- Email: [email protected]
The mechanism and control of cytokinesis
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The mechanism and control of cytokinesis Michael Glotzer Cytokinesis is under active investigation in each of the
dominant experimental model systems. During 1996 and 1997, several developments necessitated the reassessment of the prevailing model for cytokinesis. In addition, the inventory of proteins required for cytokinesis has grown considerably. However, a molecular understanding of cytokinesis still remains elusive.
Addresses Institute for Molecular Pathology, Dr Bohr-Gasse 7, 1030 Vienna, Austria; e-mail: [email protected] Current Opinion in Cell Biology 1997, 9:815-823 http :l lbiomednet.coml elecref1095506 7 4 0 0 9 0 0 8 1 5
© Current Biology Ltd ISSN 0955-0674 Abbreviation GAP GTPase-activating protein
Introduction
Cytokinesis, the division of one cell into two, is a source of fascination for cell biologists. Not only does the cell produce two entities from one, it also positions the division plane so that each daughter cell receives a full complement of chromosomes and other essential organelles. In animal cells, cytokinesis can be subdivided into five subprocesses (Figure 1) [1]. In anaphase, the mitotic spindle specifies the site at which the cleavage furrow will form. A contractile ring containing actin and myosin assembles at this site. This ring contracts, furrowing the overlying plasma membrane. A transient structure, the midbody, forms when the furrow reaches the remnants of the mitotic spindle. Finally, the common membrane that surrounds the two nascent cells is divided so that the cells may separate. There have been several notable developments in the field of cytokinesis during 1996 and 1997 since the last review in Current Opinion in Cell Biology [2]. First, there are indications that the mitotic spindle plays a role during furrow ingression beyond its critical and early role of specifying the position of the cleavage furrow. Second, a number of GTPases (and their binding partners) have been identified that are required for cytokinesis and the characterization of their role in this process has provided some new insights. Third, while myosin II plays a crucial role in cytokinesis, there is evidence that, under some circumstances, it is dispensable. Fourth, analysis of cytokinesis has begun to focus more clearly on the role of membranes in this process. Finally, and perhaps most importantly, several major model systems have emerged and a certain degree of convergence of the work in these
different systems has become clear. In this review, I will focus on these recent advances. The role of the central spindle Furrow specification
T h e role of the mitotic spindle in cytokinesis has largely been established by micromanipulation experiments performed in marine invertebrate embryos. Bipolar spindles induce furrowing, as do two asters and two poles of independent bipolar spindles [3]. After the start of furrow ingression, depolymerization of the spindle [4], or its partial or complete removal [5,6"], does not necessarily prevent the completion of cytokinesis. These and other data led to a model in which microtubules emanating from the asters of the mitotic spindle specify the position of the cleavage furrow. A second feature of this model is that, once specification has occurred, cytokinesis could proceed to completion in the absence of the spindle or other microtubular structures [7]. However, there is now reason to question the generality of certain aspects of this model. In the classic view, the asters of the mitotic spindle are the sole source of the signal that specifies the cleavage plane. However, recent experiments indicate that in some cells the 'central spindle' may determine the position of the cleavage furrow. T h e central spindle arises from the elongation and reorganization of the ellipsoid metaphase spindle during anaphase and telophase. T h e interpolar microtubules undergo extensive bundling, forming the central spindle. T h e possibility that this structure may be important for cytokinesis arose from attempts to reproduce some of the experiments on which the original model (see above) had been based, but in cultured cells rather than in a large embryo (Figure 2). To determine whether two asters that are not connected by a spindle can induce formation of an ectopic furrow, the behavior of cells containing multiple spindles has been examined [8"',9°,10"]. T h e formation of such furrows was somewhat variable and depended on the precise geometry of the spindles. However, in one study, fluorescently labeled tubulin enabled the organization of microtubules to be monitored and the variability in furrow formation could be accounted for by the organization of microtubules; when microtubule bundles formed between the asters an ectopic furrow formed, suggesting that the asters themselves were insufficient to induce furrow formation, and that a central spindle like structure is necessary [8"']. A second experiment that had implicated the asters as the source of the signal that induces furrowing consisted of placing obstructions between the spindle and the equatorial cortex. T h e obstructions, if they were of the appropriate size, would not prevent furrowing,
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Figure 1
Cleavage plane specification
Contractile ring assembly
:urrow ingression
Cell separation
C~eavage me,mDt'a~t~S
Midbody formation CurrentOpinionin CellBiology The basic subreactions of cytokinesis. Actin is shown as red, microtubules as purple, cleavage membranes as blue, and chromosomes as green
circles or elongated shapes. Pink triangles represent the hypothetical cleavage signal that specifies the position of the division plane.
presumably because the equatorial cortex remained under the influence of the asters [11]. This experiment has been adapted to cultured cells by using a blunt glass needle to fuse the dorsal and ventral plasma membranes together, producing a small hole between the central spindle region and the cell periphery (Figure 2) [12"]. A furrow formed in the cortex adjacent to the hole, deforming it, but no furrow formed in the cortex distal to the hole. Moreover, there was a correlation between the arrangement of microtubules and the contractile behavior of the cortex w in the cytoplasm distal to the hole microtubules remained unbundled and the cortex did not furrow, while in the cytoplasm proximal to the hole the microtubules became bundled and a furrow formed in the adjacent cortex. Thus, at least in cultured cells, the central spindle, as opposed to the asters, specifies the position of the furrow.
Furrow ingression
As mentioned above, in some invertebrate embryos removal of the spindle from a cell that has initiated cytokinesis does not prevent completion of that division,
suggesting that the spindle does not act during furrow ingression. However, recent observations indicate that the mitotic spindle may also function during cytokinesis, after the furrow has begun to ingress. Cultured cells that had initiated cytokinesis were treated with a microtubuledepolymerizing drug [8"1. T h e cells had been injected with ftuorescently labelled tubulin to allow monitoring of the response of the microtubular array to the drug treatment. In the majority of cells analyzed, microtubules were fully depolymerized and the cells did not complete cytokinesis. In one-quarter of the cells, some microtubule bundles remained in the central spindle; in these cases, the cells completed cytokinesis [8"°1. Thus, microtubules may in fact be required continuously during cytokinesis. How might this conclusion be harmonized with the earlier, opposite conclusion? T h e interzonal microtubules whose presence correlates well with successful completion of cytokinesis are quite insensitive to microtubule drugs and this may have misled earlier investigators. Moreover, there are indications from invertebrate embryos that displacement of the spindle can cause furrow regression [13] (furrow regression means the relaxation of a partially or fully ingressed furrow). Alternatively, the requirement
The mechanism and control of cytokinesis Glotzer
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Figure 2
(a)
(b)
Cultured cell
Emb~o
Cultured cell
Embryo Oil drop
Hole J
Oil drop
Hole
CurrentOpinionin CellBio!ogy Micromanipulation of embryos and cultured cells suggests that there are system-specific differences in the role of the central spindle in furrow specification and ingression. (a) In embryonic systems, the presence of two spindles in a single cell causes the formation of an ectopic furrow (indents in the cell membrane) between the two asters of the neighboring spindles. In cultured cells, the formation of an ectopic furrow is observed only when a bundle of microtubules forms between neighboring spindles (i.e. in the bottom right-hand cultured cell). (b) In embryos, an obstruction between the central spindle and the equatorial cortex does not inhibit furrow formation, in cultured cells, an obstruction between the central spindle and the equatorial cortex inhibits furrowing from the outer cortex of the side of the cell that contains the obstruction.
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for microtubules throughout cytokinesis may not exist in all cells.
The role of the central spindle in cytokinesis has also emerged from genetic analysis of Drosophila. Male flies homozygous for mutations in the kinesin-like protein KLP3A exhibit defects in cytokinesis during the meiotic divisions of spermatogenesis [14]. Phenotypic analysis of mutant spermatocytes reveals that although the spindles appear normal during meiotic metaphase, in anaphase the central spindle fails to form. There are no morphological signs of furrowing, indicating either that the cortex has not received the signal to furrow or that it is defective in furrow assembly or ingression. In either case, it is clear that cytokinesis requires this motor protein and it is possible that the requirement for this protein in cytokinesis may reflect its role in organizing the central spindle, which in turn may be necessary for proper furrow function. Localization of cleavage furrow markers will help to determine whether KLP3A is required to specify the position of the cleavage furrow or for its ingression. If cleavage furrow markers are found properly localized, this would indicate that KLP3A is required for furrow assembly or ingression rather than for specification.
How might the central spindle, or the microtubule bundles that make up the central spindle, participate in furrow ingression? One could speculate that the signal for furrowing is not merely a transient event, but rather that continued ingression of the furrow requires constant reinforcement by the central spindle. It is also possible that other proteins that concentrate in the central spindle, perhaps by binding to overlapping, antiparallel microtubules, are important for furrow ingression [15-17]. Alternatively, a direct interaction between the tip of the furrow and the central spindle may promote furrow ingression. In addition to actin and myosin, the furrow contains other proteins that are necessary for cytokinesis, including the septins (see [18] for a review). Septins are necessary for cytokinesis in budding yeast [19], Drosophila [20], and vertebrates ([21°']; M Glotzer, AA Hyman, unpublished data). In yeast, septins are localized to the bud neck throughout much of the cell cycle and they appear to be components of a series of 10 nm filaments [19]. Septins have been isolated from Drosophila extracts and the purified protein complex contains filamentous structures of up to -300 nm in length with a periodicity of 28 nm [22°°]. In vertebrate cells a large fraction of the septins is associated with actin-containing structures, including with the contractile ring [21°°]. In Xenopus embryos septins localize to some microtubule-containing structures and they bind to microtubules in vitro (M Glotzer, AA Hyman, unpublished data). As septins can bind both actin and microtubules, and as they are found in the cleavage furrow, perhaps they are part of a link between the cell cortex and the central spindle that promotes furrow ingression.
Regulation of furrow assembly and ingression Once the position of the furrow is specified, be it by the asters or by the central spindle, what controls the formation and constriction of the contractile ring? Several small GTPases and their interacting proteins have been implicated in cytokinesis, and these proteins are likely to play a regulatory, rather than a structural, role.
In
Schizosaccharomycespombe
A GTPase has been identified that appears to have an important regulatory function in the division of Schizosaccharomycespombe. T h e division of fission yeast is somewhat different from the division of animal cells (see [23,24] for reviews). First, the position of the division plane appears to correlate with the position of the nucleus in late Gz phase, rather than with the position of the mitotic spindle. As in animal cells, an actin ring forms in the future division plane. This ring presumably contracts, thereby pinching the membrane into two entities. Secretion of cell wall material generates the division septum. A portion of the old cell wall and a primary septum is dissolved, allowing the cells to separate. T h e gene spgl, which encodes a GTPase, has been recently isolated and implicated in cytokinesis, spglcells cannot compete cytokinesis as they are defective in septum formation [25°°]. These mutations do not affect formation of an actin ring at the correct time and place. More interestingly, mild overexpression of this GTPase induces formation of multiple septa and the formation of an actin ring, and this can occur at various cell cycle stages, thereby uncoupling the onset of cytokinesis from progression through the cell cycle. It is noteworthy that the septa induced by Spgl overexpression do not fully cleave, but the reason for this remains unclear. One testable hypothesis is that although septum formation does not need to be coordinated with the cell cycle, the later steps involved in cell separation may be cued by the cell cycle machinery. Although spgl is not required for formation of the actin ring, it can induce their formation ectopically, raising the possibility that spgl is a central regulator of cell division in S. pombe. Like several other GTPases, Spgl forms a complex with a kinase, Cdc7 [25"], and it is probably regulated by a GTPase-activating protein (GAP) and a guanine nucleotide exchange factor. Cdcl6 is a good candidate for the GAP, as it interacts genetically with Spgl as well as displaying sequence homology to known GAPs [25"]. However, Spgl is not unique in its ability to cause formation of ectopic septa. The polo-like kinase, Plol, is necessary for formation of the actin ring and for septum formation [26]. Like Spgl, overexpression of Plol induces multiple septa. A novel gene, byr4, has the opposite phenotype: loss of byr4 induces the multisepta phenotype [27°]. Further analysis is needed to characterize the relationships between Spgl, Cdc7, Cdcl6, Plol, and Byr4, in particular to determine if they comprise a single
The mechanism and control of cytokinesis Glotzer
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pathway or whether there are several pathways, each of which can induce septum formation.
[43°], Aspengillus nidulans (sepA) [44°], and Caenorhabditis elegans (cyk-1) (B Bowetman, personal communication).
Does a GTPase homologous to Spgl control cytokinesis in other organisms? It is too early to answer this question, but it is notable that the closest relatives to Spgl and its interacting kinase Cdc7 are encoded by the budding yeast genes TEM1 and CDC15, respectively, and that these genes are required for exit from mitosis [28,29]. This complicates the analysis of whether they also regulate cytokinesis, but there is as yet no evidence that they do. Moreover, the S. pombe spgl and the Saccharomycescerevisiae TEM1 gene products are not functionally interchangeable [25°°]. Thus, the Spgl pathway may exist in other organisms, but it may regulate a different cellular process.
These proteins contain a polyproline-rich region that binds to profilin, which in turn regulates actin assembly [42°',43°,45°,46°]. Formins also bind to Rho family proteins [42°°,45",46°]. Thus, this protein family may form a crucial link between these GTPases and actin. However, the precise function of the formins may vary in different cell types. In S. pombe, cdcl2 mutants fail to make an actin ring and to deposit septum material [43°], while, in C. elegans, cyk-1 mutant embryos make a furrow that ingresses to near completion and then regresses (P G6nczy, AA Hyman, unpublished data). A second class of Rho effectors has been identified that includes regulators of the phosphorylation state of myosin [47°,48°]. T h e action of Rho on these effectors may also be crucial for cytokinesis.
In Die~ostelium
A distinct GTPase, racE, is required for cytokinesis in Dictyostelium. This GTPase appears to be specifically required for cytokinesis, as the mutant exhibits no phenotypes other than an inability to undergo cytokinesis [30°]. RacE is localized to the plasma membrane, but it is not enriched in the cleavage furrow [31°]. Two GAPs have been identified which are necessary for cytokinesis, gapA and DdRasGAP1 [32°,33°]. Like racE, gapA appears to be specifically required for cytokinesis, suggesting that gapA might act in concert with racE. Although the gapA and racE mutants have slightly different phenotypes, this difference could be explained by the fact that gapA- cells could contain racE.GTP, while racE- cells would not. Deletion of DdRasGAP1, on the other hand, causes defects in development as well as in cytokinesis; thus, it is unlikely to act exclusively on racE and indeed it may have a distinct substrate. In Xenopus and other e u k a ~ o t e s
Rho protein is essential for cytokinesis in many cell types [34-36,37°°]. Detailed analysis of the cleavage of Xenopus embryos in which Rho has been inactivated indicates that Rho protein maintains the integrity of the cortical layer of actin [37°°]. This loss of cortical actin prevents the formation of an ingressing cleavage furrow. However, at least one aspect of cytokinesis, the insertion of cleavage membranes, still occurs, indicating that the failure in cytokinesis is not a consequence of blocking specification of the cleavage furrow. Perturbations of Cdc42 also cause defects in cytokinesis [37°°,38]; this GTPase also appears to act during furrow ingression rather than having a role in the specification process [37°°]. GTPase effectors
Small GTPases regulate cell behavior by x/irtue of their interactions with a number of other proteins, the so-called effector molecules (see [39] for a review). A new class of potential effector molecules has emerged, the 'formin' family (see [40] for a review). These family members are implicated in cytokinesis in Drosophila (Diaphanous) [41], S. cerevisiae (BNI1 and BNR1) [42°°], S. pombe (Cdcl2)
Myosin
Over the past three decades, multiple experimental approaches have implicated myosin as a central player in cytokinesis. T h e issue was, or so it seemed, laid to rest in 1987, when Dictyostelium cells lacking myosin II were engineered by gene knockout and antisense technology [49,50]. Myosin null cells, when grown in suspension, failed to divide and became giant multinucleate cells that eventually died. However, these cell lines could be propagated by growth on a solid support where they divided by an aberrant process that was unlinked to the cell division cycle and which was termed 'traction-mediated cytofission'. T h e division of cells lacking myosin II has recently been re-analyzed [51°°]. Surprisingly, more than 80% of adherent, mononucleated, myosin II null cells divide after mitosis in a manner that closely resembles cytokinesis of wild-type cells. When it is present, myosin II does play a role in cytokinesis of adherent Dictyostelium, as cytokinesis is faster and more efficient in its presence than in its absence, but it certainly appears to be dispensable under some circumstances. It is not known what provides the force that is necessary to deform the cleavage furrow in adherent myosin II null cells. In addition to a single myosin heavy chain gene, Dictyostelium has a plethora of 'non-conventional' myosins [52], and it is possible that one or more of them substitutes for the loss of myosin II when dividing on a substrate. Further genetic analysis will determine whether this is the case or whether an alternative, novel mechanism is at work in these cells. It is imperative to analyze whether other Dictyostelium mutants that are unable to grow in suspension, but which can grow on a solid substrate, are also able to divide in a cell cycle dependent manner or whether they are only able to divide by traction-mediated cytofission. T h e evidence implicating a role for myosin in cytokinesis in other organisms is quite strong, but the accumulated evidence does not rule out the possibility that, under some conditions, animal cells could divide in the absence of
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myosin II. This question is best addressed in genetically tractable organisms. Myosin II is required for division of the C. elegans embryo [53°°], and in Drosophila mutations in the regulatory light chain of myosin cause cytokinesis defects [54]. Although myosin II is not essential for cytokinesis in S. cerevisiae [55,56], it is essential for cytokinesis in S. pombe [57°°]. S. pombe cells lacking myosin II form actin rings at the normal site. This indicates, as does the analysis of the myosin II mutants of Dictyostelium, that myosin does not play a role in the specification of the division plane in either of these organisms (recall that division plane specification in S. pombe is distinct from that of Dictyostelium and animal cells).
M e m b r a n e dynamics T h e function of cytokinesis is to create a membranous barrier between the two daughter cells. T h e central role of the membranes has been largely ignored except in the case of higher plant cells, in which the division plane is formed by the assembly of a membranoua structure in the center of the cell that subsequently grows towards the cell periphery [58]. In plants, Golgi-derived vesicles are transported in a microtubule-dependent manner to the region between the two groups of sister chromatids. These vesicles fuse into a tubulo-vesicular network that matures into a fenestrated sheet-like structure. Cell wall materials are secreted into the lumen of this sheet and it matures further, eventually becoming a continuous membrane on either side of a cell wall [58]. Two molecules have been recently identified that may mediate the assembly of thc membranous structure. T h e Arabidopsis KNOLLE gene has found to be necessary for proper cytokinesis in the embryo [59°°]. T h e product of the KNOLLE gene is homologous to syntaxins, which are involved in membrane fusion events [60]. It appears likely that K N O L L E is involved in forming the membranous structure that gives rise to the division plate. A second protein has been discovered that could be involved in the remodelling of the membrane. A dynamin-like protein called phragmoplastin is concentrated in the cell plate [61°,62°]. Dynamin itself has been implicated in endocytosis in a wide range of organisms, where the protein has been shown to self-assemble into ring-like structures that may promote the pinching off of endocytic vesicles [63]. Phragmoplastin could perhaps function in cytokinesis by mediating the formation of the tubular vesicular network, or in the maturation of the tubular vesicular network by virtue of its ability to deform membranes. In the embryonic divisions of animal cells, new membrane is inserted just behind the leading edge of the ingressing furrow [37°°,64]. Thus, it is possible that proteins related to K N O L L E and phragmoplastin may also function in the division of animal cells. Dynamic changes in the plasma membrane might well be a general phenomenon. During division of cultured cells, there are indications that the composition of the plasma membranes changes
during cytokinesis and, furthermore, agents that disrupt the redistribution of a certain phospholipid prevent cytokinesis [65°]. Phosphatidylethanolamine (PE) is a membrane phospholipid that is asymmetrically distributed in cell membranes, being present in much greater amounts in the inner as opposed to the outer leaflet of the plasma membrane. A novel probe consisting of a tetracyclicpeptide that binds PE specifically was used to demonstrate that, during cytokinesis, the outer leaflet of the cleavage furrow contains appreciable amounts of PE [65°]. T h e source of PE in the outer leaflet is not known; it could come from the inner leaflet, it could become concentrated from elsewhere in the outer leaflet, or it could become localized t h e r e a s a result of vesicle insertion. A muhivalent derivative of this probe traps PE and prevents completion of cytokinesis; the actin ring persists long after it would normally disassemble [65°]. This study illustrates that the plasma membrane is an important player in cytokinesis. T h e composition of the membrane could be critical to the final step of cytokinesis, cell separation, which is likely to involve the regulated fusion of membranes rather than being a direct consequence of the furrowing process.
Perspectives T h e significant progress that has been made in the time period covered by this review has largely resulted from the fact that cytokinesis is emerging as an active field of study in multiple experimental systems. A number of gene products that are important for cytokinesis have been discovered by genetic analysis in fungi and Didyostelium. This effort has been complemented by studies of Drosophila and, recently, C. elegans. It is likely that this aspect of the endeavor is not yet complete. Tissue culture cells have also proven highly informative as they can be used for high resolution microscopy and can be micromanipulated to some extent. T h e analysis of cytokinesis of plant cells has provided insight into the machinery that reorganizes the plasma membrane. Ten years ago, it appeared as though there were many answers to the question, 'How is the cell cycle regulated?'. In the meantime, the combination of genetics, biochemistry, and cell biology in multiple experimental organisms has revealed that there is one essential theme to cell cycle control. T h e analysis to date of cytokinesis has suggested that there are a number of answers to the question, 'How does cytokinesis work?', yet these answers are beginning to exhibit some similarities. Perhaps we will soon know for certain how many times evolution has answered this question. T h e apparent contradictions that have surfaced during this review may reflect true differences in experimental systems: a protein (myosin) or a structure (the central spindle) that is essential for cytokinesis in one system may be partially or even fully dispensable in another system. T h e reason for this may be that a complex process like cell division could consist
The mechanism and control of cytokinesis Glotzer
of multiple, partially redundant subprocesses that vary in their degree of redundancy in different systems.
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13.
Rappaport R: Repeated furrow formation from a single mitotic apparatus in cylindrical sand dollar eggs. J Exp Zoo/1985, 234:167-171.
14.
Because of space limitations, it was not possible to discuss all of the new advances in the field. To facilitate access to this literature, the following citations are provided [66°-68°,69,70,71 °,72,73,74°,75°].
Williams BC, Riedy MF, Williams EV, Gatti M, Goldberg ML: The Drosophila kinesin-like protein KLP3A is a midbody component required for central spindle assembly and initiation of cytokinesis. J Cell B/o/1995, 129:709-723.
15.
Andreassen PR, Palmer DK, Wener MH, Margolis RL: Telophase disc: a new mammalian mitotic organelle that bisects telophase cells with a possible function in cytokinesis. J Cell Sci 1991, 99:523-534.
Acknowledgements
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JulianM, Tollon Y, Lajoie-Mazenc I, Moisand A, Mazarguil H, Puget A, Wright M: Gamma-tubulin participates in the formation of the midbody during cytokinesis in mammalian cells. J Cell Sci 1993, 105:145-156.
17.
Shu HB, Li Z, Palacios MJ, Li Q, Joshi HC: A transient association of gamma-tubulin at the midbody is required for the completion of cytokinesis during the mammalian cell division. J Cell Sci 1995, 108:2955o2962.
18.
Cooper JA and Kiehart DP: Septins may form a ubiquitous family of cytoskeletsl filaments. J Cell Bio11996, 134:13451348.
Supplementary citations
I would like to thank Suzanne Eaton, Pierre Gtinczy, Sigrid Reinsch, and Lisa Satterwhite for helpful comments on this manuscript.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • •.
of special interest of outstanding interest 19.
1.
Glotzer M: Cytokinesis. Curr Bio11997, 7:R274-R276.
2.
Fishkind DJ, Wang YL: New horizons for cytokinesis. Curr Opin Cell Biol 1995, 7:23-31.
3.
Rappaport R: Experiments concerning the cleavage stimulus in sand dollar eggs. J Exp Zoo/1961,148:81-89.
4.
Hamaguchi Y: Microinjection of colchicine into sea urchin eggs. Dev Growth Differ 1975, 17:111-117.
5.
Hiramoto Y: Analysis of cleavage stimulus by means of micromanipulation of sea urchin eggs. Exp Cell Res 1971, 68:291-298.
6. Zhang D, Nicklas RB: 'Anaphase' and cytokinesis in the • absence of chromosomes. Nature 1996, 382:466-468. Describes a micromanipulation study in grasshopper spermatocytes in which the removal of all chromosomes is found to not block anaphase B or cytokinesis. 7.
Rappaport R: Establishment of the mechanism of cytokinesis in animal cells. Int Rev Cyto/1986, 105:245-281.
Wheatley SP, Wang YL: Midzone microtubules are continuously required for cytokinesis in cultured epithelial cells, J Cell Biol 1996, 135:981-989. These three papers [8"',9",10"] analyze whether two asters are sufficient to induce an ectopic furrow in cultured cells. In some cases, furrows formed, and in others they did not. In this paper [8"'], the presence of bundled microtubules between the asters was correlated with the induction o1 furrowing. 8. .•
9. •
Rieder CL, Khodjakov A, Paliulis LV, Fortier TM, Cole RW, Sluder G: Mitosis in vertebrate somatic cells with two spindles: implications for the metaphase/anaphese transition checkpoint and cleavage [see comments]. Proc Nat/Acad Sci USA 1997, 94:5107-5112. These three papers [8"',9",10"] analyze whether two asters are sufficient to induce an ectopic furrow in cultured cells. In some cases, furrows formed and in others, they did not. In [8"'], the presence of bundled microtubules between the asters was correlated with the induction of furrowing.
Haarer BK, Pringle JR: Immunofluorescence localization of the
Saccharomyces cerevisiae CDC12 gene product to the vicinity of the lO-nm filaments in the mother-bud neck,/Vlol Cell Biol 1987, 7:3678-3687. 20.
Neufeld TP, Rubin GM: The Drosophila peanut gene is required for cytokinesis and encodes a protein similar to yeast putative bud neck filament proteins. Cell 1994, 77:371-379.
21. •-
Kinoshita M, Kumar S, Mizoguchi A, Ide C, Kinoshita A, Haraguchi T, Hiraoka Y, Noda M: Nedd5, a mammalian septin, is a novel cytoskeletal component interacting with actin-based structures. Genes Dev 1997, 11:1535-1547. Describes the immunolocalization of a septin to actin-containing structures and provides evidence that the septins are involved in cytokinesis in mammalian cells. 22. •o
Field CM, aI-Awar O, Rosenblatt J, Wong ML, Alberts B, Mitchison TJ: A purified Drosophila septin complex forms filaments and exhibits GTPase activity. J Cell Biol 1996, 133:605-616. Describes the purification of a filamentous form of a septin complex from Drosophila embryo extracts and presents a detailed analysis of the biochemical properties of the protein complex. 23.
Simanis V: The control of septum formation and cytokinesis in fission yeast. Semin Cell Biol 1995, 6:79-87.
24.
Chang F, Nurse P: How fission yeast fission in the middle. Cell 1996, 84:191-194.
25. •°
Schmidt S, Sohrmann M, Hofmann K, Woollard A, Simanis V: The S p g l p GTPese is an essential, dosage-dependent inducer of septum formation in Schizosaccharomyces pombe. Genes Dev 1997, 11:1519-1534. These references [25"',26,27"] describe novel regulators of septum formation in the fission yeast. Overexpression of spgl or plol and loss of byrl promote formation of multiple septa. 26.
Ohkura H, Hagan IM, Glover DM: The conserved
Schizosaccheromyces pombe kinase plol, required to form a bipolar spindle, the actin ring, and septum, can drive septum formation in G1 and G2 cells. Genes Dev 1995, 9:1059-1073.
10. •
Eckley DM, Ainsztein AM, Mackay AM, Goldberg IG, Earnshaw WC: Chromosomal proteins and cytokinesis: patterns of cleavage furrow formation and inner centromere protein positioning in mitotic heterokaryons and mid-anaphase cells. J Cell Biol 1997, 136:1169-1183. These three papers [8",9",10"] analyze whether two asters are sufficient to induce an ectopic furrow in cultured cells. In some cases, furrows formed and in others, they did not. In [8"}, the presence of bundled microtubules between the asters was correlated with the induction of furrowing.
Song K, Mach KE, Chen CY, Reynolds T, Albright CF: A novel suppressor of rasl in fission yeast, byr4, is • dosagedependent inhibitor of cytokinesis. J Cell Biol 1996, 133:13071319. These references [25",26,27"] describe novel regulators of septum formation in the fission yeast. Overexpression of spgl or p/ol and loss of byrl promote formation of multiple septa.
11.
28.
Schweitzer B, Philippsen P: CDC15, an essential cell cycle gene in Saccharomyces cerevisiae, encodes a protein kinase domain. Yeast 1991,7:265-273.
29.
Shirayama M, Matsui Y, Toh-E A: The yeast TEM1 gene, which encodes a GTP-binding protein, is involved in termination of M phase. Mo/ Cell Bio/1994, 14:7476-7482.
30. •
Larochelle DA, Vithalani KK, De Lozanne A: A novel member of the rho family of small GTP-binding proteins is specifically required for cytokinesis. J Cell Biol 1996, 133:1321-1329.
12. .•
Rappaport R, Rappaport BN: Cytokinesis: effects of blocks between the mitotic apparatus and the surface on furrow establishment in flattened echinoderm eggs. J Exp Zoo/1983, 227:213-227.
Cao LG, Wang YL: Signals from the spindle midzone are required for the stimulation of cytokinesis in cultured epithelial cells. Mol Biol Cell 1996, 7:225-232. The presence of a small perforation between the central spindle and the cortex prevented the formation of a furrow in the normal position. This provides evidence that the central spindle promotes furrow specification or ingression.
27. •
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This paper shows that racE is essential for cytokinesis in Dictyostelium, and mutations in the racE gene cause no other detectable phenotypes besides a cytokinesis defect. Larochelle DA, Vithalani KK, De Lozanne A: Role of Dic(yostelium racE in cytokinesis: mutational analysis and localization studies by use of green fluorescent protein. Mol Bio/Cell 1997, 8:935-944. Mutational analysis of racE (see also [30"]) suggests that the ability of race to bind GTP is essential for its function, but, surprisingly, mutations that are likely to prevent GTP hydrolysis are fully functional. 31. •
32. •
Adachi H, Takahashi Y, Hasebe T, Shirouzu M, Yokoyama S, Sutoh K: Dictyostelium IQGAP-related protein specifically involved in the completion of cytokinesis. J Cell Bio/1997, 137:891-898.
A protein with a GTPase-activating protein (GAP) domain is essential for the late stages of cytokinesis. As with racE (see [30°,31 "]), mutations in the gapA gene cause defects in cytokinesis, but do not cause other obvious phenotypes. 33. •
Lee S, Escalante R, Firtel RA: ARas GAP is essential for cytokinesis and spatial patterning in Dictyostelium. Development 1997, 124:983-996. A GTPase-activating protein (GAP) is essential for cytokinesis, but as a range of additional phenotypes are observed when this protein is mutated it probably has other roles as well. 34.
Kishi K, Sasaki T, Kuroda S, Itoh T, Take/Y: Regulation of cytoplasmic division of Xenopus embryo by rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI). J Cell Bio/ 1993, 120:1187-1195.
35.
Mabuchi t, Hamaguchi Y, Fujimoto H, Morii N, Mishima M, Narumiya S: A rho-like protein is involved in the organ/sat/on of the contractile ring in dividing sand dollar eggs. Zygote 1993, 1:325-331.
36.
MoormanJP, Bobak DA, Hahn CS: Inactivation of the small GTP binding protein Rho induces multinucleate cell formation and apoptosis in murine T lymphoma EL4. J Immune/1996, 156:4146-4153.
37. ••
Drechsel DN, Hyman AA, Hall A, GIotzer M: A requirement for Rho and Cdc42 during cytokinesis in Xenopus embryos. Curt Bio11997, 7:12-23. Analyzes the role of Rho and Cdc42 in cytokinesis of Xenopus embryos. The insertion of cleavage membranes in the presumptive furrow region, in the absence of furrow ingression, indicates that furrow specification and membrane insertion are independent of furrow ingression.
45. •
Evangelista M, Blundell K, Longtine MS, Chow CJ, Adames N, Pringle JR, Peter M, Boone C: Bnilp, a yeast formin linking cdc42p and the actin cytoskeleten during polarized morphogenesis. Science 1997, 276:118-122. These references [42"°,43°-46°] establish that the formin family is involved in cytokinesis in many organisms, and that members of the family bind to profilin and to Rho family GTPases. 46. •
Watanabe N, Madaule P, Reid T, Ishizaki T, Watanabe G, KakizukaA, Saito Y, Nakao K, Jockusch BM, Narumiya S: p140mDia, a mammalian homolog of Drosophila diaphanous, is a target protein for Rho small GTPase and is a ligand for profilin. EMBO J 1997, 16:3044-3056. These references [42°°,43"-46"] establish that the formin family is involved in cytokinesis in many organisms, and that members of the family bind to profilin and to Rho family GTPases. 47 •
KimuraK, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, YamamoriB, Feng J, Nakano T, Okawa K et al.: Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rhokinase). Science 1996, 273:245-248. A Rho effector molecule, Rho kinase, promotes myosin activation by inactivating the phosphatase that dephosphorylates the regulatory light chain (see [48"]). 48. •
Amano M, Ito M, Kimura K, Fukata Y, Chihara K, Nakano T, Matsuura Y, Kaibuchi K: Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase). J Biol Chem 1996, 271:20246-20249. A Rho effeotor molecule, Rho kinase, promotes myosin activation by phosphorylating the regulatory light chain (RLC) (see [47"]). 49.
De Lozanne A, Spudich JA: Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination. Science 1987, 236:1086-1091.
50.
Knecht DA, Loomis WF: Ant/sense RNA inactivation of myosin heavy chain gene expression in Dictyostelium discoideum. Science 1987, 236:1081-1086.
51. • ,,
NeujahrR, Heizer C, Gerisch G: Myosin II-independent processes in mitotic cells of Dictyostelium discoideum: redistribution of the nuclei, re-arrangement of the actin system and formation of the cleavage furrow. J Cell Sci 1997, 110:123137. Describes a thorough analysis of the myosin II knockout phenotype. Includes the stunning observation that the mutant cells can divide on a surface almost as well as the wild-type cells. 52.
Uyeda TQP, Titus MA: The myosins of Dictyostelium. In Dictyostelium: a Mode/System for Cell and Developmental Biology. Edited by Maeda Y, Inouye K, Takeuchi I. Tokyo: University Academic Press; 1997:in press.
38.
Dutartre H, Davoust J, Gorvel JP, Chavrier P: Cytokinesis arrest and redistribution of actin-cytoskeleton regulatory components in cells expressing the Rho GTPase CDC42Hs. J Ceil Sci 1996, 109:367-377.
39.
Ridley AJ: Rho: theme and variations. Curt Biol 1996, 6:12561264.
40.
FrazierJA, Field CM: Are FH proteins local organizers? Curr Bio/ 1997, 7:R414-R417.
41.
Castrillon DH, Wasserman SA: Diaphanous is required for cytokinesis in Drosophila and shares domains of similarity with the products of the limb deformity gene. Development 1994, 120:3367-3377.
54.
KaressRE, Chang XJ, Edwards KA, Kulkami S, Aguilera I, Kiehart DP: The regulatory light chain of nonmuscle myosin is encoded by spaghetti-squash, a gene required for cytokinesis in Drosophila. Cell 1991, 65:1177-1189.
ImamuraH, Tanaka K, H/hera T, Umikawa M, Kamei T, Takahashi K, Sasaki T, Takai Y: Bnilp and Bnrlp: downstream targets of the Rho family small G-proteins which interact with profilin and regulate actin cytoskeleton in Saccharomyces cerevisiae. EMBO J 1997, 16:2745-2755. These references [42"°,43"-46 °] establish that the formin family is involved in cytokinesis in many organisms, and that members of this family bind to profilin and to Rho family GTPases.
55.
Watts FZ, Shiels G, Orr E: The yeast MYO1 gene encoding a myosin-like protein required for cell division. EMBO J 1987, 6:3499-3505.
56.
Rodriguez JR, Paterson BM: Yeast myosin heavy chain mutant: maintenance of the cell type specific budding pattern and the normal deposition of chitin and cell wall components requires an intact myosin heavy chain gene. Cell Mot# Cytoskeleton 1990, 17:301-308.
43. •
Chang F, Drubin D, Nurse P: cdc12p, a protein required for cytokinesis in fission yeast, is a component of the cell division ring and interacts with profilin. J Cell Bio/1997, 137:169-182. These references [42",43"-46"] establish that the formin family is involved in cytokinesis in many organisms, and that members of this family bind to profilin and to Rho family GTPases.
57. •.
HarrisSD, Hamer L, Sharpless KE, Hamer JE: The Aspergillus nidulens sepA gene encodes an FH1/2 protein involved in cytokinesis and the maintenance of cellular polarity. EMBO J 1997, 16:3474-3483. These references [42"°,43=-46 `] establish that the formin family is involved in cytokinesis in many organisms, and that members of this family bind to profilin and to Rho family GTPases.
58.
Samuels AL, Giddings TJ, Staehelin I_A: Cytokinesis in tobacco BY-2 and root tip cells: a new model of cell plate formation in higher plants. J Cell Biol 1995, 130:1345-1357.
59. •-
Lukowitz W, Mayer U, Jurgens G: Cytokinesis in the Arabidopsis embryo involves the syntax/n-related KNOLLE gene product. Cell 1996, 84:61-71.
42. ••
44. •
53. •-
Guo S, Kemphues KJ: A non-muscle myosin required for embryonic polarity in Caenorhabditis elegans. Nature 1996, 382:455-458. Describes the inactivation by RNA-mediated interference of myosin II in worm embryos. Partial inactivation causes a symmetric first division, and more complete inactivation blocks cytokinesis.
Kitayama C, Sugimoto A, Yamamoto M: Type II myosin heavy chain encoded by the myo2 gene composes the contractile ring during cytokinesis in Schizosaccharomyces pombe. J Cell Bio11997, 137:1309-1319. Myosin n is required for cytokinesis in S. pombe. Green fluorescent protein-myosin II localizes to a ring that contracts during division.
The mechanism and control of cytokinesis Glotzer
These authors identified a protein that probably mediates an important vesicle fusion event during cytokinesis. 60.
Bennett MK, Garcia AJ, Elferink LA, Peterson K, Fleming AM, Hazuka CD, Scheller RH: The syntaxin family of vesicular transport receptors. Cell 1993, 74:863-8?3.
61. •
Gu X, Verma DP: Phragmoplastin, a dynamin-like protein associated with cell plate formation in plants. EMBO J 1996, 15:695-704. The gene encoding a protein with homology to dynamin has been cloned from soybean. This protein shows a striking localization to the cell plate during cytokinesis. Gu X, Verma DP: Dynamics of phragmoplastin in living cells during call plate formation and uncoupling of cell elongation from the plane of cell division. Plant Cell 1997, 9:157-169. A more detailed analysis, using green fluorescent protein technology, of the localization of phragmoplastin during cytokinesis.
cortical flowlsolation-contraction coupling during cytokinesis and cell locomotion. Mo/Bio/Cell 1996, 7:1259-I 282. This paper describes observations made with fluorescent derivatives of myosin, including a derivative whose properties are sensitive to the phosphorylation state of residue Ser19, a critical regulatory site. 69.
MartineauSN, Andreassen PR, Margolis RL: Delay of HeLa cell cleavage into interphase using dihydrocytochalasin B: retention of a postmitotic spindle and telophase disc correlates with synchronous cleavage recovery. J Cell Biol 1995, 131:191-205.
70.
Gunsalus KC, Bonaccorsi S, Williams E, Vemi F, Gatti M, Goldberg ML: Mutations in twinstar, a Drosophila gene encoding a cofilin/ADF homologue, result in defects in centrosome migration and cytokinesis. J Cell Biol 1995, 131:1243-1259.
62. •
83.
Hinshaw JE, Schmid SL: Dynamin self-assembles into rings suggesting a mechanism for coated vesicle budding. Nature 1995, 374:190-192.
64.
Bluemink JG, deLaat SW: New membrane formation during cytokinesis in normal and cytochalasin B-treated eggs of Xenopus laevis. I. Electron microscope observations. J Cell Biol 1973, 59:89-108.
65. •
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71. •
Faix J, Steinmetz M, Boves H, Kammerer RA, Lottspeich F, Mintert U, Murphy J, Stock A, Aebi U, Gerisch G: Cortexillins, major determinants of cell shape and size, are actin-bundling proteins with a parallel coiled-coil tail. Ceil 1996, 86:631-642. This paper describes the cloning of the genes encoding two isoforms of a subfamily of actin-binding proteins. The actin-binding properties of the cortexillins are characterized. Strains lacking both isoforms are defective in cytokinesis.
Emoto K, Kobayashi T, Yamaji A, Aizawa H, Yahara I, Inoue K, Umeda M: Redistribution of phosphatidylethanolamine at the cleavage furrow of dividing cells during cytokinesis. Proc Nat/ Acad Sci USA 1996, 93:12867-12872. A novel probe for phosphatidylethanolamine (PE) is used to show that this lipid is specifically exposed in the outer leaflet of the plasma membrane in the region of the cleavage furrow. Cross-linking of PE with a multivalent probe inhibits cytokinesis. 66. FishkindDJ, Silverman JD, Wang YL: Function of spindle • microtubules in directing cortical movement and actin filament organization in dividing cultured cells. J Cell Sci 1996, 109:2041-2051. Dividing cells are treated with taxol and nocodozole, and the organization of actin filaments and the changes in the behavior of surface-adhered particles are characterized.
Muto A, Kume S, Inoue T, Okano H, Mikoshiba K: Calcium waves along the cleavage furrows in cleavage-stage Xenopus embryos and its inhibition by heparin. J Cell Biol 1996, 135:181-190. The Ca2+ indicator dyes were used in conjunction with confocal microscopy to investigate the association of transient Ca2+ currents with cytokinesis. This study presents evidence that Ca2+ transients are observed during cytokinesis. A previous reference [73] describes similar observations, made in starfish embryos, that lead to qualitatively different results.
67. •
75. •
Burton K, Taylor DL: Traction forces of cytokinesis measured with optically modified elastic substrata [see comments]. Nature 1997, 385:450-454. Cells are cultured on a silicon rubber sheet which becomes deformed by cellular contractile activity. The location and extent of wrinkling provides a measure of the forces that occur during cell division. 68. •
DeBiasio RL, LaRocca GM, Post PL, Taylor DL: Myosin II transport, organization, and phosphorylation: evidence for
72.
Chang DC, Meng C: A localized elevation of cytosolic free calcium is associated with cytokinesis in the zebrafish embryo. J Cell Biol 1995, 131:1539-1545.
73.
Stricker SA: Time-lapse confocal imaging of calcium dynamics in starfish embryos. Dev Biol 1995, 170:496-518.
74. •
Asada T, Kuriyarna R, Shibaoka H: TKRP125, a kinesin-related protein involved in the centrosome-independent organization of the cytokinetic apparatus in tobacco BY-2 cells. J Cell Sci 1997, 110:179-189. Describes the cloning of the gene encoding a kinesin-related protein from the BimC family; this protein localizes to the phragmoplast microtubules during cytokinesis. Antibodies to TRKP125 inhibit the translocation of phragmoplast microtubules in a permeabilized cell system.
The mechanism and control of cytokinesis Michael Glotzer Cytokinesis is under active investigation in each of the
dominant experimental model systems. During 1996 and 1997, several developments necessitated the reassessment of the prevailing model for cytokinesis. In addition, the inventory of proteins required for cytokinesis has grown considerably. However, a molecular understanding of cytokinesis still remains elusive.
Addresses Institute for Molecular Pathology, Dr Bohr-Gasse 7, 1030 Vienna, Austria; e-mail: [email protected] Current Opinion in Cell Biology 1997, 9:815-823 http :l lbiomednet.coml elecref1095506 7 4 0 0 9 0 0 8 1 5
© Current Biology Ltd ISSN 0955-0674 Abbreviation GAP GTPase-activating protein
Introduction
Cytokinesis, the division of one cell into two, is a source of fascination for cell biologists. Not only does the cell produce two entities from one, it also positions the division plane so that each daughter cell receives a full complement of chromosomes and other essential organelles. In animal cells, cytokinesis can be subdivided into five subprocesses (Figure 1) [1]. In anaphase, the mitotic spindle specifies the site at which the cleavage furrow will form. A contractile ring containing actin and myosin assembles at this site. This ring contracts, furrowing the overlying plasma membrane. A transient structure, the midbody, forms when the furrow reaches the remnants of the mitotic spindle. Finally, the common membrane that surrounds the two nascent cells is divided so that the cells may separate. There have been several notable developments in the field of cytokinesis during 1996 and 1997 since the last review in Current Opinion in Cell Biology [2]. First, there are indications that the mitotic spindle plays a role during furrow ingression beyond its critical and early role of specifying the position of the cleavage furrow. Second, a number of GTPases (and their binding partners) have been identified that are required for cytokinesis and the characterization of their role in this process has provided some new insights. Third, while myosin II plays a crucial role in cytokinesis, there is evidence that, under some circumstances, it is dispensable. Fourth, analysis of cytokinesis has begun to focus more clearly on the role of membranes in this process. Finally, and perhaps most importantly, several major model systems have emerged and a certain degree of convergence of the work in these
different systems has become clear. In this review, I will focus on these recent advances. The role of the central spindle Furrow specification
T h e role of the mitotic spindle in cytokinesis has largely been established by micromanipulation experiments performed in marine invertebrate embryos. Bipolar spindles induce furrowing, as do two asters and two poles of independent bipolar spindles [3]. After the start of furrow ingression, depolymerization of the spindle [4], or its partial or complete removal [5,6"], does not necessarily prevent the completion of cytokinesis. These and other data led to a model in which microtubules emanating from the asters of the mitotic spindle specify the position of the cleavage furrow. A second feature of this model is that, once specification has occurred, cytokinesis could proceed to completion in the absence of the spindle or other microtubular structures [7]. However, there is now reason to question the generality of certain aspects of this model. In the classic view, the asters of the mitotic spindle are the sole source of the signal that specifies the cleavage plane. However, recent experiments indicate that in some cells the 'central spindle' may determine the position of the cleavage furrow. T h e central spindle arises from the elongation and reorganization of the ellipsoid metaphase spindle during anaphase and telophase. T h e interpolar microtubules undergo extensive bundling, forming the central spindle. T h e possibility that this structure may be important for cytokinesis arose from attempts to reproduce some of the experiments on which the original model (see above) had been based, but in cultured cells rather than in a large embryo (Figure 2). To determine whether two asters that are not connected by a spindle can induce formation of an ectopic furrow, the behavior of cells containing multiple spindles has been examined [8"',9°,10"]. T h e formation of such furrows was somewhat variable and depended on the precise geometry of the spindles. However, in one study, fluorescently labeled tubulin enabled the organization of microtubules to be monitored and the variability in furrow formation could be accounted for by the organization of microtubules; when microtubule bundles formed between the asters an ectopic furrow formed, suggesting that the asters themselves were insufficient to induce furrow formation, and that a central spindle like structure is necessary [8"']. A second experiment that had implicated the asters as the source of the signal that induces furrowing consisted of placing obstructions between the spindle and the equatorial cortex. T h e obstructions, if they were of the appropriate size, would not prevent furrowing,
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Figure 1
Cleavage plane specification
Contractile ring assembly
:urrow ingression
Cell separation
C~eavage me,mDt'a~t~S
Midbody formation CurrentOpinionin CellBiology The basic subreactions of cytokinesis. Actin is shown as red, microtubules as purple, cleavage membranes as blue, and chromosomes as green
circles or elongated shapes. Pink triangles represent the hypothetical cleavage signal that specifies the position of the division plane.
presumably because the equatorial cortex remained under the influence of the asters [11]. This experiment has been adapted to cultured cells by using a blunt glass needle to fuse the dorsal and ventral plasma membranes together, producing a small hole between the central spindle region and the cell periphery (Figure 2) [12"]. A furrow formed in the cortex adjacent to the hole, deforming it, but no furrow formed in the cortex distal to the hole. Moreover, there was a correlation between the arrangement of microtubules and the contractile behavior of the cortex w in the cytoplasm distal to the hole microtubules remained unbundled and the cortex did not furrow, while in the cytoplasm proximal to the hole the microtubules became bundled and a furrow formed in the adjacent cortex. Thus, at least in cultured cells, the central spindle, as opposed to the asters, specifies the position of the furrow.
Furrow ingression
As mentioned above, in some invertebrate embryos removal of the spindle from a cell that has initiated cytokinesis does not prevent completion of that division,
suggesting that the spindle does not act during furrow ingression. However, recent observations indicate that the mitotic spindle may also function during cytokinesis, after the furrow has begun to ingress. Cultured cells that had initiated cytokinesis were treated with a microtubuledepolymerizing drug [8"1. T h e cells had been injected with ftuorescently labelled tubulin to allow monitoring of the response of the microtubular array to the drug treatment. In the majority of cells analyzed, microtubules were fully depolymerized and the cells did not complete cytokinesis. In one-quarter of the cells, some microtubule bundles remained in the central spindle; in these cases, the cells completed cytokinesis [8"°1. Thus, microtubules may in fact be required continuously during cytokinesis. How might this conclusion be harmonized with the earlier, opposite conclusion? T h e interzonal microtubules whose presence correlates well with successful completion of cytokinesis are quite insensitive to microtubule drugs and this may have misled earlier investigators. Moreover, there are indications from invertebrate embryos that displacement of the spindle can cause furrow regression [13] (furrow regression means the relaxation of a partially or fully ingressed furrow). Alternatively, the requirement
The mechanism and control of cytokinesis Glotzer
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Figure 2
(a)
(b)
Cultured cell
Emb~o
Cultured cell
Embryo Oil drop
Hole J
Oil drop
Hole
CurrentOpinionin CellBio!ogy Micromanipulation of embryos and cultured cells suggests that there are system-specific differences in the role of the central spindle in furrow specification and ingression. (a) In embryonic systems, the presence of two spindles in a single cell causes the formation of an ectopic furrow (indents in the cell membrane) between the two asters of the neighboring spindles. In cultured cells, the formation of an ectopic furrow is observed only when a bundle of microtubules forms between neighboring spindles (i.e. in the bottom right-hand cultured cell). (b) In embryos, an obstruction between the central spindle and the equatorial cortex does not inhibit furrow formation, in cultured cells, an obstruction between the central spindle and the equatorial cortex inhibits furrowing from the outer cortex of the side of the cell that contains the obstruction.
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for microtubules throughout cytokinesis may not exist in all cells.
The role of the central spindle in cytokinesis has also emerged from genetic analysis of Drosophila. Male flies homozygous for mutations in the kinesin-like protein KLP3A exhibit defects in cytokinesis during the meiotic divisions of spermatogenesis [14]. Phenotypic analysis of mutant spermatocytes reveals that although the spindles appear normal during meiotic metaphase, in anaphase the central spindle fails to form. There are no morphological signs of furrowing, indicating either that the cortex has not received the signal to furrow or that it is defective in furrow assembly or ingression. In either case, it is clear that cytokinesis requires this motor protein and it is possible that the requirement for this protein in cytokinesis may reflect its role in organizing the central spindle, which in turn may be necessary for proper furrow function. Localization of cleavage furrow markers will help to determine whether KLP3A is required to specify the position of the cleavage furrow or for its ingression. If cleavage furrow markers are found properly localized, this would indicate that KLP3A is required for furrow assembly or ingression rather than for specification.
How might the central spindle, or the microtubule bundles that make up the central spindle, participate in furrow ingression? One could speculate that the signal for furrowing is not merely a transient event, but rather that continued ingression of the furrow requires constant reinforcement by the central spindle. It is also possible that other proteins that concentrate in the central spindle, perhaps by binding to overlapping, antiparallel microtubules, are important for furrow ingression [15-17]. Alternatively, a direct interaction between the tip of the furrow and the central spindle may promote furrow ingression. In addition to actin and myosin, the furrow contains other proteins that are necessary for cytokinesis, including the septins (see [18] for a review). Septins are necessary for cytokinesis in budding yeast [19], Drosophila [20], and vertebrates ([21°']; M Glotzer, AA Hyman, unpublished data). In yeast, septins are localized to the bud neck throughout much of the cell cycle and they appear to be components of a series of 10 nm filaments [19]. Septins have been isolated from Drosophila extracts and the purified protein complex contains filamentous structures of up to -300 nm in length with a periodicity of 28 nm [22°°]. In vertebrate cells a large fraction of the septins is associated with actin-containing structures, including with the contractile ring [21°°]. In Xenopus embryos septins localize to some microtubule-containing structures and they bind to microtubules in vitro (M Glotzer, AA Hyman, unpublished data). As septins can bind both actin and microtubules, and as they are found in the cleavage furrow, perhaps they are part of a link between the cell cortex and the central spindle that promotes furrow ingression.
Regulation of furrow assembly and ingression Once the position of the furrow is specified, be it by the asters or by the central spindle, what controls the formation and constriction of the contractile ring? Several small GTPases and their interacting proteins have been implicated in cytokinesis, and these proteins are likely to play a regulatory, rather than a structural, role.
In
Schizosaccharomycespombe
A GTPase has been identified that appears to have an important regulatory function in the division of Schizosaccharomycespombe. T h e division of fission yeast is somewhat different from the division of animal cells (see [23,24] for reviews). First, the position of the division plane appears to correlate with the position of the nucleus in late Gz phase, rather than with the position of the mitotic spindle. As in animal cells, an actin ring forms in the future division plane. This ring presumably contracts, thereby pinching the membrane into two entities. Secretion of cell wall material generates the division septum. A portion of the old cell wall and a primary septum is dissolved, allowing the cells to separate. T h e gene spgl, which encodes a GTPase, has been recently isolated and implicated in cytokinesis, spglcells cannot compete cytokinesis as they are defective in septum formation [25°°]. These mutations do not affect formation of an actin ring at the correct time and place. More interestingly, mild overexpression of this GTPase induces formation of multiple septa and the formation of an actin ring, and this can occur at various cell cycle stages, thereby uncoupling the onset of cytokinesis from progression through the cell cycle. It is noteworthy that the septa induced by Spgl overexpression do not fully cleave, but the reason for this remains unclear. One testable hypothesis is that although septum formation does not need to be coordinated with the cell cycle, the later steps involved in cell separation may be cued by the cell cycle machinery. Although spgl is not required for formation of the actin ring, it can induce their formation ectopically, raising the possibility that spgl is a central regulator of cell division in S. pombe. Like several other GTPases, Spgl forms a complex with a kinase, Cdc7 [25"], and it is probably regulated by a GTPase-activating protein (GAP) and a guanine nucleotide exchange factor. Cdcl6 is a good candidate for the GAP, as it interacts genetically with Spgl as well as displaying sequence homology to known GAPs [25"]. However, Spgl is not unique in its ability to cause formation of ectopic septa. The polo-like kinase, Plol, is necessary for formation of the actin ring and for septum formation [26]. Like Spgl, overexpression of Plol induces multiple septa. A novel gene, byr4, has the opposite phenotype: loss of byr4 induces the multisepta phenotype [27°]. Further analysis is needed to characterize the relationships between Spgl, Cdc7, Cdcl6, Plol, and Byr4, in particular to determine if they comprise a single
The mechanism and control of cytokinesis Glotzer
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pathway or whether there are several pathways, each of which can induce septum formation.
[43°], Aspengillus nidulans (sepA) [44°], and Caenorhabditis elegans (cyk-1) (B Bowetman, personal communication).
Does a GTPase homologous to Spgl control cytokinesis in other organisms? It is too early to answer this question, but it is notable that the closest relatives to Spgl and its interacting kinase Cdc7 are encoded by the budding yeast genes TEM1 and CDC15, respectively, and that these genes are required for exit from mitosis [28,29]. This complicates the analysis of whether they also regulate cytokinesis, but there is as yet no evidence that they do. Moreover, the S. pombe spgl and the Saccharomycescerevisiae TEM1 gene products are not functionally interchangeable [25°°]. Thus, the Spgl pathway may exist in other organisms, but it may regulate a different cellular process.
These proteins contain a polyproline-rich region that binds to profilin, which in turn regulates actin assembly [42°',43°,45°,46°]. Formins also bind to Rho family proteins [42°°,45",46°]. Thus, this protein family may form a crucial link between these GTPases and actin. However, the precise function of the formins may vary in different cell types. In S. pombe, cdcl2 mutants fail to make an actin ring and to deposit septum material [43°], while, in C. elegans, cyk-1 mutant embryos make a furrow that ingresses to near completion and then regresses (P G6nczy, AA Hyman, unpublished data). A second class of Rho effectors has been identified that includes regulators of the phosphorylation state of myosin [47°,48°]. T h e action of Rho on these effectors may also be crucial for cytokinesis.
In Die~ostelium
A distinct GTPase, racE, is required for cytokinesis in Dictyostelium. This GTPase appears to be specifically required for cytokinesis, as the mutant exhibits no phenotypes other than an inability to undergo cytokinesis [30°]. RacE is localized to the plasma membrane, but it is not enriched in the cleavage furrow [31°]. Two GAPs have been identified which are necessary for cytokinesis, gapA and DdRasGAP1 [32°,33°]. Like racE, gapA appears to be specifically required for cytokinesis, suggesting that gapA might act in concert with racE. Although the gapA and racE mutants have slightly different phenotypes, this difference could be explained by the fact that gapA- cells could contain racE.GTP, while racE- cells would not. Deletion of DdRasGAP1, on the other hand, causes defects in development as well as in cytokinesis; thus, it is unlikely to act exclusively on racE and indeed it may have a distinct substrate. In Xenopus and other e u k a ~ o t e s
Rho protein is essential for cytokinesis in many cell types [34-36,37°°]. Detailed analysis of the cleavage of Xenopus embryos in which Rho has been inactivated indicates that Rho protein maintains the integrity of the cortical layer of actin [37°°]. This loss of cortical actin prevents the formation of an ingressing cleavage furrow. However, at least one aspect of cytokinesis, the insertion of cleavage membranes, still occurs, indicating that the failure in cytokinesis is not a consequence of blocking specification of the cleavage furrow. Perturbations of Cdc42 also cause defects in cytokinesis [37°°,38]; this GTPase also appears to act during furrow ingression rather than having a role in the specification process [37°°]. GTPase effectors
Small GTPases regulate cell behavior by x/irtue of their interactions with a number of other proteins, the so-called effector molecules (see [39] for a review). A new class of potential effector molecules has emerged, the 'formin' family (see [40] for a review). These family members are implicated in cytokinesis in Drosophila (Diaphanous) [41], S. cerevisiae (BNI1 and BNR1) [42°°], S. pombe (Cdcl2)
Myosin
Over the past three decades, multiple experimental approaches have implicated myosin as a central player in cytokinesis. T h e issue was, or so it seemed, laid to rest in 1987, when Dictyostelium cells lacking myosin II were engineered by gene knockout and antisense technology [49,50]. Myosin null cells, when grown in suspension, failed to divide and became giant multinucleate cells that eventually died. However, these cell lines could be propagated by growth on a solid support where they divided by an aberrant process that was unlinked to the cell division cycle and which was termed 'traction-mediated cytofission'. T h e division of cells lacking myosin II has recently been re-analyzed [51°°]. Surprisingly, more than 80% of adherent, mononucleated, myosin II null cells divide after mitosis in a manner that closely resembles cytokinesis of wild-type cells. When it is present, myosin II does play a role in cytokinesis of adherent Dictyostelium, as cytokinesis is faster and more efficient in its presence than in its absence, but it certainly appears to be dispensable under some circumstances. It is not known what provides the force that is necessary to deform the cleavage furrow in adherent myosin II null cells. In addition to a single myosin heavy chain gene, Dictyostelium has a plethora of 'non-conventional' myosins [52], and it is possible that one or more of them substitutes for the loss of myosin II when dividing on a substrate. Further genetic analysis will determine whether this is the case or whether an alternative, novel mechanism is at work in these cells. It is imperative to analyze whether other Dictyostelium mutants that are unable to grow in suspension, but which can grow on a solid substrate, are also able to divide in a cell cycle dependent manner or whether they are only able to divide by traction-mediated cytofission. T h e evidence implicating a role for myosin in cytokinesis in other organisms is quite strong, but the accumulated evidence does not rule out the possibility that, under some conditions, animal cells could divide in the absence of
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myosin II. This question is best addressed in genetically tractable organisms. Myosin II is required for division of the C. elegans embryo [53°°], and in Drosophila mutations in the regulatory light chain of myosin cause cytokinesis defects [54]. Although myosin II is not essential for cytokinesis in S. cerevisiae [55,56], it is essential for cytokinesis in S. pombe [57°°]. S. pombe cells lacking myosin II form actin rings at the normal site. This indicates, as does the analysis of the myosin II mutants of Dictyostelium, that myosin does not play a role in the specification of the division plane in either of these organisms (recall that division plane specification in S. pombe is distinct from that of Dictyostelium and animal cells).
M e m b r a n e dynamics T h e function of cytokinesis is to create a membranous barrier between the two daughter cells. T h e central role of the membranes has been largely ignored except in the case of higher plant cells, in which the division plane is formed by the assembly of a membranoua structure in the center of the cell that subsequently grows towards the cell periphery [58]. In plants, Golgi-derived vesicles are transported in a microtubule-dependent manner to the region between the two groups of sister chromatids. These vesicles fuse into a tubulo-vesicular network that matures into a fenestrated sheet-like structure. Cell wall materials are secreted into the lumen of this sheet and it matures further, eventually becoming a continuous membrane on either side of a cell wall [58]. Two molecules have been recently identified that may mediate the assembly of thc membranous structure. T h e Arabidopsis KNOLLE gene has found to be necessary for proper cytokinesis in the embryo [59°°]. T h e product of the KNOLLE gene is homologous to syntaxins, which are involved in membrane fusion events [60]. It appears likely that K N O L L E is involved in forming the membranous structure that gives rise to the division plate. A second protein has been discovered that could be involved in the remodelling of the membrane. A dynamin-like protein called phragmoplastin is concentrated in the cell plate [61°,62°]. Dynamin itself has been implicated in endocytosis in a wide range of organisms, where the protein has been shown to self-assemble into ring-like structures that may promote the pinching off of endocytic vesicles [63]. Phragmoplastin could perhaps function in cytokinesis by mediating the formation of the tubular vesicular network, or in the maturation of the tubular vesicular network by virtue of its ability to deform membranes. In the embryonic divisions of animal cells, new membrane is inserted just behind the leading edge of the ingressing furrow [37°°,64]. Thus, it is possible that proteins related to K N O L L E and phragmoplastin may also function in the division of animal cells. Dynamic changes in the plasma membrane might well be a general phenomenon. During division of cultured cells, there are indications that the composition of the plasma membranes changes
during cytokinesis and, furthermore, agents that disrupt the redistribution of a certain phospholipid prevent cytokinesis [65°]. Phosphatidylethanolamine (PE) is a membrane phospholipid that is asymmetrically distributed in cell membranes, being present in much greater amounts in the inner as opposed to the outer leaflet of the plasma membrane. A novel probe consisting of a tetracyclicpeptide that binds PE specifically was used to demonstrate that, during cytokinesis, the outer leaflet of the cleavage furrow contains appreciable amounts of PE [65°]. T h e source of PE in the outer leaflet is not known; it could come from the inner leaflet, it could become concentrated from elsewhere in the outer leaflet, or it could become localized t h e r e a s a result of vesicle insertion. A muhivalent derivative of this probe traps PE and prevents completion of cytokinesis; the actin ring persists long after it would normally disassemble [65°]. This study illustrates that the plasma membrane is an important player in cytokinesis. T h e composition of the membrane could be critical to the final step of cytokinesis, cell separation, which is likely to involve the regulated fusion of membranes rather than being a direct consequence of the furrowing process.
Perspectives T h e significant progress that has been made in the time period covered by this review has largely resulted from the fact that cytokinesis is emerging as an active field of study in multiple experimental systems. A number of gene products that are important for cytokinesis have been discovered by genetic analysis in fungi and Didyostelium. This effort has been complemented by studies of Drosophila and, recently, C. elegans. It is likely that this aspect of the endeavor is not yet complete. Tissue culture cells have also proven highly informative as they can be used for high resolution microscopy and can be micromanipulated to some extent. T h e analysis of cytokinesis of plant cells has provided insight into the machinery that reorganizes the plasma membrane. Ten years ago, it appeared as though there were many answers to the question, 'How is the cell cycle regulated?'. In the meantime, the combination of genetics, biochemistry, and cell biology in multiple experimental organisms has revealed that there is one essential theme to cell cycle control. T h e analysis to date of cytokinesis has suggested that there are a number of answers to the question, 'How does cytokinesis work?', yet these answers are beginning to exhibit some similarities. Perhaps we will soon know for certain how many times evolution has answered this question. T h e apparent contradictions that have surfaced during this review may reflect true differences in experimental systems: a protein (myosin) or a structure (the central spindle) that is essential for cytokinesis in one system may be partially or even fully dispensable in another system. T h e reason for this may be that a complex process like cell division could consist
The mechanism and control of cytokinesis Glotzer
of multiple, partially redundant subprocesses that vary in their degree of redundancy in different systems.
821
13.
Rappaport R: Repeated furrow formation from a single mitotic apparatus in cylindrical sand dollar eggs. J Exp Zoo/1985, 234:167-171.
14.
Because of space limitations, it was not possible to discuss all of the new advances in the field. To facilitate access to this literature, the following citations are provided [66°-68°,69,70,71 °,72,73,74°,75°].
Williams BC, Riedy MF, Williams EV, Gatti M, Goldberg ML: The Drosophila kinesin-like protein KLP3A is a midbody component required for central spindle assembly and initiation of cytokinesis. J Cell B/o/1995, 129:709-723.
15.
Andreassen PR, Palmer DK, Wener MH, Margolis RL: Telophase disc: a new mammalian mitotic organelle that bisects telophase cells with a possible function in cytokinesis. J Cell Sci 1991, 99:523-534.
Acknowledgements
16.
JulianM, Tollon Y, Lajoie-Mazenc I, Moisand A, Mazarguil H, Puget A, Wright M: Gamma-tubulin participates in the formation of the midbody during cytokinesis in mammalian cells. J Cell Sci 1993, 105:145-156.
17.
Shu HB, Li Z, Palacios MJ, Li Q, Joshi HC: A transient association of gamma-tubulin at the midbody is required for the completion of cytokinesis during the mammalian cell division. J Cell Sci 1995, 108:2955o2962.
18.
Cooper JA and Kiehart DP: Septins may form a ubiquitous family of cytoskeletsl filaments. J Cell Bio11996, 134:13451348.
Supplementary citations
I would like to thank Suzanne Eaton, Pierre Gtinczy, Sigrid Reinsch, and Lisa Satterwhite for helpful comments on this manuscript.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • •.
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9. •
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10. •
Eckley DM, Ainsztein AM, Mackay AM, Goldberg IG, Earnshaw WC: Chromosomal proteins and cytokinesis: patterns of cleavage furrow formation and inner centromere protein positioning in mitotic heterokaryons and mid-anaphase cells. J Cell Biol 1997, 136:1169-1183. These three papers [8",9",10"] analyze whether two asters are sufficient to induce an ectopic furrow in cultured cells. In some cases, furrows formed and in others, they did not. In [8"}, the presence of bundled microtubules between the asters was correlated with the induction of furrowing.
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Cao LG, Wang YL: Signals from the spindle midzone are required for the stimulation of cytokinesis in cultured epithelial cells. Mol Biol Cell 1996, 7:225-232. The presence of a small perforation between the central spindle and the cortex prevented the formation of a furrow in the normal position. This provides evidence that the central spindle promotes furrow specification or ingression.
27. •
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This paper shows that racE is essential for cytokinesis in Dictyostelium, and mutations in the racE gene cause no other detectable phenotypes besides a cytokinesis defect. Larochelle DA, Vithalani KK, De Lozanne A: Role of Dic(yostelium racE in cytokinesis: mutational analysis and localization studies by use of green fluorescent protein. Mol Bio/Cell 1997, 8:935-944. Mutational analysis of racE (see also [30"]) suggests that the ability of race to bind GTP is essential for its function, but, surprisingly, mutations that are likely to prevent GTP hydrolysis are fully functional. 31. •
32. •
Adachi H, Takahashi Y, Hasebe T, Shirouzu M, Yokoyama S, Sutoh K: Dictyostelium IQGAP-related protein specifically involved in the completion of cytokinesis. J Cell Bio/1997, 137:891-898.
A protein with a GTPase-activating protein (GAP) domain is essential for the late stages of cytokinesis. As with racE (see [30°,31 "]), mutations in the gapA gene cause defects in cytokinesis, but do not cause other obvious phenotypes. 33. •
Lee S, Escalante R, Firtel RA: ARas GAP is essential for cytokinesis and spatial patterning in Dictyostelium. Development 1997, 124:983-996. A GTPase-activating protein (GAP) is essential for cytokinesis, but as a range of additional phenotypes are observed when this protein is mutated it probably has other roles as well. 34.
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Drechsel DN, Hyman AA, Hall A, GIotzer M: A requirement for Rho and Cdc42 during cytokinesis in Xenopus embryos. Curt Bio11997, 7:12-23. Analyzes the role of Rho and Cdc42 in cytokinesis of Xenopus embryos. The insertion of cleavage membranes in the presumptive furrow region, in the absence of furrow ingression, indicates that furrow specification and membrane insertion are independent of furrow ingression.
45. •
Evangelista M, Blundell K, Longtine MS, Chow CJ, Adames N, Pringle JR, Peter M, Boone C: Bnilp, a yeast formin linking cdc42p and the actin cytoskeleten during polarized morphogenesis. Science 1997, 276:118-122. These references [42"°,43°-46°] establish that the formin family is involved in cytokinesis in many organisms, and that members of the family bind to profilin and to Rho family GTPases. 46. •
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57. •.
HarrisSD, Hamer L, Sharpless KE, Hamer JE: The Aspergillus nidulens sepA gene encodes an FH1/2 protein involved in cytokinesis and the maintenance of cellular polarity. EMBO J 1997, 16:3474-3483. These references [42"°,43=-46 `] establish that the formin family is involved in cytokinesis in many organisms, and that members of this family bind to profilin and to Rho family GTPases.
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42. ••
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Guo S, Kemphues KJ: A non-muscle myosin required for embryonic polarity in Caenorhabditis elegans. Nature 1996, 382:455-458. Describes the inactivation by RNA-mediated interference of myosin II in worm embryos. Partial inactivation causes a symmetric first division, and more complete inactivation blocks cytokinesis.
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The mechanism and control of cytokinesis Glotzer
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61. •
Gu X, Verma DP: Phragmoplastin, a dynamin-like protein associated with cell plate formation in plants. EMBO J 1996, 15:695-704. The gene encoding a protein with homology to dynamin has been cloned from soybean. This protein shows a striking localization to the cell plate during cytokinesis. Gu X, Verma DP: Dynamics of phragmoplastin in living cells during call plate formation and uncoupling of cell elongation from the plane of cell division. Plant Cell 1997, 9:157-169. A more detailed analysis, using green fluorescent protein technology, of the localization of phragmoplastin during cytokinesis.
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Gunsalus KC, Bonaccorsi S, Williams E, Vemi F, Gatti M, Goldberg ML: Mutations in twinstar, a Drosophila gene encoding a cofilin/ADF homologue, result in defects in centrosome migration and cytokinesis. J Cell Biol 1995, 131:1243-1259.
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Faix J, Steinmetz M, Boves H, Kammerer RA, Lottspeich F, Mintert U, Murphy J, Stock A, Aebi U, Gerisch G: Cortexillins, major determinants of cell shape and size, are actin-bundling proteins with a parallel coiled-coil tail. Ceil 1996, 86:631-642. This paper describes the cloning of the genes encoding two isoforms of a subfamily of actin-binding proteins. The actin-binding properties of the cortexillins are characterized. Strains lacking both isoforms are defective in cytokinesis.
Emoto K, Kobayashi T, Yamaji A, Aizawa H, Yahara I, Inoue K, Umeda M: Redistribution of phosphatidylethanolamine at the cleavage furrow of dividing cells during cytokinesis. Proc Nat/ Acad Sci USA 1996, 93:12867-12872. A novel probe for phosphatidylethanolamine (PE) is used to show that this lipid is specifically exposed in the outer leaflet of the plasma membrane in the region of the cleavage furrow. Cross-linking of PE with a multivalent probe inhibits cytokinesis. 66. FishkindDJ, Silverman JD, Wang YL: Function of spindle • microtubules in directing cortical movement and actin filament organization in dividing cultured cells. J Cell Sci 1996, 109:2041-2051. Dividing cells are treated with taxol and nocodozole, and the organization of actin filaments and the changes in the behavior of surface-adhered particles are characterized.
Muto A, Kume S, Inoue T, Okano H, Mikoshiba K: Calcium waves along the cleavage furrows in cleavage-stage Xenopus embryos and its inhibition by heparin. J Cell Biol 1996, 135:181-190. The Ca2+ indicator dyes were used in conjunction with confocal microscopy to investigate the association of transient Ca2+ currents with cytokinesis. This study presents evidence that Ca2+ transients are observed during cytokinesis. A previous reference [73] describes similar observations, made in starfish embryos, that lead to qualitatively different results.
67. •
75. •
Burton K, Taylor DL: Traction forces of cytokinesis measured with optically modified elastic substrata [see comments]. Nature 1997, 385:450-454. Cells are cultured on a silicon rubber sheet which becomes deformed by cellular contractile activity. The location and extent of wrinkling provides a measure of the forces that occur during cell division. 68. •
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74. •
Asada T, Kuriyarna R, Shibaoka H: TKRP125, a kinesin-related protein involved in the centrosome-independent organization of the cytokinetic apparatus in tobacco BY-2 cells. J Cell Sci 1997, 110:179-189. Describes the cloning of the gene encoding a kinesin-related protein from the BimC family; this protein localizes to the phragmoplast microtubules during cytokinesis. Antibodies to TRKP125 inhibit the translocation of phragmoplast microtubules in a permeabilized cell system.