K. Jeon, ed.), Vol. 100, pp. 49-92. Academic Press, San Diego. Mazia, D., Paweletz, N., Sluder, G., and Finze, E.-M. (1981). Proc. Nail. Acad. Sci. U.S.A. 78, 377-38 1. McCully, E. K., and Robinow, C. F. (1972a). J. Cell Sci. 10, 857-88 I . McCully, E. K., and Robinow, C. F. (1972b). J. Cell Sci. 11. 1-31. McNeill, P. A., and Berns, M. W. (198 I). J. Cell Biol. 88, 543-553. Meeks-Wagner, D., Wood, J. S., Garvik, B., and Hartwell, L. H. (1986). Cell (Cambridge, Muss.) 44,53-63. Miake-Lye, R., and Kirschner, M. W. (1985). Cell (Cambridge, Muss.) 41, 165-175. Minshull, J., Pines, J., Golsteyn, R., Standart, N., Mackie, S., Colrnan, A,, Blow, J., Ruderman, J. V.,Wu, M., and Hunt, T. (1989). J. Cell Sci. Suppl. 12, 77-97. MobBajer, J . (1958). Chromosoma 9, 332-358. Moms, N. R. (1976a). Exp. Cell Res. 98, 204-210. Moms, N. R. (1976b). Genet. Res. 26, 237-254. Motokura, T., Bloom, T., Kim, H. G., Jiippner, H., Ruderman, J.V., Kronenberg, H. M., and Arnold, A. (1991). Nature (London) 350,512-515. Murray, A. W., and Kirschner, M. W. (1989). Science 246,614-621. Nagl, W. (1981). In “International Review of Cytology” (G. Bourne el al., eds.), Vol. 73, pp. 21-53. Academic Press, San Diego. Naha, P. (1969). Nature (London) 233, 1380-1381. Navas. P., Hidalgo, A., and Garcia-Herdugo, G. (1986). Experientiu 42, 437-439. Newmayer, D. D., Lucocq, J. M., Burglin, T. R., and De Robertis, E. M. (1986). EMBO J . 5, 501-510. Newport, J. W., and Kirschner, M. W. (1984). Cell (Cambridge, Mass.) 37,731-745. Newport, J., and Spann, T. (1987). Cell (Cambridge, Mass.) 48, 219-230. Nishimoto, T., Ajiro, K., Hirata, M., Yamashita, K., and Sekiguchi, M. (1985). Exp. Cell Res. 156, 351-358. Nishimoto, T., Eilen E., and Basilico, C. (1978). Cell (Cambridge, Mass.) 15, 475-483. North, G. (1991). Nature (London) 351,604-605. Nurse, P., and Thuriaux, P. (1980). Genetics 95, 627-637. Nurse, P., Thuriaux, P., and Nasrnyth, K. (1976). Mol. Gen. Genet. 146, 167-178. Nuti Ronchi, V., Avanzi, S., and D’Amato, F. (1965). Curyologiu 18, 599-617. Oakley, B. R., and Dodge, J. D. (1974). J. Cell Biol. 63, 322-328. Oakley, B. R., and Moms, N. R. (1981). Cell (Cambridge, Mass.) 24, 837-841. Obara. Y., Weinfeld, H., and Sandberg, A. A. (1975). J . Cell B i d . 64, 378-388. Ohkura, H., Adachi, Y., Kinoshita, N., Niwa, 0.. Toda, T., and Yanagida, M. (1988). EMBO J . 7, 1465-1473. Ohkura, H., Kinosita, N., Miyatani, S., Toda, T.,and Yanagida, M. (1989). Cell (Cambridge, Muss.) 57,997-1 007. Osmani. S . A., Engle, D. B., Doonan, J. H., and Moms, N. R. (1988). Cell (Cambridge, Mass.). 52, 241-25 1. Paul, D. C., and Gaff, C. W. (1973). Exp. Cell Res. 78, 399-413. Paulus, J. (1968). Exp. CellRes. 53, 310-313. Paweletz, N. (1974). NaticM,issenschufiliche Rundschau 27, 359-370. Paweletz, N. (1981). Cell B i d . Inr. Rep. 5 , 323-336.

MITOTIC EVENTS

257

Paweletz, N. (1987). In “Biomechanics of Cell Division” (N. M a s ed.), pp. 97-122. Plenum, New York. Paweletz, N., and Lang, H. (1988). Eur. J. CellBiol. 47, 334-345. Paweletz. N., Mazia, D., and Finze, E. M. (1984). Exp. Cell Res. 152, 47-65. Pederson, T. (1972). Proc. Natl. Acad. Sci. U.S.A.69, 2224-2228. Peterson, J. B., and Ris. H. (1976). J. Cell Sci. 22, 219-242. Peterson, S. P.. and Berns, M. W. (1979). Exp. Cell Res. 120, 223-236. Pickett-Heaps, J. (1974). Biosvstems 6, 37-48. Pickett-Heaps, J. D., and Tippit. D. H. (1978). Cell (Cambridge, Mass.) 14, 455-467. Pines, J., and Hunt, T. (1987). EMBO J. 6, 2987-2995. Quinlan, R. A.. Pogson, C. I., and Gull, K. (1980). J . Cell Sci. 46, 341-352. Rao, P. N., and Johnson, R. T. (1970). Nature (London) 225, 159-164. Rao, P. N., Wilson, B., and Puck, T. T. (1977). J. Cell Physiol. 91, 131-142. Rattner, J. B., Kingwell, B. G., and Fritzler, M. G. (1988). Chromosoma 96, 360-367. Rattner, J. B., and Phillips, S. G. (1973). J . Cell B i d . 57, 359-372. Reed. S. I., Harwiger, J. A., and Lorincz, A. T. (1985). Proc. Natl. Acad. Sci. U.S.A.82, 4055-4059. Rieder. C. L. (1982). In “International Review of Cytology” (G. Bourne et al., eds.), Vol. 79, pp. 1-58. Academic Press, San Diego. Ripoll. P., Pimpinelli, S., Valdivia, M. M., and Avila, J. (1985). Cell (Cambridge, Mass.) 41, 907-9 12. Ris, H., and Kubai, D. F. (1974). J . Cell B i d . 60, 702-720. Rizzoni, M., and Palitti, E (1973). E.tp. Cell Res. 77, 453-458. Roos, U.-P. (1973). Chromosoma 41, 195-220. Ross, I. K. (1968). Protoplasma 66, 173-184. Russel, P., and Nurse, F? (1987a). Cell (Cambridge, Mass.) 49, 569-576. Russel, P., and Nurse, P. (1987b). Cell (Cambridge, Mass.) 49, 559-567. Sahasrabuddhe, C. G., Adlakha. R. C., and Rao, P. N. (1984). Exp. Cell Res. 153, 439-450. Schiff. P. B., Fant, J., and Horowitz, S. B. (1979). Nature (London) 277, 665-667. Schlegel. R., and Pardee, A. B. (1986). Science 232, 1264-1266. Schwarzacher, H.-G., and Schnedl, W. (1965). Cytogenetics 4, 1-18. Siebert. A. E., and West, J. A. (1974). Protoplasma 81, 17-35. Simanis, V., and Nurse, P. (1986). Cell (Cambridge, Mass.) 45, 261-268. Shiomi, T., and Sato, K. A. (1976). Exp. CeNRes. 100, 297-302. Sluder. G., Miller, F. G., and Rieder, C. L. (1986). J . Cell Biol. 103, 1873-1881. Smith, B. J.. and Wigglesworth, N. M. (1972). J . Cell Physiol. 80, 253-260. Smith, D. A,, Baker, B. S., and Gatti, M. (1985). Genetics 110, 647-670. Smith. G. E. Riddler, M. A. C., and Faunch, J. A. (1967). Nature (London) 216, 1134-1 135. Solomon, M.. Booher, R., Kirschner, M., and Beach, D. (1988). Cell (Cambridge. Mass.) 54, 738-739. Soyer, M.-0. (1969). J. Microscopie 8, 709-720. Speit, G., Mehnert, K., and Vogel, W. ( 1984). Chromosoma 89, 79-84. Spurck, T. P.. Pickett-Heaps, J. D., and Klymkowsky, M. W. (1986). Protoplasma 131.47-59. Stafstrom, J. P., and Staehelin, L. A. (1984). Eur. J. Cell Biol.34, 179-189. Standart. N., Ninshull, J., Pines, J., and Hunt, J. (1987). Dev. Biol. 124, 248-258. Stick, R. (1987). In “Molecular Regulation of Nuclear Events in Mitosis and Meiosis” (R. A. Schlegel, M. S. Halleck, and P. N. Rao, eds.), pp. 43-66. Academic Press, New York. Sunkara, P. S.. Wright. D.-A,, and Rao, P. N. (1976). J. Supramol. Sfrucr. 11, 189-195. Sunkel, C. E., and Glover, D. M. (1988). J. Cell Sci. 89, 25-38. Suprynowicz, E A,, and Gerace, L. (1986). J . Cell Biol. 103, 2073-2081. Surana, U., Robitsch, H., Price, C., Schuster, T., Fitch. I., Bruce Futcher, A,, and Nasmyth, K. (1991). Cell (Cambridge. Mass.) 65, 145-161.

258

SIBDAS GHOSH AND NEIDHARD PAWELETZ

Swenson, K. I., Forrell, K. M., and Rudermann, I. V. (1986). Cell (Cambridge, Mass.) 47,861-870. Szollosi, D., Czolowska, R., Sottynska, M. S., and Tarkowski, A. K. (1986). Biol. CeN 56,239-250. Takanari, A,, and Izutsu, K. (1981). Cytogenet. Cell Genet. 29, 77-83. Takanari, A., and Izutsu, K. (1983). Mutut. Res. 107,297-306. Thomas, J. H., and Bottstein, D. (1986). Cell (Cambridge, Mass.) 44,65-76. Thompson, L. H., and Lindl, P. A. (1976). Somatic. Cell Genet. 2, 387-400. Toda, T., Adachi, Y., Hiraoka, Y., and Yanagida, M. (1984). Cell (Cambridge. Muss.)37, 233-242. Uemura, T., and Yanagida, M. (1986). EMBO J . 5, 1003-1010. Vickerman, K., and Preston, T. M. (1970). J. Cell Sci. 6, 365-384. Wagenaar, E. B. (1983a). Exp. Cell Res. 144, 393-403. Wagenaar, E. B. ( 1 983b). Cell Biol. Int. Rep. 7,827-833. Wang, R. J. (1974). Nature (London) 248, 76-78. Wang, R. J. (1976). Cell (Cambridge, Mass.) 8, 257-261. Wang, R. J., Wissinger, W., King, W. J., and Wang, G. (1983). J. Cell B i d . 96, 301-306. Wang, R. J., and Yin, L. (1976). Exp. Cell Res. 101, 331-336. Wang, R. J., Yin, L.,DuMontier, A.. and Sheridan, W. (1974). J . CeN Biol. 63, 365a. Weil, C. F., Oakley, C. E., and Oakley, B. R. (1986). Mol. Cell Biol. 6, 2963-2969. Weintraub, H., Buscaglia, M., Ferrez, M., Weiller, S., Boulet, M., Fabre, F., and Baulieu, E. E. (1982). C. R. Acad. Sci. (Paris) 295,787-790. Wick, S . M. (1985). Cell Biol. Int. Rep. 9, 357-371. Wilson, I. B. (1925). “The Cell in Development and Heredity,’’ 3rd ed. McMillan Co., New York. Wissinger, W., and Wang, R. J. In “International Review of Cytology,” Suppl. 15, pp. 91-113. Academic Press, San Diego. Wittenberg, C., and Reed, S. I. (1988). Cell (Cambridge, Mass.) 54, 106-1072. Wolf, R. (1980). Wilhelm RouxS Arch. 188, 65-73. Wood, J. S., and Hartwell, L. H. (1982). J. Cell Biol. 94, 718-726. Woodgett, J. R. (1991). Curr. B i d . 1, 106-107. Wright, S. J., and Schatten, G . (1990). Dev. Biol. 142, 224-232. Wu, N., and Gerhart, J. C. (1980). Dev. Biol. 79, 465-477. Yamashita, K., Yasuda, H., Pines, J., Yasumoto, K., Nishitani, H., Ohtsubo, M., Hunter, T., Sugimura, T.,and Nishimoto, T. (1990). EMBO J. 9, 4331-4338. Zoalkar, M., and Erk, I. (1976). J . Microsc. B i d . Cell 25, 97-106.