On the evolutionary origin and possible mechanism of colchicine-sensitive mitotic movements

B&Sysrems6 (1974) 16-36 0 NORTH-HOLLAND PUBLISHlNG COMPANY

ON THE EVOLUTIONARY

ORIGIN AND POSSIBLE MECHANISM

OF COLCHICINE-SENSITIVE MITOTIC Ea4OVEMENTS

LynnMARGULIS Departmznt

of Biolo~

Boston, Massachusetts

Boston

Univem’ty,

02215 U.S.A.

is pbin that the increpe of length ofthe central spindle results from the increase in length, by ,Towth, of the fibers that compose it and that this W&ease in length functiuns to push daughter nuclei farther apart. ” L.R. Cleyeland ” It

1. Introduction

I. I. Slow intracellular movements Eukaryotic cell& unl;kc those of the prokaryotic bacteria and blue green algae, typically display intracellular movements. Such movement includes, for example, Pagellar an ciliar beating, cy.toplasmic streaming, cell extension and contraction, at least. As many as four types of intracellular movement can be distinguished in a single cell (see, for example, Table II, pa 34 in Margulis, 1973). Ce!l motility often can be cor&ated with the presence of intact cytoplasmic structures, commonly either hollow microtubules, 25QA in diameter or one of several types of smaller microfilaments. Where known, it seems that these ulirastructures are composed of one of two major classes of so-called “motile proteins”; the microtubules ade of tubulin proteins (Stephens, 1970; gz2dand Borisy, 1973) and at least certa,‘; types o made of aetin <&iohri, 1%8; momura, 19Ti3).It is possible that all forms of

eukaryotic intracellular motility will be provthat individual en ultimat homologous be relatable by tubulin an ctin proteins conservation of primary amino acid sequences. (For the current state of the art: see Elzinga et al., 1973 for the complete primary amino acid sequence of rabbit muscle actin, and Luduena an3 Woodward, 1974, for a partial primary amino acid sequence of alpha and beta tubulins from two animal sources.) This paper will not attempt to discuss the evolution of all intracellular motility but only one aspect: the possible evolutionary origin and mechanism of the slow intracehular movement that is arrested by low concentrations of the plant alkaloid colchicine (Eigsti and Dustin, 1968). This slow movement, of the order of a micron per minute in velocity, is characteristic of the movement of chromosomes in mitosis (Nicklas, 1971) and the outgrowth of flagella. The original concepts of L.R. Cleveland ( 1934, 1953) concerning the mechanism of chromosome movement in eukaryotes and t.he evolution of this rne~~~~isrn in protists wiII be developed in more modern terms. That is, ss Cleveland recognized, it will be argued that ent is esse~ti~ly

L. Margulis. Colchine-sensitive mitoiic movements

phogenetic movement : chromosomes move because they are attached to a mitotic spindle structure which forms between the chromosome sets (Fig! :). 1.2. Mitosis: a late precambriun adaptive radiati 9” Since all eukaryotes are vsentially aerobic, it has been argued that sometime either during or after the transitio to the oxidizing atmo? sphere classical standard “textbook” mitosis evolved. Mitosis, such as that characteristic of

all metazoan animals and embryo@ytic green plants must have originated in eukaryotic protists, ancestral to metazoans and metaphytes. Many protists show no mitosis or extremely idiosyncratic mitotic behavior (Wilson, 1925; Marguhs, 1970). Mitosis itself, never encountered in obligate anaerobic organisms, is a process requiring oxygen. For example, 8y varying the amounts of oxygen in gas mixtures, Amoore ( 196 1a,b) determined that oxygen concentrations greater thm %S% are required for euka Fj-Gtic cells to complete normally t!?iecell division cyc&. At lower concen-

Pig. 1, Ivbsis in a hypsrmastigote ~erno~st~ati~gspindle ~~~n~ationbetween spiral 1953).

17

18

L. hforpulis, Colchine-sensitive milotic movements

tratjons pea cells in different stages of mitotic division were differently affected. Amoore conchded “All stages of [eukaryoticl cell division depend on the presence of oxygen, but the visible stages of mitosis are less dependent than is the stage of entering mitosis” (p. 107, hnoore, 196 1a). Stabilization of standard $totic behaviour is no doubt a prerequisite for the origin of meiosis and hence meiotic sex (Margulis, 1970; Schopf and Blacic, 1971). The atmospheric transition has been d:ted at about 1.8 f: 0.2 billion years ago (Cloud, 1968a); this sets a lower limit on the date for the origin of eukairyote sex. By approximately 0.75 billion years fossils that can be interpreted as the remains of soft-bodied multicellular metazoans (animals, sensu strictu) are relatively abundant in the record (Glaessner, 1968; Cloud, 1968b). It can be deduced that mitosis and ) meiosis evolved within that billion year peri d!,of the late preP Cambrian but any more preci$z statement is difficult to make on the basis of firm evidence. It is not known for certain if eukaryotic organisms of any kind are present in the record prior to the deposition of the Ediacaran sandstone fossils of Australia (Glaessner, 1968). The possibility that the tetrahedral structure in the 0.9 billion year old Bitter Springs chert is a meiotic tetrad has been raised (Schopf and Blacic, 197 1); it seems to me remote. Such tetragonal tetrads are representative of the more advanced red algae Floridales, which most likely are evolutionarily derived from the cruciate red algae. The tetrads tend to be retained within the parent thallus and released as single tetraspores (Norris, R., 1973, pers. comm.). The preservation of the more delicate tetrad in the total absence of either pare& thallus or predecessor red algal genera is highly suspect. For example, nlone of ihe more prim’tive Bangiales are found in the fossil record that early. Although

rare, tetragonal asexual division products are known among blue green algae (Golubic, pers. comm., 1973). If the beautifully preserved tetrad (Schopf and Blacic, 1971) represents the remains of eukaryote cells, a species of tetrasporales green algae or even yeast ascospore tetrads provide closer comparisons than red algal reproductive structures. The tetrad form, far more characteristic of eukaryotes than prokaryotes is often produced by mitotic divisions and certainly does not necessarily imply meiosis. Furthermore many of the supposed eukaryotic microstructures, i.e., “fossil organelles” (e.g. Schopf, 1972) can result from partial cell degradation and/or desiccation of blue green algal cells at death. Such morphological alterations have been seen clearly in recent algal mat-forming blue green algae from the Persian Gulf and Western Australia (Awramik, et al., 1972 and pers. comm.). Comparable stable blue green algal “pseudoorganeiles” czn also be artificically produced in the laboratory (Barghoorn and Margulis, 1974, in preparation). Regular intracellular granular inclusions, superficially resembling eukaryotic organelles, can easily be observed with light microscopy in blue green algae grown under certain physiological conditions (Allen, MM., pers. comm. 1973). Thus, in spite of claims of some paleontologists (Cloud et al., 1969; Schopf, 1972), the micromorphological fossil data are equivocal. The identification of a single “statocyst” of a chrysophyte alga in the Beck Springs dolomite of eastern California (older than a billion years; Cloud et al., 1969) is perhaps the most provocative suggestion of a middle Precambrian eukaryote and deserves concerted attention. However, it seems more likely that the protrusions (Figs. 10 and 11 in Cloud et al., 1969) are not cell processes but glass chards or other artifacts (Barghoorn, 1974, pers. comm.). Wilfi regard ‘to Cloud’s (et al., 1969) figure of a “fossil crysopihyte” R. Norris ( 1974) has writ-

L. Murgutis,Colchine-sensitivemitotic movements

ten “I don’t know of any present-day statospores that have such a long and conspicuous plug in the cyst. Extant statospores usually have a neck into which the plug fits, and the neck of the spore is more conspicuous, usually, than the plug.., Plugs in extant statospores are quite small and inconspicuous and i know of no plug in statospores that I have seen that have such a long tapered form. Usually the plug is tapered in the opposite direction from the one shown by Cloud. Very little is known about the chemical composition of the plugs, but they have been described as containing silicopectin. 1 would seriously doubt that they would be preserved in such a fossil. I have looked at many statospores that were found in bores of lake sediments and I have never found a statospore in which the plug was intact. 1 think that it must be of a material that does not fossilize readily which would cast more doubt on Cloud’s (et al., 1969) interpretation” (letter of January 24, 1974). Thus, it is my opinion that both the Beck Springs dolomite and the Bitter Springs chert nannofossils represent highly diversified prokaryotic microbiota dominated by blue green algae (Schopf and lacic, 197 1) and that there is not yet any definitive evidence for fossil eukaryotes prior to the Ediacaran metazoans. The question of the xenogenous (symbiotic) or endogenous origin of the cilia-flagella system has been argued ai length (see Margulis, 1970; Younger et al., 1972 and Taylor, 1974 for discussion). Regardless of the ultimate origin of complex eukaryotic motility it seems clear that the tubulin-protein based system of intracel ular motility must eiotic sexualit realized that the origin’ and stabi~zation of pIex processes of meiosis

relationships between such organisms (e.g., nucleate algae, ~agel~ate fungi, protozoa and

19

their allies) are greatly clarified. Presumably because they are a product of that late preCambrian adaptive radiation that for example, the following “lower eukaryotes” among others, show significant variations upon the standard theme of mitosis: amoebae (Goode, 1971), red algae (MacDonald, 1972), oumycetes (Heath and Greenwood, 197l), green phytoflagellate algae (Norris, 1974), euglenoids (Leedale, 1967), dinoflagellates (Kubai and Ris, 1969; Soyer, 1971, 19721, ciliates (Paulin, 1974) and so forth. See Pick&t-Heaps I1 969, 1972, and this volume) for discussion of mitotic variations in “plant cells”.

It is assumed, in this paper, that the acquisition (or endogenous evolution) of the complex 9+2 flagellar system of eukaryotic cells preceded the evolution of eumitosis, (See Pickett-Heaps, p. 37, this volume, for an alternative view). The presence of motile flagclla/cilia preadapted eukaryotes for the origin of mitosis and meiosis, providing the regular synthesis and polymerization of microtubule protein. This assumption is useful for the central concept developed here: that mitotic chromosome movement in eukaryotes is morphogenatic movement. Hnother words, mitotic movement is thought to involve the growth of microtubule-based structures homologous to the flagellar system. These structures elongate by controlled polymerization of microtubule protein; tubules form between chromatin DNA-containing material and hence separate

20

L. Mlugulis, Colchine.sensitivemitotic movements

havior can be observed, the hypothesis developed here is that there is a unitary mechanism of eukaryotic mitosis: in every case chromc-

socnes (or chromatin) separate because of the growth of a flagellarderivative system bet’Neen chromosome masses. Usually, but not always. that system is composed of many elongating 25OA colchicine-sensitive’microtubules comprising the classical mitotic spindle. If the symbiosis hypothesis is correct, these morphogenetic movements of the mitotic apparatus will be found to be .homologous to prokaryote cell elongation of the ancestral microbial symbiont. If the ancestral microbial symbiont was a spirochaete-like microbe, homology between tlie growth of the relevant spirochaete axial filaments and elongating microtubules may eventually be established. However, independent of the symbiotic theory of the origin of the system, this paper attempts to translate the Cleveland growth concept (Fig. 1) into explicit biochemical hypotheses whicn can be tested. The concept of force-production directly by polymerization of tubulin into elongating microtubules is applied to the chromosome movements in mitosis and briefly to other cellular phenomena, including colchicine-sensitive morphogenesis in ciliates and some microtubule-based processes in animals. Inoue’ and Sato (,1967) and Sato et al. ( 197 1) have presented convincing arguments for the concept that spindle birefringence is due to longitudinally aligned microtubules. Chromosomal movements are thought by these workers to be due to alterations in the conditions that determine,. the dynamic equilibrium between “monomer” and “polymer” (e.g., subunit tubulin and microtubules), It has been shown that entropy increases accomp:my spindle formation (Carolan, 1966; Stephens, 1973), presumably due to disorganization of bound water. Although the seminal contribution of Inou6’s work is acknowledged

and the involvement of tubules in both chromosome movements and the generation of birefringence is entirely accepted here, there is a fundamental difference between Inoub’s model and this one. Inoue suggests anaphase chromosomal movement is due to loss of “monomers’* from the chromosomal spindle fibers, i.e. shortening of these fibers. I will argue that anaphase chromosomal movements result from the same process that prophase movement does: the “growth” (tubule elongation) of spindle fibers. McIntosh, Hepler and Van Wie (1969) have also presented a provocative and fascinating model for mitosis. This modification of their attempt to explain mitotic chromosome movements is also indebted to their work and its antecedents (e.g., Ino& and Sato, 1967; Mazia, 196 1). However, application of this theory of colchicine-sensitive movement also differs in substantial ways from theirs and other current views (e.g., Forer, 1969; Dietz, Luykx; see Luylcx, 1970; Bajer, 1973) in that the forces that move chromosomes are thought to be independent of any lateral interactions between microtubules; they are thought to be generated directly by the spontaneous polymerization of tubulin dimer into microtubules. For clarity, first the essential points of this model as applied to mitosis will be outlined. Although many of these postulates are extremely similar, if not identical, to suggestions of previous authors, especially McIntosh et al., 1969; because of voluminous citations that would result no attempt to credit all previous workers will be made (but see Mazia, 1961; Luykx, 1970; and Nicklas, 1971 for comprehensive references). 2.1. Mitosispostulates 2.1.7. Microtubule organising ten ters ~MTOC’S). Arguments, accepted and utilized

further here, for the existence of microtubule

L. Mar&s.

Colchine-scnsizive mitotic movements

organizing centers (MTOC’s) of the cell have been presented by Pickett-Heaps ( 197 1, 1972). These MTOC’s are not centrioles, basal bodies, kinetochores, kinetosomes or axopods per se but are associated with them. The classical (9+2) centriole (or centriolar pinwheel, Fulton, 1970) is considered necessary only as the nucleating center for later flagellum or cilium formation and a manifestation of the MTOC rather than the MTOC itself (PickettHeaps, 197 1). A morphological identification of the MTOC’s has not been definitively made, but Picket+Heaps suggests they may be the ubiquitous granular-fibrillar material associated with developing microtubule structures. Pickett-Heaps has made comprehensible the normal mitotic movement of plant cells in the absence of centrioles, sensu strictu. By implication, of course, MTOC’s are present not only at the poles in acentriolar plant mitosis but in every biological circumstance where microtubules form. In this model the microtubule-organizing centers (MTOC’s) of Pickett-Heaps are hypothesized to be composed of (at least) two essential components: (1) an enzyme, perhaps a “tpbulin dimerase” or other membrane bound factor responsible for formation of active dimer (either from inactiver dimer or from monomer) and 2) “nucleating centers” comparable to material found by Stephens (1968, 1970b) and the disks of Olmsted and Borisy (1973). These are presumably required for in vitro polymerization of tubulin. Nucleating centers may be simply tubulin protein alone or, in the case of basal bodies and more complex structures, they may contain RNA (Hartman and Gurney, 1974). In any case nucleating centers determine the type of final product composed of microtubules that will be formed. In this model there are no fundamental differences in any mitotic tubules and all that come into close enough contact will interact

21

in the same way regardless of the organizing center from which they originated. Observed differences of sensitivity of mitotic tubules to cold and coichicine (Brinkley, 1970) are not interpreted to be differences in materials from which these are composed. It is thought that tubules in the process of being formed are more sensitive than those older tubules already formed because the latter are stabilized by interactions with themselves such as bridge formation (Tilney, 1968, 1971). Stabilization is also likely to involve interaction with other cell proteins in many cases. Evidence for equality of spindle microtubules has been presented by Lam bert and Bajer ( 1973). 2. I. 2. Microtubule pro teitz. Protein monomer (tubulin) is synthesized off cytoplasmic ribosomes continuously throughout the interphase but remains constant ‘n total amount throughout mitosis. Tubulin (but not the larger organized tubules) free!y passes throughout the cell. Monomer (or possibly inactiv.. dimer), upon conversion to active dimer, per haps by the reactions in Fig. 2, spontaneously po!ymerizes into the visible 250A diameter, glutaraldehyde fixable microtubules (Tilney, 1971). The hypothesized sequence of steps in the formation and action of the spindle are described below in connection with the relevant standard animal and plant mitotic stages.

? r+GPP

%y+m?n&#s mommar-

GDP-

omer

micro

4 ATTP

Fig. 2. Hypothetical tubulirl protein reactions: the formation of dimer and its spontaneous polymerization into tubules. Abbreviations: ADP, ATP adenosine di- and triphosphate; GDP, GTP guanosiue di- and tr~pbosphat~.

L. Maqulis, t3l&ine-se&five

22

2.1.3. Ruks for the formation of microtu-

bules. The typical eukaryotic cell contains two sets of MTOC’s for microtubule protein

polymerization. These are associated with (1) the chromosomal kinetochores (in animals, plants, fungi and many protists) and (2) ccntrioles and their homologues: centrioles sensu strictu in animals (Fulton, 1970); clear zone caps of microtubules in many higher plants (Ledbetter and Porter, 1970), “centriolar plaques” in fungi (Motta, 1969) or “centrosomal plates” in diatoms (Manton et al., 1969a,b), the rostral areas of hypermastigote flagellates (Grimstone and Gibbons, 1966), and so forth. When tubulin dimer is produced by the exposure, activation or other action of the factor involved in its formation from monomer (or inactive dimer) it spontaneously forms microtubules. The structure of the resul ting microtubular complex is determined by the “nucleating center” property of the MTOC. As long as intracellular conditions are appropriate and tubulin monomer is available dimer is polymerized and microtubules continue to form from the tip distal to th.e MTOC (Fig. 3). 2.1.4. Early prophase. The transition from interphase to prophase is marked either by the appearance of the tubulin formation factor, or a change in intracellular conditions to side arms become b:‘dges

mitutic movements

which tubules are sensitive. Thus dimer is produced and tubule formation begins from the MTOC’s: the kinetochores on the outer surfaces of the chromosomes and the cytoplasmic centrioles of animal cells or centiolar regions in plants. Monomer is removed from a common cytoplasmic and in some cases (e.g. Tokcphyra, Millecchia and Rudzinska, 197 1) nuclear pool of protein. The ccntrioles themselves or MTOC’s associated with centrioles in animals, which are paired at each pole, organize them outward in both dire”ctions generating the asters and the central spindle but moSt kinetochores generate microtubules only in an outward direction perpendicular to their SULface, forming chromosomal fibers (Fig. 4a). The functioning of these two sets of MTOC’s (polar and kinetochore) does not necessarily

(plant)

I(a)

free hrbulin dimer

\

side arm

Fig. 3. Key to diagrams(Figs. 3-9). Arrows represent.microtubule protein tina. Linear arrays of arrows represent microtubules. MTOCs (microtubxlc organizing centers; dots) may be at kinctochores, on nuclear or endoplasmic membrane or at poles. Darker arrows were most recent arrivals since growth is from the tip (Allen and Borisy, 197J).

Fig. 4. Prophase in (a) plant and (b) animal cells. Dipoid chromosome number of 4, NM is nuclearmembrane.

L. Margulis,Coldtine-sensitive mitotic movements

occur simultaneously in all systems (Wilson, 1925). In plants the organizing center, on the inside or just outside the nuclear membrane, generate microtubules outward in all unimpeded directions (Fig. 4a). 2.1.5. Middle prophase. The nuclear membrane and other organelles excluded from the regions of growing tubules offer resistance to the growing tubules. When the membrane or other organelles are contacted by growing tubules polymerization in their direction is impeded, therefore a net force is generated and chromosomes move back, ultimately toward the equator (Fig. 5). This is congressional movement (Nicklas, 1971 j. Middle prophase is characterized by the build-up of a pressure due to the microtubules on the inner surface of the nuclear membrane. A cytoplasmic pressure is generated as well in many ceils. it is caused by polar microtubules (originating in animals from MTOC’s associated with centrioles) that, in the process of polymerizing, exclude cytoplasmic organelles. In some plant

Fig. 5. In prophase ~~~a~ce by nuclear membrane generates net force toward the equator of the cell &rk arrow represents net force).

23

cells the microtubular pressure is only intranuclear, in others extranuclear polar zones of microtubules are MTOC’s analogous to animal centrioles (Fig. 6). Because tubuie production is equal and in opposite directions, and restrained by spherical nuclear membrane or by cytoplasmic organelles such as ER and mitochondria, the approximate equatorial position of the chromosomes is achieved. The spindle shape, as McIntosh et al. (1949) point out, results from the microtubules and their interactions via cross-bridges. 2.1.6. Late prophase. In “open mitoses” late prophase is marked by the appearance of something, totally unknown, that dissolves the nuclear membrane. The interacting microtubular structure previously impeded by membrane can now lengthen. The release of pressure on the inner surface of the nuclear

Fig. 6. In organisms with ““closedmitosis” the confjg~t~o~ of the tubules and c~orno~orn~ in prophase just prior to brea~~~ow~of nuclear membrane. In the “open mitosis” of (a) animals and (b) plants this would be the metaphase con~gu~tion but the tubules woul be impeded by fragments of nuclear membrane and other cytoplasmic components.

24

L. Margdis, Colchine-sensitive mitotic movements

membrane is seen in live cells with light microscopy as spindle nearly fills the entire cell. Cytoplasmic tubules enter the nuclear area freely. The polar region MTOC-generated microtubules associated with centrioles are now lengthened sufficiently so that some contact between microtubules generated from the opposite ends of the cell may be made. These are the “interpolar” (Nick& 197 1) or “continuous” fibers (Luykx, 1970). (Most polegenerated microtubules probably do not extend all the way from one pole to the other, but overlap at the equator. See Nicklas ( 197 1) for discussion of this issue and comparison between different organisms.) Kinetochore tubules become extended enough to interact with pole generated tubules. 2.1.7. Metaphase. Since all microtubules continue to lengthen until impeded by membranes or organelles and all interact via bridges with each other, the stable dynamic metaphase spindle configuration results directly from the production in equal amounts but opposite directions of microtubules (McIntosh et al., 1969). 2.1.8. New MTOC’s in metaphuse and anaphase mowments. Before anaphase movements begin new sister kinetochores in some organisms and nuclear membrane MTOC’s in other organisms are developed. The chemical composition of the kinetochore or other MTOC’s is not known. It is possible that the process of new MTOC formation involves a very limited amount of protein synthesis off small (streptomycin and chloramphenicol sensitive) ribosomes, although this point is not central to the argument (see p. 25 for discussion). Because of the standard ski-configuration of chromosomes treated with low concentrations of colchicine, new sister kinetochores probably cannot be produced in the presence of the drug and therefore the kineto-

chore is at least partly tubulin. Clearly the arrangement of kinetochoric material varies with species (Schrader, 1953; see Went, 1966). In metaphase new kinetochores are formed and therefore new MTOC’s appear, presumably next to old kinetochores in the surface of each metaphase chromosome. This new MTOC formation involves the process of “splitting” of chromatids to become chrome somes. Deducible from the fact that the new kinetochore is an MTOC, tubulin activation and nucleating center production accompany its production. As new tubules polymerize from new MTOC’s interzonal microtubules elongate causing anaphase poleward movements (Fig. 7). In some cells the spindle tubules most likely elongate by intercalation of new tubulin near the poles. In such cases the chromosomes retain their connection with the poles but the spindle elongates (or bulges when physical constraints prevent elongation as in Allenspach and Roth, 1967). In other cases new tubule elongation may be generated from nuclear membrane-attached MTOC’s. The net effect is to move the chromosomes toward the poles because of the elongation of spindle microtubules between the chromosome masses. The movement of chromatin correlated with the elongat&n of intranuclear spindle tubules is most clearly seen in ciliate diploid micronuclei and polyploid macronuclei (Millecchia and Rudzinska, 197 1; Raikov, 1969). Central spindle tubule elongation in animals and green plants is usually at the expense of breakdown of chromosome-to-pole tubules as emphasized by Inoud and Sato (1967) in their dynamic equilibrium model.

Fig, 7. New kinetochores formed, bottom view, anaphase.

L. Margulis, Cblchine-sensitive mitotic movements

The concept developed here of redeployment of tubulin from kinetochore-pole tubules to interzonal tubules accounts for the data (Jenson and Bajer, 1973) as well as does Bajer’s ( 1973) own concept of mitosis. Although for the general animal-plant mitotic system he stated it obliquely, this is essentially the concept of Cleveland derived from years of observation of live material (Cleve‘land et al., 1934 and Fig. 1, redrawn from Cleveland, 1953). “In other words, once the proper chromosomal connections are made, movement of the chromosomes to the poles and the division of the nucleus is brought about by the increase in size and the tendency of the developing daughter cells to move apart in opposite directions. Although the increase in size and elongation of the central spindle may play a role in pulling the chromosomes farther apart and finally pulling the nucleus in two, the main role of the central spindle in Barbulanympha is that of a stabilizer which holds the two symmetrical halves of the dividing cell together until each developing daughter cell is capable of leading an independent existence or until the proper distribution of the chromosomes has occurred and the required development of the extranuclear organelles has been carried out.” (Also see Cleveland title quote, p. 16. Mitosis is thus thought to consist of the sequential activity of MTOC’s associated with these structures usually in the following order: (1) centrioles, and their equivalents in

25

plants and fungi; (2) old kinetochores; (3) newly forming centrioles (animals) or phragmoplasts (many plants). The total amount of tubulin remains constant but there is seqtientiai competition for it around the MTOC’s as microtubules elongate and mitosis proceeds. Chromosomes are thus segregated toward opposite poles by the lengthening of spindle microtubules (Fig. 8). As Cleveland emphasized, tElere can be no &lJch thing as a functional half spindle if this type of model is correct. In the absence of elongating microtubules anaphase movement would not be possible. 2.1.9. Telophase. Chromosome movement slows and then stops because newly forming MTOC’s in telophase provide new competitive nucleation centers. The new MTOC’s vary. In animal cells these MTOC’s generally are newly forming daughter centrioles (Fig. 9a), in some plants the MT00 reside in the newly forming phragmopiast (Fig. 9b). In the hypermastigote flagellates the new MTOC’s correspond to the rostra1 area with its flagella bands (Grimstone and Gibbons, 1966). In higher plants and some green algae new MTOC’s remove the spindle protein to form a matrix of tubules upon which to lay down cellulose fibers that will become the new primary cell

(a)

om0some

Fig* 8. chromosome

phase mo~rn~t shown).

(only one

netochor~ and sism

0mOsome

26

L. Murgulis,Cblchine-sensitivemitotie movements

wall (Hepler and Jackson, 1968). These cen-

ters may be attached to vesicles that are remnants of nuclear membrane-associated centers of earlier stages, as vaguely suggested by electron micrographs (Ledbetter and Porter, 1970). 2.1.10. Late telophase and return to inierphase. Some signal in the cell calls for the

return of microtubules (unstabilized microtubules, i.e. those not in the new centrioles or phragmoplast) to the dimeric tubulin state (InouC and Sato, 1967). As suggested by the sensitivity of tubule polymerization to Ca++ (Olmsted and Borisy, 1973) perhaps change in the intracellular concentration of this divalent ion is involved. Alternatively changes in intracellular ATP may be involved. ATP which is produced continuously by mitochondria but not utilized in biosynthetic reactions during mitosis (Taylor, 1965) may shift the equilibrium of some reaction such as that shown in Fig. 1 to the left. The involvement of the nucleotide triphosphates in the tubulin-tubule balance is implicated in several studies (Sawada and Rebhun, 1969; Avivi et al., 1970b; Olson, 1970; Margulis, 1973). 2.2. Concepts of the effects of mitotic spindle

inhibitors based on this model

2.2.1. Colchicine-like inhibitors. To form

microtubules, tubulin dimer binds to itself at specific surface binding sites. Colchicine (Borisy and Taylor, 1967) and the functionally analogous alkaloid podophyllotoxin (Wilson, 1970) compete by binding at those same sites. It is reasonable to assume that in fully formed microtubules, such as those offlagellar axonemes, these sites are not exposed. In microtubules, in the process of forming, these binding sites nray only be exposed at the growing tips. Since mitosis intrin-

sically involves microtubule formation, it is intrinsically sensitive to colchicine., By hypothesis, at low concentrations only the active tubule forming dimer protein and therefore the growing tips of microtubules bind colchicine. It is thought that tubulin monomer is insensitive to colchicine; this would explaph why the drug can be easily tolerated at certain life cycle stages at concentrations inhibitory to subsequent atages of microtubular formation. There is much evidence indicating the extreme importance of timing in colchitine experiments (da&and and Hecht, 1967; Banerjee et al., 1971; Eigsti and Dustin, 1955; Millecchia and Rudzinska, 197 1). 2.2.2. Nonspecific inhibitors. The assembly of tubulin dimer into microtubules is considered to be the specific colchicine-sensitive step (Borisy and Taylor, f967), presumably other spindle poisons are not specific for this step but simply bind to tubulin protein. Nonspecific agents such as D20 and isopropyl-nphenyl carbamate (IPC) bind to monomer, dimer, tubules or other receptors resulting in nonspecific effects including the depression of the mitotic index (Deysson, 1968). Apparently cold temperature (4” C) and high hydrostatic pressures simply favor the tubulin dimer subunit over the formed microtubule and hence lead to the rapidly reversible disappearance of the spindle or the axopod (Tilney, 197 1). These agents are to be distinguished from the “mitostatic” agents podophyllotox. vinblastine, colchicine and Co&mid i:eysson, 1968) which prevent assembly of dimer and therefore formation of new microtubules but at low concentrations do not affect already formed tubules, chromosome coiling or other mitotic processes. The role of various inhibitors hypothesized on the basis of this model is depicted in Fig. 10.

L. Mu&is,

Colchine-sendtive mitotic movements

27

klitoctlondrl~l

inhibitore (DHP) chlorctllphanicol, oulfhydryl

Protein rynthaah and norrpcciflc inhibitor8 that kill c.Slr md/ot dopaw th mltotLc tndo*.

atents

“t&a

dimeram”

botrhl .ynth.*1&i

----+-w

3.

rubulka*

mowme* OTPMDP

I

cotd.preosun,Ca* \ tubulln* c-----------_ ~pontaroua / dimer @m~~x-CDl’-~r) + ATP + COlCdiCfru

I 4 colhhfoim-tullu11*

*

bound $0

**

colchicim,

rrplaeerblo

oolcoaid,

D0 *2 podophyllotonios by

wfrtonio

aad others

Oeyrrf~a,

1968; XWPJL~O.1973)

Fig. 10. Hypothesized effects of inhibitors on mitosis.

2.3. Problems and predictions This view of mitotic “pushing” rather than the standard “pulling” force generated at the poles has been suggested and rejecte times in the cytology Jiterature. (See Luykx, 1970 and Nicklas, 1971 for review.) On what evidence? Both Forer ( 1968) and Mole-Bajer (1970) have microbeam the regions of the anaphase cell between the pole and chromosome and found this applieation of irradiation to stop movement. Movement is not necessarily concurrently halted in the sister chromatids of the distal half spindle. Furthermore comparable interpolar region irradiation did not stop ovement and some chromosomes on the i diated half spindle even m 1970). ~‘pus~ng” force is consi

cromanipulation (Ni las, 197 1) is considered favorable for the ” Sing” hypothesis. None of these experiments seem to me definitive enough to rule out “pushing forces” i al nor the theory in this paper in particular. the microtubular connections betwe and chromosome are broken by uv the spindle would be expected to expand leavhind the severed chromosome. Contact be reestablished by UV-induced poleward growth of new tubules from the old kinetochores which might then even cause the observed backwards movement. The movement of manipulated chromosomes back to the spindle has the same explanation: microtubules are generated from he kinetochore outward in both directions.

28

L. Margds, Colct;iffe-sensitivemitotic movements

proper pole (Nicklas, 1971). Presumably if the chromosome is manipulated into a region where no tubulin is available kinetosomal MTW,‘s are prevented from generating their new microtubular connections and relocation on the spindle is prohibited. However, if further microbeam experihents entirely eliminate interpolar microtubules and movement still continues, clearly the theory in this paper must be wrong. In fact, such an experimental result would strongly support the model of McIntosh et al. (1969). Likewise the discovery of a functional half spindle would support the model of Bajer (1973). The literature contains many unexplained data that at least do not contradict the theory as presented here. The many observations of lengthening tubules and the absence of any evidence for changes in microtubule diameter with mitotic stages seem consistent (Allenspach and Roth, 1967; Nicklas, 197 1). Autonomy of chromosome movement in poorly defined anaphase of fungal nuclei are associated with elongating spindle (Aist, 1969). The differences between “open” mitosis in which the p&ear membrane ruptures and “closed” mitosis in which the membrane remains intact must not be fundamental since both types of mitosis are observed at different stages of the life cycles of plasmodial slime molds such as askins, 1973, pers+ cornin.). nt chromosome velocities in fotic and meiotic cells quite independent of chromosome masses moved seem consistent with the idea that the rate of movement of the elongating spindle is determined by intrinsic polyme~zation that either

seem more consistent wit

McIntosh et al. ( 1969) can be involved in force generation. It is much more likely that elongation of very few intranuclear microtubules separate chromatin and subsequent elongating extranuclear microtubules acting on nuclear membrane separate daughter nuclei (see Heath and Greenwood, 1968, 1970, 197 1). This second process is comparable to macronuclear node segregation dependent upon bundles of macronuclear microtubules in Stentor (Paulin, 1974). The lengthening microtubule structures of some ciliate nuclei have even been called “pushing bodies” (Raikov, 1969). In the small green phytoflagellate lfwamimomzs division structures are seen that apparently grow through the nucleus and plastids. In one species this structure seems to be an extension of the flagellar rootlet system, in a more specialized species a definite unique division “microbody” is observed. There are very few, or no intranuclear microtubules at this stage (Ndrris and son, 1974, and Norris, 1973, pers. comm.). The toxic effect of chloramphenicol on mitosis unrelated to its effect on protein and RNA synthesis ylor, 1965) is tho be due to the s ribosomal contrib TOC’s: the synthesis of some might be the rate limiting ste

Taylor (1965) as necessary for the initiation. of mitosis. This is consistent with observation of the sensitivity of cilia and flagella to streptomycin (A&man et al., 1974; Kerr, 1965, 1972). The complex curve of the of colchicine on dividing eggs of Arba ITSecht, 1967) may be explained as

L. Margulis.Colchine-sensitivem&xic movements

Proof (or disproof) of this theory will come in several forms: characterization of the hypothetic tubulin dimerization factor (which may be a GTP-ATP transphosphorylase, Tilney, 197 I). Isolation of a colchicine insensitive tubulin monomer during interphase is predicted. Morphologically authen’k nucleating centers (such as cartwheels of basal bodies) that retain in vitro activity and sensitivity to colchicine should be isolated; when used as “crystallizatiort nuclei” tubular structures (such as cilia axonemes) should polymerize in vitro. &$ost definitive would be a method of labeling growing microtubules for pulse experiments during mitotic stages.

3. ~o~ch~c~nesensitive morp

29

and Bussey, 1971) are associated with the stage (stage 3 of Tartar, 1960) of maximal sensitivity of the band movement to mitotic spindle inhibitors such as Colcemid and Marguiis, 1971). According to t presented here the band movement which proceeds at the same rate and is sensitive to the same agents as chromosome movements is caused directly by the continued lengthening of the accessory tubules. presumably at later stages (stage 5-8 of Tartar, 1960) microtubule elongation continues in the cortical root fiber system, positioning the band at the anterior end of the cell. This concept of the homology between chromosome movement and Stentor oral band migration has proved fruitful. The specific ~o~chicine-type of sensitivity band migration to melato

Slow morphogenetic movements (comparable to the rates of chromosome movements in mitosis, 0. I-4 p/min, Nicklas, 197 1) involving structures c of mi~rotubu~es a by hypothesis, o caused directly by polymerization of preformed tubulin dimer

~~cine-sensitive morphoge the he~~ozoa~ w

TokQph_vru, suctoriim te

trichous ciliate

inhibition of oral membranellar band

Stentar, heterotrichous it presumably inhibits formation of macronuclear microtubules in these large spindle-less nuclei prevents proliferation of new intranuclear microtubules reformation can occur in the presence of inhibitors of protein synthesis. Reviews discuss ro!e of microtubules in formation and maintenance of cell shape spindle disappearance, inhibition of orderly cell wall formation also noted

Breakdown of microtubules of retractable axonemes

inhibits normal sperm morphogenesis, branched tlagella induced, ordered microtubules required for nurmal morphogenesis

delay and sensitivity function of developmental stage and concentration

correlated with lack of development of stalk microtubules

Comments

inhl%itsmacsonu clear div%ion and cell growth inhibits micronucleat and macrorvuciear division

morphugenesis

prevents stalk developmeiit

Description

i2&tesium, colonial, peritI2chous ciliate

System

Table 1 Examples of Colchicine Sensitive Processes in Protists, Animals and Plants (after Margulis, 1973).

s 2 s 2

4_s

:

Turner, 1970

Tilney, 1968 Tilney, 1971

Mel&&ii and Rudzinska, 1971 2Ip

Haight and Burchill, 1970 Banerjee and biarguiis, 197 f Margulis, 1973 Wunderlich and Peyk, 1969

Rabat et al., 1974

Reference

_.__.____

somatic association of homologous chromosomes inhibited

inhibits spindle formation

inhibits formation of continuous spindle fibers

many chemical derivatives tested, some more potent inhibitors than colchicine plants containing different doses of somatic association suppressor genes show different colchicine sensitivities ____^_ ___-

at low doses (0.06 rcg/ml) centrioles not affected

cytoplasmic 250A microtubuies of gliding cells disappear, 1OOA filaments induced correlated with altered movement does not inhibit cone growth function and elongation and cytochalasin B sensitive processes

correlated with microtubule disruption

prevents outgrowth of lens primordium induces pseudopodlike processes and correlated amoeboid motion in gliding cells inhibits axonal re-elongation

Avivi et al., 1970a, b

Deysson, 1968

Brinkley et at., 1967

Wessellset al., 197 1

Arnold, 1966 (in Warren, 1967) Bhisney and Freed, 197 1

Moran and Varela, 1971

correlated disassembly of sensory disruption _

induces loss of response to stimuii after l-2 hrs

Table 4, pp. 348 - 349. Margulis, 8973)

_I_^. _._ _..__--___-_--_---.-

Triricum, wheat

root rn~t~m

in collie

tactile sp s (mechanoreceptors1 Rho&&s, squid, dev~¶o~~g eye

Cockroach, campa

Reference

Comments

cription

32

L. Margulis, Colchine-sensftfvemitotic movemmts

4. Microtubule systems in animals

The original microtubular system has been considered to have been coded for by a genetic system of exogenous origin that became involved in the origin of mitosis (Margulis, 1970). From the extreme dependence of basal bodies and centrioles and other microtubular systems on the nuclear-gene based metabolism of the cell, it is obvious (despite observations of genetic autonomy of ciliate microtubule systems, see Sonneborn, 1970 and Fulton, 1970 for reviews) that even if once genetically separate the two systems are entirely merged in modern eukaryotes (Younger et al., 1972). Mitosis, as a process, depends on the complete temporal and spatial integration of chromatin-based nuclear (i.e., original host) and MTOC-microtubular (original sym biont, by hypothesis, Margulis, 1970) events. In multicellular organisms all cells except gametes are ultimately somatic. Further cell division is not necessaril v required of differentiated cells. Therefore the elaborately integrated mitotic apparatus-centriolar MTOC system can br utilized fi>r special function as long as the mitotic prot:ess is preserved in the perpetuating germ line. Consistent with the model of mitosis presented in this paper is a further conceptualization of the varied diversification of the microtubular systems in the soma of animals, modern investigation of which is only just begining. The varieties of somatic uses by animals ol the microtubular based system will merely be mentioned. The receptor cells of visual, auditory and mechanoreceptors contain basal body derivatives. In a well-argued paper Atema (1973) proposes that the ‘“ciliar and microtubule apparatus in sensory cells hold the key to their receptor and conductor properties”. He suggests that impulses are not conducted by membrane-mediated processes as commonly assumed, but by rapidly propagating confor-

mational changes in microtubule protein in situ. Small molecule interaction with the microtubule system has led to the panoramic extension of the sensory system of animals. These concepts (Atema, 1973) which require further development and experimental verification are strengthened by observations that sensory impulse conduction can be inhibited by bathing insect mechanoreceptors in colchitine (Moran and Varela, 1971). It is thus no coincidence that nerve cells that do not divide are rich sources of colchicine-binding microtubule protein (Dutton and Barondes, 1969). Presumably the protein originally synthesized in connection with mitotic division is secondarily organized and utilized in connection with normal neuron function. From an evolutionary point of view then, the presence of tubulin-based mitosis preadapted metazoans for the origin of neuron and sensory cell tubulins. It is predicted that several genetically distinct but related tubulins will be isolated from animal cells (Winston, 1973). The tubulins of the melanocytes of animal skin are y to have a high affinity for melatonin; it ssible that anaesthetic receptors are (presumably membrane-bound) tubulin proteins (Waschke et al., 1974).

5. Summary and implications This model for colchicine sensitive movements is based on the premise that mechanical force can be generated directly by tubulin protein polymerization into microtubules. If true, the force should be directly measurable. The assetibly of dimer into tubules, demonstrable in vitro and in~bited by co1 ~Olmsted and Borisy, 19?3), should be to be sufficient mosome movement 51 vitro. Particular s must be isolated and used as “‘nucleating centers” in of specific micro~ubular structures.

L. Margulis, Colchine-sensitive mitotic movements

symbiosis hypothesis, MTOC’s should ultimately prove to contain protein and RNAs homologous with that from the approf;riate exogenous prokaryote microbes. Cessation of chromosome movement must accompany cessation of tubule assembly and destruction of interpolar spindle microtubules, (a prediction that differs from t3at made by McIntosh et al., 1969). There should be no such entity as a unipolar spindle, as Cleveland (1955) claimed. The development of the colchicine-sensitive mitotic system is thought to have been a necessary precursor to the origin and diversification of protists, fungi, animals and green plants. After its stabilization mitotic microtubular systems were involved in the evolution of certain differentiated systems that depend on functioning of tubulin protein, e.g. the animal nervous system, which is thought to have evolved in late Precambrian times. Because all multicellular eukaryote organisms depend on microtubule-based mitotic cell division, these ideas imply that environmental oxygen was necessary but not sufficient for the impressive rise of multicellular forms in the late precambrian. If new observations are not consistent with the concepts diagrammed :!.he model clearly is at fault; its virtue will have been in the research and discussion stimulated.

Acknowledgements I am grateful to S. Banerjee, S. Bropst, K. Altman, M. Winston, R.E. Stephens, LB. Heath, R.B. Nicklas, L.W. Olson, V. Tartar, R. Norris, D. Garland, J. Atema,

these issues. %thank

33

References Allen,C.

and Borisy, G.G., 1973, Fiagellar assembly in Chiamydomonas: in vitro addition of microtubule sub. units to isolated axoncmes cabs.), J. Cell BioL., 59, Sa. Allenspach, A.L. and Roth, LX., 6967, Structural variatious during mitosis in the chick embryo, J. Cell BioL, 33, 179. ARman, K., Banerjee, S., Keheher, J. and Margulis, L., 1974, Cilia membrane abnormalities induced by amirroglycoside antibodies in Stentor coeruleus 1974 (Abs.) J. Protozoolo gy Supplement. Amoore, J.E., 1961a, Arrest of mitosis by oxygen-lack or cyanide, Proc. Royal Sot. B., 154, 9.5. Amoore, J.E., 1961b, Dependence of mitosis and respiration in IOOtS upon oxygen tension. Proc. Royal Sot. B., 154, 109. Atema, J., 1973, Microtubule theory of sensory transduction, J. Theoret. Biol., 38, 181. Aist, J.R., 1969, The mitotic apparatus in fungi, Ceratoqstis, Fagaceantm and Fusarium oxysporum, J. CeB Biol., 40. 120. Avivi, L., Feldman, M. and Bushuk, W., 1970a, The mechanism of somatic association in common wheat, Triticum aestivum L. ii. Differential affinity for colchicine for spindle microtubules of plants having different doses of the somatic association suppressor,Genetics, 65,585. Avivi, L, Feldman, M. and Bushuk, W., 1970b, Tine mechanism of somatic association in common wheat, Triticum aestivum E. Ill. Differential affinity for nucleotides of spindle microtubules of plants having different doses of the somatic-association suppressor, Genetics, 66,449. Awramik, S.M.. Golubic, S. and Barghoorn, ES.. 1972, Blue green algal cell degradation and its implication fltr the fossil record, Geol. Sot. Amer. Annual Meeting, Minneapolis, Mmn. Abs. with programs, Vol. 4, Abs. 7,438. Bajer, A. and Mole-Bajer, J., 1970, 7th Congres Internatl. de Microscopic Electronique, Grenoble, France. Bajer, A., 1973, How microtubules pull chromosomes, tabs.), J. Cell Biol., 59, 14a. Banerjee, S. and MarguJis, L., 1971, inlnbition of cilia regeneration by antineoplastic agents, Cant. Chemother. Reports, 55,531 Banerjee, S. and Margulis, L., 1973, Mitotic arrest by meiatonin. Exp. Cell Res., 78,314. Banerjee, S., Kerr, V., Winston, WI.,KeUeher, J.K. and Margulis, L., 1972, Melatonin: inhibition of microtubule-based oral morphogenesis in Sten:or coepuleus. J. Protozool., 19,108. , J.J., 1978, Ameboid movement inhages by colchicme or vi 9. ., 1967, The mec~la~is

34

L. @rrgufis, Colchine-sensitive mitotic movements

Brinkley, B.R., Stubblefield, E. and HSU,T.C., 1967, The effects of CoRemid inhibition and reversal on the fine structure of the mitotic apparatus of Chinese hamster cells in vitro, J. Ultrastruct. Res., 17,1card&t, R.M., Sate, H. and Inoue, S., 1966, Further observations on the thermodynamics of the living spindle, BioL Bull, (Woods Hole) 131,385. Cleveland, L.R., 1953, Studies on chromosomes and nuclear division. I-IV Trans. Amer. Phil. Sot., 43,809. Cleveland, L.R., 1955, Hormone-induced sexual cycles of flagellates. XIII. Unusual behavior of gametes and centrioles of Earbr&nympha, J. Morph., 97,511. Cleveland, L.R., Hall, S.R., Sanders, E.P. and Collier, J., 1934, The wood feeding roach Cryptocercus, its protozoa, and the symbiosis between protozoa and roach. Mem. Amer. Acad. Arts Sci., 17,185 Cloud, P.E., Jr., 1968a, Atmospheric and hydrospheric evoiution on the primitive earth, Science, 160,729. Cloud, P.E., Jr., 1968b, Prcmetazoa evolution and the origin of metazoa. In: Evolution and Environment, E.T. Drake, e.d. (Yale Univ. Press, New Haven, Corm.) pp. l-72. Cloud, P.E., Jr., Licari, G.R., Wright, L.A. and Troxel, B.W., 1969, Proterozoic eucaryotes from Eastern California, Proc. Nat. Acad. Sci., 62,623. Deysson, G., 1968, Antimitotic substances, Int. Rev. Cytol., 24,99. Dutton, G.R. and Barondes, S., 1969, Microtubule protein: Synthesis and metabolism in developing brain, Science, 166,1637. Eigsti, O.J. and Dustin, P., 1955,Colchicine. In: Agriculture, Medicine, Biology and Chemistry. (Xerox reprint, University Microfti, Ann Arbor, Michigan, 1968). Elzinga, M., Collins, J.H., Keuhal, W.M. and Adelstein, R.S., 1973, Complete amino acid sequence of actin of rabbit skeletal muscle. Proc. Natl. Acad. Sci. U.S., 70,2687. Forer, A., 1969, Chromosome movements during cell division. In: Handbook of Molecular Cytology, LimadePa& A., ed. (North-Holland, Amsterdam) pp. 553-601. Franke, W.W., Stadler, J. and Krein, S., 1972, Autoradiographic localization of colchicine binding sites, Beitr. Path, Bd., 146,289. F&on, C., 1970, Results and Problems in Cell Diiferentiation. In: Grigin and Continuity of Cell Organelles (Springer-Verlag, Berlin) pp. 470-221. Glaessner, M.F., 1968, Biological evenfs and the Precambrian time scale, Canad. J. Earth Sci., 5,585. Goode, M.D., 1971, Ph.D. Thesis, Iowa State University, Ames, Iowa. Grimstone, A.V. and Gibbons, I.E., 1966, Fine structure of the centrioiar appgratus and associated structures in the complex flagellates l’kichonympha and PseudotrichonymPhrr, phi. Trans. Roy. Sot. B, 250,215. Haight, J.M., Jr. and Burchill, B.R., 1970, The effects of colchicine and CoRemid on oral differentiation in Stentor @@&us, J. Protozool., I7.139. Hartman, H. and Gurney, T., Jr., 1974, Evidence for th,e association of RNA with ciliary basal bodies of Z&nhyrnena, in press.

Haschke, R.H., Byers, M.R. and Fink, B.R., 1974, Effect of local anesthetics on microtubule rcpolymerization, J. Neurochem., in press. Haskins, E., 1973, Pets. cqmm. Heath, I.B. and Greenwood, A.D., 1968, Electron microscopic observations of dividing somatic nuclei in Saprolegnia, I. Gen. Microbial., 53,287. Heath, I.B. andGreenwood, A.D., 1970, Centriole replication and nuclear division in Akprolegnia, J. Gen. Microbial., 62,139. Heath, I.B. and Greenwood, A.D., 1971, Ultrastructural observations on the kinetosomsb and Golgi bodies during the asexual life cycle of Saprolegnti, Z. Zellforsch Mikrosk. Anat., 112,371. Hepler, P.K. and Jackson, W.T., 1969, rsopropyi-n-phenyl carbamate affects spindle microtubule orientation in dividing endosperm cells of Huemanthus kuthetinae Baker. J. Cell Sci., 5,727. Hepler, P.K., Mclntosh, J.R. and Cleland, S., 1970, Intermicrotubule bridges in mitotic spindle apparatus, J. Cell Biol., 45,438. Inoud, S. and Sato, H., 1967, Cell motility by labile association of molecules. The nature of mitotic spindle fibers and their role in chromosome movement, J. Gen. Physiol., 50,259. Jensen, C. and Bajer, AS., 1973, Kinetochore microtubules of Huemanthus endosperm during mitosis (ahs.), J. Cell Biol., 59, 156a. Kerr, N.S., 1965, Inhibition by streptomycin of flagella formation in a true slime mold, J. Protozool., 12,276. Kerr, S.J., 1972, Flagellum growth and regeneration in the true slime mould Didymium nigripes, J. Gen. Microbial., 72,429. Kubai, D. and Ris, H., 1969, Division in the dinoflagellate Gyrodinium cohnii (Schiller). A new type of nuclear reproduction, J. Cell Biol., 40,508. Lambert, A-M. and Bajer, A.S., 1973, Lateral activity of chromosome fibers and chromosome movement (Abs.) J. Cell Biol., 59,184a. Ledbetter, MC and Porter, K.R., 1970, introduction to the Fine Structure of Plant Cells (Springer-Verlag, Berlin). Leedale, G., 1967, Euglenoid Fiagellates. (Prentice-Hall, Englewood Cliffs, N.J.) Luduena, R.F. and Woodward, D.O., 1974, Primary structure of tubulin protein, in press (See abs. J. Cell Biol., 59, 203a. Nov. 1973). Luykx, P., 1970, Cellular Mechanisms of Chromosome Distribution (Academic Press, New York). MacDonald, K., 1972, The ultrastructure of mitosis in the marine red alga, ~en~brffnopter~ p~typ~y~~, 3. Phycol,, 8,156. Malawista, S., Sato, H. and Bcnsch, K.G., 1968, Vinblastine and griseofulvin reversibly disrupt the living mitotic spin* dle, Science, 160,770. Manton, I., Kowaliik, K. and van Stosch, HA. 1969a, Observations on the fine structure of a marine centric tom, J. Microscopy, 89,317.

L. i#fargulis,Colchine-sensitivemitotic movements Manton, I., Kowallik, K. and von Stosch, H.A., 1969b, Observations on the fine structure development of the spindle at mitosis and meiosis in a marine centric diatom (Lirhodesmium undulittum). II. The early meiotic stages in the male gametogenesis, J. Cell Sci., 5,271. Margulis, L., 1970, Origin of Eukaryotic Cells (Yale Univ. Press, New Haven, Corm.). Marguiis, L., 1971, Whittaker’s live kingdoms of organisms: Minor revisions suggested by consideration of the origin of mitosis, Evolution, 25,242. Margulis, L., 1973, Colchicine-sensitive microtubules. In: int. Rev. Cytology, B. Boume and 3.F. Daniel& eds., (Academic Press, New York) pp. 333-361. . MarsIand, D. am! Hecht, R., 1967, Cell division: Combined antimitotic effects of colchicine and heavy water on first cleavage in the eggs of Arbacia punctulata, Exp. Cell Res., 51,602. Mazia, A., 1961, Mitosis and the Physiology of Cell Division. In: The Cell, J. Brachet and A.E. Mirsky, eds. (Academic Press, New York) Vol. III. McIntosh, J.R., Hepler, P.K. and Van Wie, D.G., 1969, Model for mitosis, Nature, 224,659. Millecchia, L.L. and Rudzinska, M.A., 1971, The ultrastructure of nuclear division in a suctorian, Tokophyra in& sionum, Z. ZelIforsch., 115,149. Mohri, H., 1968, Amino acid composition of “tubulin” constituting microtubules of sperm flagella, Nature, 217, 1053. Mshri, H. and Shimomura, M., 1973, Comparison of tubulin and actin, J. Biochem., 74,209. Moran, D.T. and Varela, F.G., 1971, Microtabules and sensory transduction. Proc. Natl. Acad. Sci. U.S., 68,757. Motta, J.J., 1967, A note on the mitotic apparatus in the rhizomorph meristem ofArmtilan2 mellea. Mycologia, 59, 370. Nick&, R.B., 1971, Mitosis. In: Advances in Cell Biology, D.M. Prescott, L. Goldstein and E.H. McConkey, eds., (Appleton-Century-Crofts, New Yang) pp. 225 -297. Norris RE. and Pearson, 8. 1974, Ultrastructure of cell division in ~mmimonas (in preparation). Pers. comm. Olmsted, 9.5. and Borisy, E.G., 1973, Microtubules, Ann. Rev. Biocheu;., 507. Olson, LW., 1970, Microtubule morphogenesis in Allomyces (Ph.D. dissertation, Dept. of Botany, II. of Calif., Berkeley). Paulin, J.J., 1974, Macronuclear microtubules in S?en:or (in preparation). Paulin, J.J. and Bussey, J., 1971, Oral regeneration in the cilia Stentor coeruleus: A scanning and transmission electron optical study, J. Protozool., ! 8,201. Pickett-Heaps, B.D., 1978, The autonomy of the centriob: fact or fallacy? Cytobios, 1,285. Pickets-Heaps, J.Q., 1972, Vanation in mitosis and cyto’kinesis in plant ce : its significance . e phylogeny and evolution of ultrastructuraI systems, Cytobios, 5,59. Ratov, I.B., 1969, Macronucleus of Protozoology, T-T. Cben, ed., ( York) pp. I-128.

35

Raikgv, I.B., 1973, Mitose intranucl&re acentrique du micronoyau de Loxodes magnus, C%EHolotriche. Etude ultrastructurale. CR. Acad. Sci. Paris, 276,238s. Rahat, M., Friedlaender, H., Pripaz, Y. and Parnas, I., 1974, The contractile stalk of Carchesium,an elementary muscle? Proc. 4th Internatl. Cong. Protozool., CbrmontFerrand, France. Sept., 1973. Ris, W.H., 1949, The anaphase movement of chromosomes in the spermatocytes of the grasshopper., Biol. Ball., 96,90. Rosenbaum, J.L. and Carlson, K., 1969, Cii regeneration in Tetruhymena and its inhibition by colchicine. J. Cell Biol., 40,415. Sato, H., hod, S. and Ellis, G.W., 1971, The microtubule origin of spindle birefringence: experimental verifications of Wiener’s equations. Abs. 11th Annual Meeting, Amer. Sot. Cell Biol., New Orlea Sawada, N. and Rebhun, L.I., 1969, The effect of dinitrophenol and other phosphorylation uncouplers on the birefringence of the mitotic apparatus of marine eggs, Exp. Cell Res., 55, 33. Schopf, J.W., 1972, Precambrian paleobiology. In: Exobiology, C. Ponnamperuma, ed. (North-Holland, Amsterdam). Schopf, 3-W. and Blacic, J.M., 1971, New microorganisms from the Bitter Springs Formation (Late Precambrian) of the North-Clentral Amadeus Basin, Australia, P. Paleontology, 45,925. Sonneborn, T.M., 1970, Determination, Development and Inheritance of the Structure of the Cell Cortex. In: Symposia of the Society for Developmental Biology [Academic Press, New York). Soyer, M-O., 1971, Structure du noyau des Blastodinium (dinoflagellds parasites). Division et condensation chromaChromosoma, 33,70. .-O., 1972, Les ultrast~uct~res nucltires de al Noctiluque (DinoiIagelIe’ libre) au cours de la spoaogen&e, Chromosoma, 39,419. Stadler, J. and Franke, W.W., 1972, Colchicine-binding proteins in chromatin and membranes, Nature New Biol., 237,237. Stephens, R.E., 1968, Reassociation of microtubule protein, J. Molec. Biol., 33,517. Stephens, R.E., 1970, h%zrotubules. In: Biological Macromolecules, S.N. Timaheff and G.D. Fasman eds. (Marcel Dekker, New York) pp. 355-391. Stephens, R.E., 1973, A thermodynamic anaIysis of mitotic spindle eq~~~brjurn at active metaphase, J. Cell BioE.,57, 133. Tartar, V., 1961, The Biology of Stentor. (Pergamon Press, 1974, ~rn~~~t~ons an

biosis theory of the

es, Taxon, in press. TiIney, L.G., 1968, The assembly of role iu the development of cell form. In: 27th iology , SuppIement 2, of the Study for 63.