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Cytokinesis: an emerging unified theory for eukaryotes? Karen G Hales*†, Erfei Bi‡, Jian-Qiu Wu*, Jennifer C Adam*, I-Ching Yu* and John R Pringle*§ In animal and fungal cells, cytokinesis involves an actomyosin ring that forms and contracts at the division plane. Important new details have emerged concerning the composition, assembly, and dynamics of these contractile rings. In addition, recent advances suggest that targeted membrane addition is a central feature of cytokinesis in animal cells — as it is in fungi and plants — and the coordination of actomyosin ring function with targeted exocytosis at the cleavage plane is being explored. Important new information has also emerged about the spatial and temporal regulation of cytokinesis, especially in relation to the function of the spindle midzone in animal cells and the control of cytokinesis by GTPase systems. Addresses *Department of Biology and Program in Molecular Biology and Biotechnology, University of North Carolina, 421 Fordham Hall, Chapel Hill, NC 27599-3280, USA ‡ Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA 19104-6058, USA † e-mail: [email protected] §e-mail: [email protected] Current Opinion in Cell Biology 1999, 11:717–725 0955-0674/99/$ — see front matter © 1999 Elsevier Science Ltd. All rights reserved. Abbreviations FH formin homology GAP GTPase-activating protein KLP kinesin-like protein RMLC myosin regulatory light chain SPB spindle-pole body

Introduction Cytokinesis is the final step in cell division during which the daughter nuclei are invested with their own plasma membranes and cytoplasms (see [1–9] for other reviews). Traditionally, the mechanisms of eukaryotic cytokinesis have been viewed as falling into two categories. In animal cells and cellular slime molds, cytokinesis has appeared to depend on the formation of a cortical actin- and myosinbased (‘actomyosin’) contractile ring, which constricts and pinches off the membrane to form the two daughter cells. In contrast, fungal and plant cells have appeared to require the deposition of cell wall material (via the secretory pathway) at the cleavage plane to form a septum or cell plate between the daughters. In plants, but not in fungi, a specialized cytoskeletal structure called the phragmoplast directs the delivery of cell-plate material [3,7]. A number of observations now suggest a more unified view of cytokinesis. For example, actomyosin contractile rings have been found to be present in fungi; moreover, targeted vesicle fusion at the cleavage plane is now known to play an important role in animal cell cytokinesis, with phragmoplast-like microtubule arrays perhaps helping to

direct vesicle transport. Membrane addition may be coordinated with and/or guided by contraction of the actomyosin ring, with these two processes together forming the foundation of cytokinesis in both fungal and animal cells. In addition, several conserved protein families — including the septins, the Cdc15p/PSTPIP proteins, and GTPases of the Rho/Rac/Cdc42 family — are now known to act quite broadly in cytokinesis and could direct or coordinate aspects of actomyosin-ring function, membrane addition, or both in various organisms. In this review, we focus on advances that have added significantly to our understanding of cytokinesis since the two most recent reviews in this journal [5,8]. In the first section, we discuss mechanisms of cytokinesis. In the second section, we discuss the spatial and temporal regulation of cytokinesis.

Mechanisms of cytokinesis Composition, assembly, and function of contractile rings

A large number of proteins are associated with actomyosin rings, including many that are known to bind actin and regulate its polymerization (reviewed in [8]). Ongoing efforts are elucidating the intricacies of ring formation and subsequent contraction. The identification of actomyosin rings in the fission yeast Schizosaccharomyces pombe (reviewed in [4,6,9]) and the budding yeast Saccharomyces cerevisiae [10–12] has not only blurred the traditional view of cytokinetic mechanisms but also allowed the beginnings of a detailed genetic analysis of ring formation and function. Surprisingly, recent studies in the two yeasts have indicated major differences in the timing and order of addition of various components. In S. cerevisiae, Myo1p (the single type II myosin) forms a ring just before bud emergence in the late G1 or early S phase of the cell cycle; this is dependent on the presence of the septin neck-filament proteins but is not dependent on actin [11,12]. Later, after spindle elongation in anaphase, the IQGAP-like protein Iqg1p and then actin are localized to the ring, which then almost immediately contracts [10–12]. In contrast, in S. pombe, F-actin forms a ‘medial ring’ early in M phase and is required for the near-simultaneous localization to the ring of Myo2p and Myo3p/Myp2p (a pair of type II myosins), the myosin regulatory light chain (RMLC) Cdc4p, tropomyosin, α-actinin, fimbrin, and the IQGAP-related protein Rng2p ([4,6,9,13,14]; J-Q Wu, J Bähler, JR Pringle, unpublished data). The ring does not contract until later, after anaphase. The continued maintenance of Myo2p in the S. pombe medial ring does not depend on actin [14], and the S. pombe septins do not appear to play a role in medial-ring assembly or maintenance ([15]; OS Al Awar et al., unpublished data).

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IQGAPs are multidomain proteins associated with the contractile ring and implicated in cytokinesis in a number of organisms [8,16]. Recently, the roles of the S. cerevisiae IQGAP Iqg1p (or Cyk1p) were dissected in some detail [17]. A structure-function study suggested that separate domains within the protein direct localization to the bud neck (analogous to the cleavage furrow), participate in recruitment of actin to the ring, and promote subsequent ring contraction. In particular, the carboxy-terminal GTPase-activating protein (GAP)related domain of Iqg1p appears to be required to trigger ring constriction. The corresponding domains of mammalian IQGAPs have been shown to interact with Rho-family GTPases, thereby suggesting a possible molecular link between signaling and the regulation of actomyosin function (reviewed in [16]; see also below). The S. pombe IQGAP Rng2p is similar to S. cerevisiae Iqg1p in that it directs proper actin organization within the contractile ring; however, it differs in that actin is required for Rng2p localization, whereas Iqg1p is required for actin localization [10,12,13,17]. Remarkably, although Iqg1p is required for cytokinesis in S. cerevisiae [10,12], the actomyosin ring is not ([11]; WS Korinek, JA Epp, J Chant, personal communication). Thus, Iqg1p must participate in an alternative cytokinesis pathway (see below). In animal cells, actin and myosin are detectable at the cleavage furrow only in late anaphase as furrowing begins [18]. In at least some fungal and animal cells, formin homology (FH) proteins help recruit actin to the ring (reviewed in [8]). The Drosophila protein anillin binds actin [19] and might play a role in assembly or maintenance of the contractile ring. In spermatocytes undergoing meiotic divisions, anillin is localized to the cleavage furrow independently of actin and before actin is detectable there [20•]; anillin subsequently colocalizes with the contractile ring during its constriction [20•]. Animal septins, which localize to the cleavage furrow and are required for cytokinesis in at least some cell types [8,15,21,22], might also have a role in localizing components of the contractile ring, as they do in S. cerevisiae. Further genetic studies of anillin and the septins in animal cells should be informative. Meanwhile, the very existence of a contractile ring in the cellular slime mold Dictyostelium has been called into question by studies of myosin and actin distribution in dividing cells [23,24]. Myosin II, but not actin, is enriched in regions flanking the cleavage furrow in cells grown under physiological conditions. Moreover, myosin II is not absolutely required for cytokinesis in cells grown on a solid substrate, and in general may only help to maintain differential cortical stability [23–26]; the actin-binding proteins called cortexillins are also enriched in the furrow and could have similar function [26,27]. Studies of membrane dynamics may help to explain cytokinetic mechanisms in Dictyostelium (see below).

Function of the phragmoplast

The phragmoplast — a structure required for cytokinesis in plant cells — appears in late anaphase and is an array of two sets of oppositely oriented microtubules, the plus ends of which interdigitate (reviewed in [3,7]). Vesicles carrying cell plate material are thought to travel down these microtubules to the plane of cell division, where vesicle fusion gives rise to the growing cell plate [3,7]. Actin filaments are also present both in the phragmoplast and at the cell plate, although their role has not been clarified [7,28]. Recently, novel MAP kinases have been identified in alfalfa and tobacco that localize to the phragmoplast (in anaphase) and the growing cell plate (in telophase) [29,30]; the substrates and roles of these kinases are unknown. Little is known about the molecular mechanisms of phragmoplast assembly and function; however, it is becoming clear that animal cells may have an analogous structure, the spindle midzone (see below), and that the vesicle transport and fusion seen in plant cell division is likely to be a universal aspect of eukaryotic cytokinesis. Membrane dynamics in cytokinesis

Membrane transport and fusion have long been viewed as central to fungal as well as plant cytokinesis. For example, in S. cerevisiae, the bud is connected to the mother cell by a narrow (1 µm) neck that is surrounded by a ring of chitin in the cell wall and the septin ring on the cytoplasmic face of the plasma membrane. During cytokinesis, this neck is filled in by secretion of cell wall material to form a septum, the central portion of which is eventually digested to result in separation of the daughter cells [31]. S. pombe cells also form a septum between the nascent daughter cells but must construct it across the entire width of the cell (3 µm) instead of just across a narrow neck [6,9]. In both yeasts, actin patches cluster near the site of septum construction, indicating that secretion is polarized to this region [4,6,9,31]. The S. pombe actomyosin ring forms at the cell middle and appears to help guide septum formation (reviewed in [4,6,9]); the recently discovered S. cerevisiae actomyosin ring may have a similar role [10–12,32•]. In S. cerevisiae, Myo1p (and thus the actomyosin ring) is nonessential in some (if not all) wild type strain backgrounds ([11]; WS Korinek, JA Epp, J Chant, personal communication). However, septins, which are required for localization of Myo1p [11,12], are essential for cell division, indicating that they have an additional function during cytokinesis. Cell wall synthesis appears to be required for cytokinesis in S. cerevisiae, as mutations in CHS2 and CHS3, which encode the catalytic subunits of two chitin synthases, are synthetically lethal [31,33]. Septins anchor chitin synthase subunits to the plasma membrane at the bud neck ([34]; I Yu, JR Pringle, unpublished results) and thus appear to act as scaffolds for two different protein complexes involved in cytokinesis: the actomyosin ring and the septum-construction machinery. Although the actomyosin ring may normally help to guide septum formation, the bud neck may be narrow enough

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that localized cell wall deposition alone can produce efficient cytokinesis. In contrast, in S. pombe, the need to fill a larger gap may make guidance by the actomyosin ring essential, accounting for the essentiality of myosin II for cytokinesis in this species. In animal cells, the ingression of the cleavage furrow has commonly been attributed to force from contraction of the actomyosin ring, which has been thought to pull a passive plasma membrane centripetally into the furrow, eventually pinching it off between the two daughter cells. However, as noted previously [8,35], the topology of forming two cells from one requires the addition of surface area. Experiments with Xenopus embryos have indicated that membrane addition occurs specifically at the cleavage plane. When the contractile ring was disrupted by mutations or a chemical inhibitor, no cleavage furrow formed, but membrane material was inserted in a microtubuledependent manner where a cleavage furrow would have ingressed [35–37]. Without an intact contractile ring, the new membrane spread out on the surface of the embryo, suggesting that a normal role of the ring is to guide newly added membrane into the cleavage furrow. If membrane addition is a central aspect of animal-cell cytokinesis, one would expect the involvement of the many proteins that mediate vesicle fusion in other contexts. Indeed, several recent studies have indicated a cytokinetic role for syntaxins, which are t-SNAREs that play a central role in the docking and fusion of secretory vesicles [38,39]. Early Drosophila development involves 13 syncytial nuclear divisions, after which each nucleus is enveloped in its own membrane during a specialized cytokinetic process called cellularization [1]. Mutations in the Drosophila gene syntaxin 1 cause defective cellularization with an accumulation of vesicles at the sites of intended furrow ingression [40]. Similarly, the Caenorhabditis elegans syntaxin Syn-4 is enriched in cleavage furrows, and experiments using RNA interference have shown that it is required for furrow ingression during early embryonic cytokineses [41•]. In addition, inhibition of sea urchin syntaxin by antibodies or Botulinum neurotoxin C1 resulted in failure of cell division [42•]. Syntaxins and other components needed for exocytosis also help deliver the materials and machinery for septum formation in S. cerevisiae [43,44] and Arabidopsis [45]. Consistent with a role for membrane dynamics in animal cytokinesis, the Drosophila gene four wheel drive (fwd) encodes a predicted phosphatidylinositol 4-kinase (PI 4-K) required for male meiotic cytokinesis [46]. Spermatocytes from fwd mutant flies initiate cleavage furrows that subsequently regress; actomyosin rings form but are disorganized. Phosphatidylinositides regulate vesicle formation and targeted exocytosis [47,48], and the fwd mutant phenotype might result from failed membrane delivery to the cleavage furrow. Alternatively, or perhaps in addition, the defects in fwd spermatocytes could be

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explained by phosphatidylinositide effects on actin filament polymerization [47–49]. Thus, it now seems possible that the role of the contractile ring is not solely (or may not be at all) to generate force to deform the plasma membrane; instead, ring contraction may provide spatial guidance for the arrangement of new added membrane. If this were the case, might we explain the lack of necessity for actomyosin rings in some cell types by the existence of different means for directing membrane addition? The answer for plants is clearly yes. Golgi-derived vesicles accumulate and fuse in the middle of the cell to form the cell plate, which grows centrifugally, as opposed to the centripetal movement of furrows and septa in other cell types (reviewed in [3,7]). Cell plate growth is guided by cues established earlier in the cell cycle, when a ring of microtubules and actin (the preprophase band) encircles the cell cortex at the future site of cell plate fusion (reviewed in [50]). The preprophase band disappears during mitosis, but the phragmoplast orients in response to spatial cues left behind (reviewed in [3]). The rigidity of the material secreted in the cell plate might also contribute to successful cytokinesis, as the Arabidopsis cyt1 mutation causes cell plate accumulation of callose, a fluid glucose polymer, instead of the more rigid cellulose; this is accompanied by cytokinesis failure [51•]. Dictyostelium amoebae can divide without myosin II when adhering to a solid support [24–26]. A recent study indicates that some mammalian cells can also divide without functional actomyosin rings when attached to a solid substrate [52•]. As discussed above, cytokinetic membrane addition appears not to require contractile ring function; perhaps the cell cortex of adherent Dictyostelium and mammalian cells is stabilized by the solid support in such a way that even without actomyosin contraction, membrane added at the cleavage plane remains roughly in a furrow conformation instead of spreading out on the surface of the cell. In S. cerevisiae cells lacking myosin II, the cortical support at the narrow bud neck provided by septins and associated proteins, and/or by the cell wall, may function analogously such that newly added membranes do not absolutely require additional spatial guidance. Coordination of ring contraction with membrane addition

If the actomyosin ring functions to guide ingression of newly added membrane at the cleavage plane, then ring contraction must be coordinated with membrane dynamics. There are some hints as to which protein families might regulate this coordination. As mentioned above, the septins localize to the site of cell division and function in cytokinesis in many organisms [8,15,21,22], influencing both actomyosin ring formation and septum construction in S. cerevisiae. In mating and sporulating S. cerevisiae cells and in various animal cell types, the localization of septins is consistent with a general role for these proteins in targeted membrane insertion [15,21,22]. Accordingly, septins have been found to interact both with syntaxin [22,53•]

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and with the exocyst complex [54], which is required for exocytosis. Thus, septins might serve to integrate actomyosin contraction and membrane addition during cytokinesis in some cells. Another protein family that could be involved in coordinating these two aspects of cytokinesis includes Cdc15p and Imp2p in S. pombe [55,56], PSTPIP in mouse [57], and Hof1p (Cyk2p) in S. cerevisiae [32•,58,59]. Cdc15p/PSTPIP family members are Src homology 3 (SH3) domain-containing phosphoproteins that localize to the contractile ring. S. pombe cdc15 mutants are defective in septation; although they form actomyosin rings, they do not reorganize their actin patches to the middle of the cell [6,55,60]. Because the reorganization of actin patches is thought to contribute to the directed secretion of septal material, the cytokinesis defect of cdc15 mutants has been suggested to result from a defect in targeting vesicles to the growing septum [60]. S. cerevisiae Hof1p also appears to play a role in septum formation. Neither the assembly [32•,59] nor the later contraction [32•] of the actomyosin ring is significantly affected in a hof1 deletion strain, but the mutant displays a temperature-sensitive defect in cytokinesis and septum construction [32•,58,59]. Proteins involved in actomyosin function or in septum construction in S. cerevisiae are anchored to the septin ring [11,12,15,21,34], and Hof1p also depends on the septins for proper localization to the neck [32•,59]. Consistent with the hypothesis that Hof1p functions primarily in septum formation rather than in actomyosin dynamics, the hof1 deletion phenotype is distinct from, and much more severe than, the myo1 deletion phenotype [32•]. In addition, the hof1 and myo1 deletions are synthetically lethal at a temperature permissive for the hof1 single mutant [32•], suggesting that the products of these genes have parallel roles in cytokinesis. Moreover, a hof1 deletion is also synthetically lethal with mutations in BNI1, which encodes an FH protein that itself affects actomyosinring contraction [32•,58]. Further synthetic-lethality and two-hybrid studies place Myo1p and Bni1p in one functional group and Hof1p in a second group with Bnr1p, the other S. cerevisiae FH protein [32•,58]. As with the hof1 deletion strains, a bnr1 deletion strain has a septum formation phenotype but apparently normal actomyosin dynamics. Interestingly, these data show functional differentiation within the FH protein family, the original members of which (including Bni1p) appeared to affect actomyosin dynamics early in cytokinesis (reviewed in [8,32•,61]). These observations could explain why the C. elegans FH protein Cyk-1 appears to act late in cytokinesis [62]: it might be more related to Bnr1p-like FH proteins than to Bni1p-like proteins, and may thus coordinate membrane addition rather than the formation of the actomyosin ring. Taken together, the data suggest that Hof1p may function in coupling the secretory machinery to the actomyosin ring. It is not yet clear whether the other members of the Cdc15p/PSTPIP protein family have similar functions. An

initial study found that an S. pombe imp2 deletion strain formed abnormal septa as well as actomyosin rings that did not disassemble properly after contraction [56]. Actin patch distribution was affected in cells overexpressing Imp2p but not in cells with imp2 deleted [56]. Mouse PSTPIP is associated with the actin cytoskeleton in interphase cells and inhibits septation when overexpressed in S. pombe [57]. The tyrosine phosphorylation of PSTPIP might affect cytokinesis [63] and could correspond to the phosphorylation of Cdc15p and Hof1p seen during cell division [32•,55]. Additional studies of this protein family should be very revealing. Midzone microtubules and vesicle delivery

In plant cells, the phragmoplast microtubules appear to be derived from the mitotic spindle in late anaphase and mediate delivery of vesicles carrying cell wall material to the cell plate (see above). Animal cells have a seemingly analogous structure, termed the spindle midzone (or central spindle) that contains an antiparallel array of bundled microtubules derived from the spindle interzone after chromosome segregation [8]. The spindle midzone may also mediate vesicle delivery during cytokinesis, as mutations in several associated proteins prevent the completion (although not the initiation) of cleavage furrow ingression [64,65,66•–68•]. In at least some cell types, the spindle midzone also appears to have an earlier role in determining the cleavage plane, as discussed below.

Regulation of spatial and temporal aspects of cytokinesis The spindle midzone and cleavage-plane specification

The molecular strategies for spatial control of cytokinesis vary in different organisms (reviewed in [8]). In animal cells, spindle orientation determines the cleavage plane, and the role of the spindle midzone has recently become appreciated [5,8,69]. During anaphase, the antiparallel interzonal microtubules become bundled with their plus ends interdigitating; the bundles are associated with many proteins, including some ‘chromosomal passenger proteins’ originally bound to centromeres as well as proteins found earlier at the spindle poles and/or centrosomes. The cleavage furrows that form between asters of different spindles in the same cell were originally thought to be specified by an astral signal [70]. It now appears that such ectopic furrows correlate with the presence of interastral antiparallel microtubules that are associated with the chromosomal passenger protein INCENP and the kinesin family member CHO1, at least in mammalian cells [71•]. Moreover, rat epithelial cells treated with topoisomerase inhibitors display misplaced interzonal microtubule bundles that appear to induce ectopic cleavage furrows in adjacent cortical regions [72•]. Structure-function studies of INCENP have shown that its ability to bind centromeres is crucial for its transfer to the spindle midzone at anaphase and thus for promotion of cytokinesis [65]; genetic analyses of the C. elegans and Drosophila CHO1 homologues MKLP1/ZEN4 and Pav-KLP support the

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idea that proteins in this family bundle midzone microtubules [64,66•,67•]. Cell-cycle kinases also appear to be important for spindle midzone construction. The Drosophila Polo kinase and the C. elegans AIR-2 (Aurora/Ipl1-related) kinase are present at the spindle midzone, and each is required for midzone localization of CHO1-family microtubulebundling proteins [68•,73•]. Recently, the minus-end-directed microtubule motor dynein and members of its activating dynactin complex were also localized to the spindle midzone in both mammalian cells [74] and C. elegans embryos [75]. The motor activity of dynein (as well as of the midzone chromosomal passenger protein CENP-E) could help to maintain microtubule interdigitation in the interzone, since the presence of only plus-end motors like those of the MKLP1 family might tend to push apart microtubules of opposite polarity. Dynactin subunits in mammalian cell extracts interact physically with the EB1 protein [76]; the S. cerevisiae EB1 homologue Bim1p is also found at the spindle midzone and appears to function as part of a checkpoint that delays cytokinesis in response to spindle misorientation [77,78•]. The significance of the dynactin–EB1 interaction has yet to be determined. Although many components of the spindle midzone have been identified, it is not known how this structure signals the cell cortex to determine the cleavage plane. Mutations affecting INCENP, AIR-2, or some CHO1 family members do not prevent the initiation of furrowing, indicating that none of these proteins serves as the spatial signal [65,66•–68•]. Studies of Drosophila spermatocytes undergoing meiotic divisions have suggested a cooperative interaction between the spindle midzone and the contractile ring, wherein disruption of one by mutation or drugs affects formation of the other [79], and a recent study using mammalian cells appears to confirm this [80]. In the absence of properly localized Polo kinase at the spindle midzone in Drosophila, neither the septin Pnut nor the contractile ring component anillin is recruited to the division site [64,73•]. The kinesin-like protein KLP3A is also present in the spindle midzone in Drosophila and is required for actomyosin-ring formation and initiation of cytokinesis [79,81]. However, at least one component of the contractile ring, anillin, can localize normally to a cortical ring at the cell equator in spermatocytes mutant for KLP3A [20•]. These data suggest, firstly, that a spatial signal defines the cleavage plane even in the absence of KLP3A and an actomyosin ring, and secondly, that anillin is an upstream component in the establishment of the contractile ring and appears to depend (at least indirectly) on Polo kinase activity for its localization. Anillin is nuclear throughout interphase and forms a cortical ring during mitosis [19,20•], similar to S. pombe Mid1p, which appears to serve as a cortical marker defining the cleavage plane and which has some structural similarity to anillin [6,9,82•]. Interestingly, one of the several

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functions of the S. pombe Polo kinase is to promote (by phosphorylation) the movement of Mid1p from the nucleus to the cell cortex [82•]. Thus, although the positioning of the S. pombe cleavage plane appears to be determined by nuclear position early in mitosis [4,6,8,9] rather than by the position of the spindle midzone late in mitosis, the mechanisms might have enough in common that continued genetic analysis in S. pombe will help illuminate the stillmysterious mechanisms by which the spindle midzone influences cleavage plane specification. Temporal control of cytokinesis by GTPase systems

The initiation of cytokinesis is a crucial point at which temporal coordination with the rest of the cell cycle must be regulated. In S. pombe, several components of the signaling pathway that triggers cytokinesis have been identified, primarily through screens for septation mutants that assemble their cytokinesis machinery but fail to divide. Central to the pathway is the small GTPase Spg1p, which localizes to spindle-pole bodies (SPBs) throughout the cell cycle [83,84]. Spg1p becomes GTP-bound at mitotic entry, allowing the kinase Cdc7p (which is not on SPBs during interphase) to associate with it [84]. GAP activity for Spg1p is provided by Byr4p and Cdc16p acting together; these proteins localize to only one of the two SPBs late in mitosis, causing the Spg1p at that pole to hydrolyze GTP and release Cdc7p [85•,86•]. This asymmetric localization of Cdc7p is correlated with septation initiation, but Cdc7p itself does not appear to go to the septation site [85•]. Recently, an additional protein that functions in a late step of this signaling pathway, Sid2p, was characterized. Sid2p is a protein kinase that is required for the initiation of septation; it is associated with the SPB throughout the cell cycle and is also detectable at the cell division site during cytokinesis, when its kinase activity peaks [87•]. Function of all the other known septation-initiation genes is required for Sid2p to have kinase activity and localize properly to the division site. Future experiments should determine whether Sid2p is directly activated by Cdc7p or whether there are intermediate molecular steps. In addition, identifying the downstream target(s) of Sid2p kinase activity will be most important for elucidating how cytokinesis is initiated in S. pombe. Components of this signaling pathway have homologues in S. cerevisiae that might also function in cytokinesis [88]. In particular, the S. cerevisiae Spg1p homologues Tem1p binds the contractile ring IQGAP Iqg1p much more tightly than does Cdc42p, which was originally thought to be the molecule that signals to Iqg1p [17,89]. Iqg1p could thus be a critical factor in receiving a temporal signal for cytokinesis and initiating actomyosin ring contraction. In animal cells, initiation of cytokinesis is thought to be triggered by activation of Rho-family GTPases [37,52•,90,91] and probably involves the concerted effort of a number of downstream targets (reviewed in [92,93]), including IQGAPs, FH proteins, and RMLCs. The

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Rho effectors Rho-kinase and citron kinase localize to the cleavage furrow in mammalian tissue culture cells [94,95], and citron kinase activity appears to be important for proper actomyosin contractility and furrow progression [94]. Overexpression of mutant Rho-kinase was reported not to affect cytokinesis [94], a result that appears to conflict with an independent report that dominant-negative Rho-kinase inhibited cytokinesis in both Xenopus embryos and mammalian cells [96]. Rho-kinase phosphorylates intermediate filaments in the cleavage furrow, affecting their separation to daughter cells [95,96]. Whereas Rho-kinase and citron kinase seem to be involved in progression of an established cleavage furrow, mutation of a putative Drosophila Rho1 guanine nucleotide exchange factor prevented the initiation of cytokinesis [97•], suggesting that other Rho effectors act earlier during cell division. Identification of additional Rho effectors and their downstream targets, as well as targets of Rho-kinase and citron kinase, should help unravel the molecular complexities of cytokinesis initiation and progression. Another set of proteins downstream of Rho includes the myosin-light-chain kinases and myosin phosphatases. By analogy to actomyosin function in smooth muscle [98], the phosphorylation state of RMLC is thought to regulate contractility of the actomyosin ring during cytokinesis. A recent study suggests that the regulation of myosin phosphatase activity is critical for controlling phosphorylation of the activating Ser19/Thr18 sites on RMLC during the onset of cytokinesis [99•]; upregulation via phosphorylation of the phosphatase during mitosis may prevent the activating phosphorylation of RMLC from accumulating before completion of mitosis. Subsequently, myosin phosphatase is dephosphorylated and becomes less active, allowing myosin-light-chain kinase activity to predominate and activate myosin contractility. Meanwhile, the role of inhibitory phosphorylation of RMLC Ser1/2 by p34cdc2, once widely accepted as important for delaying cytokinesis onset [100,101], has been cast into doubt, because cortically associated RMLC showed no sign of Ser1/2 phosphorylation even in the presence of active p34cdc2 [102•]. Future work should fill in the current gaps in the signaling pathway(s) linking cell-cycle kinases, Rho, myosin phosphatase, and myosin itself.

Conclusions The past year has seen substantial progress in several areas of cytokinesis research, perhaps most importantly in the widening support for the idea that cytokinesis is an event that coordinates actomyosin contractility with targeted membrane addition in animals, fungi, and slime molds. At its core, cytokinesis appears to be a membrane targetingbased event in eukaryotes. Plants have evolved one means for spatial regulation of vesicle fusion during cell division, and animals, fungi, and slime molds have evolved another. In the latter groups, the major role of the contractile ring may be to guide newly added membrane into furrows, although in some cell types and under some conditions this

guidance appears to be unnecessary. Future experiments should shed light on how secretion is directed to the plane of division, how some cells can divide without myosin II, and how actomyosin contraction and secretion are coordinated. Present indications are that the septins, the Cdc15/PSTPIP-family proteins, a subset of FH proteins, and perhaps IQGAPs may all contribute to this coordination. There has also been progress in our understanding of the spatial and temporal control of cyokinesis. The spindle midzone appears to specify the cleavage plane in many cell types, and GTPases feature prominently in pathways that initiate cytokinesis at the proper time in the cell cycle in both fission yeast and animal cells. Exciting areas for future exploration are plentiful, including identification of the spindle midzone components that signal the placement of a cleavage furrow, dissection of the interplay between spindle midzone components and actomyosin dynamics, and filling in the signaling pathways that originate with cellcycle kinases and, via different GTPases, ultimately control actomyosin ring contraction and membrane insertion.

Acknowledgements We thank J White, J Chant, and other cytokinesis workers for stimulating discussions. Work in our laboratory is supported by National Institutes of Health grant GM31006. KGH is supported by National Institutes of Health post doctoral fellowship 1F32GM19981-01.

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10. Epp JA, Chant J: An IQGAP-related protein controls actin-ring formation and cytokinesis in yeast. Curr Biol 1997, 7:921-929. 11. Bi E, Maddox P, Lew DJ, Salmon ED, McMillan JN, Yeh E, Pringle JR: Involvement of an actomyosin contractile ring in Saccharomyces cerevisiae cytokinesis. J Cell Biol 1998, 142:1301-1312. 12. Lippincott J, Li R: Sequential assembly of myosin II, an IQGAP-like protein, and filamentous actin to a ring structure involved in budding yeast cytokinesis. J Cell Biol 1998, 140:355-366. 13. Eng K, Naqvi NI, Wong KCY, Balasubramanian MK: Rng2p, a protein required for cytokinesis in fission yeast, is a component of the actomyosin ring and the spindle pole body. Curr Biol 1998, 8:611-621.

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35. Danilchik MV, Funk WC, Brown EE, Larkin K: Requirement for microtubules in new membrane formation during cytokinesis of Xenopus embryos. Dev Biol 1998, 194:47-60. 36. Bluemink JG, deLaat SW: New membrane formation during cytokinesis in normal and cytochalasin B-treated eggs of Xenopus laevis. J Cell Biol 1973, 59:89-108. 37.

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19. Field CM, Alberts BM: Anillin, a contractile ring protein that cycles from the nucleus to the cell cortex. J Cell Biol 1995, 131:165-178.

40. Burgess RW, Deitcher DL, Schwarz TL: The synaptic protein syntaxin 1 is required for cellularization of Drosophila embryos. J Cell Biol 1997, 138:861-875.

20. Giansanti MG, Bonaccorsi S, Gatti M: The role of anillin in meiotic • cytokinesis of Drosophila males. J Cell Sci 1999, 112:2323-2334. The authors show that the localization of anillin to the site of the cleavage furrow in Drosophila spermatocytes precedes and is independent of the actomyosin ring, suggesting that anillin may have an early role in assembly of the contractile ring.

41. Jantsch-Plunger V, Glotzer M: Depletion of syntaxins in the early • Caenorhabditis elegans embryo reveals a role for membrane fusion events in cytokinesis. Curr Biol 1999, 9:738-745. Provides evidence that vesicle fusion events are crucial to animal cell cytokinesis. The C. elegans syntaxin Syn-4 is enriched at cleavage furrows; Syn-4 depletion by RNA interference inhibits embryonic cytokinesis.

21. Longtine MS, Pringle JR: Septins. In Guidebook to the Cytoskeletal and Motor Proteins. Edited by Kreis T, Vale R. Oxford: Oxford University Press; 1999:359-363.

42. Conner SD, Wessel GM: Syntaxin is required for cell division. Mol • Biol Cell 1999, 10:2735-2743. Further evidence for the role of vesicle fusion in animal cytokinesis — the inactivation of sea urchin syntaxin using drugs or antibodies inhibited cell division.

22. Trimble WS: Septins: a highly conserved family of membraneassociated GTPases with functions in cell division and beyond. J Membrane Biol 1999, 169:75-81. 23. Neujahr R, Heizer C, Albrecht R, Ecke M, Schwartz J-M, Weber I, Gerisch G: Three-dimensional patterns and redistribution of myosin II and actin in mitotic Dictyostelium cells. J Cell Biol 1997, 139:1793-1804. 24. Neujahr R, Heizer C, Gerisch G: Myosin II-independent processes in mitotic cells of Dictyostelium discoideum: redistribution of the nuclei, rearrangement of the actin system and formation of the cleavage furrow. J Cell Sci 1997, 110:123-137. 25. De Lozanne A, Spudich JA: Disruption of the Dictyostelium myosin heavy chain by homologous recombination. Science 1987, 236:1086-1091. 26. Gerisch G, Weber I: Cytokinesis without myosin II. Curr Opin Cell Biol 2000, 12: in press. 27.

Weber I, Gerisch G, Heizer C, Murphy J, Badelt K, Stock A, Schwartz J-M, Faix J: Cytokinesis mediated through the recruitment of cortexillins into the cleavage furrow. EMBO J 1999, 18:586-594.

28. Endle MC, Stoppin V, Lambert AM, Schmit AC: The growing cell plate of higher plants is a site of both actin assembly and vinculin-like antigen recruitment. Eur J Cell Biol 1998, 77:10-18. 29. Calderini O, Bogre L, Vincente O, Binarova P, Heberle-Bors E, Wilson C: A cell cycle regulated MAP kinase with a possible role in cytokinesis in tobacco cells. J Cell Sci 1998, 111:3091-3100. 30. Bögre L, Calderini O, Binarova P, Mattauch M, Till S, Kiegerl S, Jonak C, Pollaschek C, Barker P, Huskisson NS et al.: A MAP kinase is activated late in plant mitosis and becomes localized to the plane of cell division. Plant Cell 1999, 11:101-113. 31. Cid VJ, Durán A, del Rey F, Snyder MP, Nombela C, Sánchez M: Molecular basis of cell integrity and morphogenesis in Saccharomyces cerevisiae. Microbiol Rev 1995, 59:345-386. 32. Vallen EA, Caviston J, Bi E: Roles of Hof1p, Bni1p, Bnr1p and • Myo1p in cytokinesis in Saccharomyces cerevisiae. Mol Biol Cell 2000, 11:in press. Genetic analysis in S. cerevisiae indicates that Hof1p (a Cdc15p/PSTPIP family member) and Bnr1p (a formin homology family member) act in a cytokinesis pathway parallel to actomyosin ring function, perhaps involving targeted exocytosis of septal materials and machinery at to the bud neck.

43. Holthuis JCM, Nichols BJ, Pelham HRB: The syntaxin Tlg1p mediates trafficking of chitin synthase III to polarized growth sites in yeast. Mol Biol Cell 1998, 9:3383-3397. 44. Carr CM, Grote E, Munson M, Hughson FM, Novick PJ: Sec1p binds to SNARE complexes and concentrates at sites of secretion. J Cell Biol 1999, 146:333-344. 45. Lauber MH, Waizenegger I, Steinmann T, Schwarz H, Mayer U, Hwang I, Lukowitz W, Jürgens G: The Arabidopsis KNOLLE protein is a cytokinesis-specific syntaxin. J Cell Biol 1997, 139:1485-1493. 46. Brill JA, Hime GR, Scharer-Schuksz MQ, Fuller MT: The Four Wheel Drive PI 4-kinase regulates intercellular bridge formation during Drosophila male meiosis. Mol Biol Cell 1999, 10: in press. 47.

Martin TFJ: Phosphoinositides as spatial regulators of membrane traffic. Curr Opin Neurobiol 1997, 7:331-338.

48. Corvera S, D’Arrigo A, Stenmark H: Phosphoinositides in membrane traffic. Curr Opin Cell Biol 1999, 11:460-465. 49. Toker A: The synthesis and cellular roles of phosphatidylinositol 4,5 bisphosphate. Curr Opin Cell Biol 1998, 10:254-261. 50. Mineyuki Y: The preprophase band of microtubules: its function as a cytokinetic apparatus in higher plants. Int Rev Cytol 1999, 187:1-49. 51. Nickle TC, Meinke DW: A cytokinesis-defective mutant of • Arabidopsis (cyt1) characterized by embryonic lethality, incomplete cell walls, and excessive callose accumulation. Plant J 1998, 15:321-332. The results reported suggest that cell plate rigidity is important for plant cytokinesis. The Arabidopsis cyt1 mutant accumulates callose instead of cellulose in the cell plate, resulting in defective cell wall architecture and failure of cell division. 52. O’Connell CB, Wheatley SP, Ahmed S, Wang Y: The small GTP • binding protein Rho regulates cortical activities in cultured cells during division. J Cell Biol 1999, 144:305-313. Shows that some types of animal tissue culture cells can divide without a functional contractile ring when adhering to a solid substrate, as can Dictyostelium amoebae. Suggests that Rho regulates cell division through complex patterns of cortical stabilization.

33. Shaw JA, Mol PC, Bowers B, Silverman SJ, Valdivieso MH, Durán A, Cabib E: The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle. J Cell Biol 1991, 114:111-123.

53. Beites CL, Xie H, Bowser R, Trimble WS: The septin CDCrel-1 binds • syntaxin and inhibits exocytosis. Nat Neurosci 1999, 2:434-439. Suggests a possible role for septins in membrane addition during animal-cell cytokinesis. The septin CDCrel-1 bound syntaxin and, when overexpressed, inhibited secretion. Raises the possibility that vesicle fusion at the cleavage furrow may be at least in part regulated by septin–syntaxin interactions.

34. DeMarini DJ, Adams AEM, Fares H, Virgilio CD, Valle G, Chuang JS, Pringle JR: A septin-based hierarchy of proteins required for localized deposition of chitin in the Saccharomyces cerevisiae cell wall. J Cell Biol 1997, 139:75-93.

54. Hsu S-C, Hazuka CD, Roth R, Foletti DL, Heuser J, Scheller RH: Subunit composition, protein interactions, and structures of the mammalian brain sec6/8 complex and septin filaments. Neuron 1998, 20:1111-1122.

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55. Fankhauser C, Reymond A, Cerutti L, Utzig S, Hofmann K, Simanis V: The S. pombe cdc15 gene is a key element in the reorganization of F-actin at mitosis. Cell 1995, 82:435-444. 56. Demeter J, Sazer S: Imp2, a new component of the actin ring in the fission yeast Schizosaccharomyces pombe. J Cell Biol 1998, 143:415-427. 57.

Spencer S, Dowbenko D, Cheng J, Li W, Brush J, Utzig S, Simanis V, Lasky LA: PSTPIP: a tyrosine phosphorylated cleavage furrowassociated protein that is a substrate for a PEST tyrosine phosphatase. J Cell Biol 1997, 138:845-860.

58. Kamei T, Tanaka K, Hihara T, Umikawa M, Imamura H, Kikyo M, Ozaki K, Takai Y: Interaction of Bnr1p with a novel Src homology 3 domaincontaining Hof1p. Implication in cytokinesis in Saccharomyces cerevisiae. J Biol Chem 1998, 273:28341-28345. 59. Lippincott J, Li R: Dual function of Cyk2, a cdc15/PSTPIP family protein, in regulating actomyosin ring dynamics and septin distribution. J Cell Biol 1998, 143:1947-1960. 60. Balasubramanian MK, McCollum D, Chang L, Wong KCY, Naqvi NI, He X, Sazer S, Gould KL: Isolation and characterization of new fission yeast cytokinesis mutants. Genetics 1998, 149:1265-1275. 61. Wasserman S: FH proteins as cytoskeletal organizers. Trends Cell Biol 1998, 8:111-115. 62. Swan KA, Severson AF, Carter JC, Martin PR, Schnabel H, Schnabel R, Bowerman B: cyk-1: a C. elegans FH gene required for a late step in embryonic cytokinesis. J Cell Sci 1998, 111:2017-2027. 63. Angers-Loustau A, Côté J-F, Charest A, Dowbenko D, Spencer S, Lasky LA, Tremblay ML: Protein tyrosine phosphatase-PEST regulates focal adhesion disassembly, migration, and cytokinesis in fibroblasts. J Cell Biol 1999, 144:1019-1031. 64. Adams RR, Tavares AAM, Salzberg A, Bellen HJ, Glover DM: pavarotti encodes a kinesin-like protein required to organize the central spindle and contractile ring for cytokinesis. Genes Dev 1998, 12:1483-1494. 65. Mackay AM, Ainsztein AM, Eckley DM, Earnshaw WC: A dominant mutant of inner centromere protein (INCENP), a chromosomal protein, disrupts prometaphase congression and cytokinesis. J Cell Biol 1998, 140:991-1002. 66. Powers J, Bossinger O, Rose D, Strome S, Saxton W: A nematode • kinesin required for cleavage furrow advancement. Curr Biol 1998, 8:1133-1136. These three papers [66•–68•] examine the roles of two C. elegans proteins that localize to the spindle midzone, ZEN-4/MKLP-1 and AIR-2, both of which are dispensable for early furrow formation (and thus cleavage plane specification) but required for continued furrow ingression. 67. •

Raich WB, Moran AN, Rothman JH, Hardin J: Cytokinesis and midzone microtubule organization in Caenorhabditis elegans require the kinesin-like protein ZEN-4. Mol Biol Cell 1998, 9:2037-2049. See annotation [66•]. 68. Schumacher JM, Golden A, Donovan PJ: AIR-2: an Aurora/Ipl1• related protein kinase associated with chromosomes and midbody microtubules is required for polar body extrusion and cytokinesis in Caenorhabditis elegans embryos. J Cell Biol 1998, 143:1635-1646. See annotation [66•]. 69. Oegema K, Mitchison TJ: Rappaport rules: cleavage furrow induction in animal cells. Proc Natl Acad Sci USA 1997, 94:4817-4820. 70. Rappaport R: Experiments concerning the cleavage stimulus in sand dollar eggs. J Exp Zool 1961, 148:81-89. 71. Savoian MS, Earnshaw WC, Khodjakov A, Rieder CL: Cleavage • furrows formed between centrosomes lacking an intervening spindle and chromosomes contain microtubule bundles, INCENP, and CHO1 but not CENP-E. Mol Biol Cell 1999, 10:297-311. Reports the correlation of ectopic furrow formation between asters of different spindles with the presence of interastral microtubules associated with spindle midzone components. 72. Wheatley SP, O’Connell CB, Wang Y: Inhibition of chromosomal • separation provides insights into cleavage furrow stimulation in cultured epithelial cells. Mol Biol Cell 1998, 9:2173-2184. Further evidence for the role of the spindle midzone in cleavage plane specification is provided. Misplaced spindle midzone arrays (induced by topoisomerase inhibitors) are shown to correlate positionally with misplaced furrow formation.

73. Carmena M, Riparbelli MG, Minestrini G, Tavares AM, Adams R, • Callaini G, Glover DM: Drosophila Polo kinase is required for cytokinesis. J Cell Biol 1998, 143:659-671. Genetic analysis of the Drosophila Polo kinase was performed, indicating a role for Polo in septin localization to the cleavage plane and assembly of the contractile ring in spermatocytes. These results could explain why Pav-KLP mutants (which fail to localize Polo) also fail to localize septins and contractile ring components [64]. 74. Karki S, LaMonte B, Holzbaur ELF: Characterization of the p22 subunit of dynactin reveals the localization of cytoplasmic dynein and dynactin to the midbody of dividing cells. J Cell Biol 1998, 142:1023-1034. 75. Skop AR, White JG: The dynactin complex is required for cleavage plane specification in early Caenorhabditis elegans embryos. Curr Biol 1998, 8:1110-1116. 76. Berrueta L, Tirnauer JS, Schuyler SC, Pellman D, Bierer BE: The APC-associated protein EB1 associates with components of the dynactin complex and cytoplasmic dynein intermediate chain. Curr Biol 1999, 9:425-428. 77.

Schwartz K, Richards K, Botstein D: BIM1 encodes a microtubulebinding protein in yeast. Mol Biol Cell 1997, 8:2677-2691.

78. Muhua L, Adames NR, Murphy MD, Shields CR, Cooper JA: • A cytokinesis checkpoint requiring the yeast homologue of an APC-binding protein. Nature 1998, 393:487-491. Bim1p, the S. cerevisiae homologue of the human EB1 protein, is required for a cytokinesis delay in cells that have misoriented spindles due to a lack of dynein. 79. Giansanti MG, Bonaccorsi S, Williams B, Williams EV, Santolamazza C, Goldberg ML, Gatti M: Cooperative interactions between the central spindle and the contractile ring during Drosophila cytokinesis. Genes Dev 1998, 12:396-410. 80. Cimini D, Fioravanti D, Tanzarella C, Degrassi F: Simultaneous inhibition of contractile ring and central spindle formation in mammalian cells treated with cytochalasin B. Chromosoma 1998, 107:479-485. 81. 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 Biol 1995, 129:709-723. 82. Bähler J, Steever AB, Wheatley S, Wang Y, Pringle JR, Gould KL, • McCollum D: Role of polo kinase and Mid1p in determining the site of cell division in fission yeast. J Cell Biol 1998, 143:1603-1616. This work shows that the S. pombe Polo kinase Plo1p helps to regulate cleavage plane specification by promoting phosphorylation and subsequent nuclear exit of Mid1p during mitosis. Mid1p forms a cortical ring at the cell middle and influences placement and organization of the medial ring. 83. Schmidt S, Sohrmann M, Hofmann K, Woollard A, Simanis V: The Spg1p GTPase is an essential, dosage-dependent inducer of septum formation in Schizosaccharomyces pombe. Genes Dev 1997, 11:1519-1534. 84. Sohrmann M, Schmidt S, Hagan I, Simanis V: Asymmetric segregation on spindle poles of the Schizosaccharomyces pombe septum-inducing protein kinase Cdc7p. Genes Dev 1998, 12:84-94. 85. Furge KA, Wong K, Armstrong J, Balasubramanian MK, Albright CF: • Byr4 and Cdc16 form a two-component GTPase-activating protein for the Spg1 GTPase that controls septation in fission yeast. Curr Biol 1998, 8:947-954. For the initiation of cytokinesis to occur in fission yeast, the Spg1p GTPase must hydrolyze GTP and release the kinase Cdc7p from one of the two spindle pole bodies. These papers [85•,86•] show that GTP hydrolysis by Spg1p specifically requires two proteins, Cdc16p and Byr4, which together have GAP activity and which are localized asymmetrically to the spindle pole bodies. 86. Cerutti L, Simanis V: Asymmetry of the spindle pole bodies and • Spg1p GAP segregation during mitosis in fission yeast. J Cell Sci 1999, 112:2313-2321. See annotation [85•]. 87. •

Sparks CA, Morphew M, McCollum D: Sid2p, a spindle pole body kinase that regulates the onset of cytokinesis. J Cell Biol 1999, 146:777-790. This paper reports the identification of a kinase, Sid2p, that is a downstream component of the pathway that initiates septation in S. pombe. In response to signals from the other septation initiation genes, Sid2p moves from the spindle pole body to the cell division site at the time of medial ring constriction and septum formation; in addition, the kinase activity of Sid2p peaks at this time.

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interacts genetically with rho1 but not with other rho family members. These results suggest that Rho1, via an unknown effector, specifically functions in the initiation of cytokinesis.

89. Osman MA, Cerione RA: Iqg1p, a yeast homologue of the mammalian IQGAPs, mediates Cdc42p effects on the actin cytoskeleton. J Cell Biol 1998, 142:443-455.

98. Somlyo AP, Somlyo AV: Signal transduction and regulation in smooth muscle. Nature 1994, 372:231-236.

90. Kishi K, Sasaki T, Kuroda S, Itoh T, Takai Y: Regulation of cytoplasmic division of Xenopus embryo by rho p21 and its inhibitory GDP/GTP exhange protein (rho GDI). J Cell Biol 1993, 120:1187-1195. 91. Mabuchi I, Hamaguchi Y, Fujimoto H, Morii N, Mishima M, Narumiya S: A rho-like protein is involved in the organisation of the contractile ring in dividing sand dollar eggs. Zygote 1993, 1:325-331. 92. Hall A: Rho GTPases and the actin cytoskeleton. Science 1998, 279:509-514. 93. Aspenström P: Effectors for the Rho GTPases. Curr Opin Cell Biol 1999, 11:95-102. 94. Madaule P, Eda M, Watanabe N, Fujisawa K, Matsuoka T, Bito H, Ishizaki T, Narumiya S: Role of citron kinase as a target of the small GTPase Rho in cytokinesis. Nature 1998, 394:491-494. 95. Kosako H, Goto H, Yanagida M, Matsuzawa K, Fujita M, Tomono Y, Okigaki T, Odai H, Kaibuchi K, Inagaki M: Specific accumulation of Rho-associated kinase at the cleavage furrow during cytokinesis: cleavage furrow-specific phosphorylation of intermediate filaments. Oncogene 1999, 18:2783-2788. 96. Yasui Y, Amano M, Nagata K, Inagaki N, Nakamura H, Saya H, Kaibuchi K, Inagaki M: Roles of Rho-associated kinase in cytokinesis; mutations in Rho-associated kinase phosphorylation sites impair cytokinetic segregation of glial filaments. J Cell Biol 1998, 143:1249-1258. 97. •

Prokopenko SN, Brumby A, O’Keefe L, Prior L, He Y, Saint R, Bellen HJ: A putative exchange factor for Rho1 GTPase is required for initiation of cytokinesis in Drosophila. Genes Dev 1999, 13:2301-2314. The Drosophila gene pbl, which is required for initiation of cytokinesis, encodes a putative Rho1 guanine nucleotide exchange factor. The pbl gene

99. Totsukawa G, Yamakita Y, Yamashiro S, Hosoya H, Hartshorne DJ, • Matsumura F: Activation of myosin phophatase targeting subunit by mitosis-specific phosphorylation. J Cell Biol 1999, 144:735-744. Shows that during mitosis, the myosin regulatory light chain (RMLC) is kept inactive by highly active myosin phosphatase, which prevents accumulation of activating phosphorylation. At the onset of cytokinesis, myosin phosphatase becomes less active, allowing the RMLC to become phospharylated and promote contractile activity. Along with [102•], this paper suggests that regulation of actomyosin contractility during mitosis and cytokinesis is primarily through the control of RMLC phosphorylation on activating sites. 100. Satterwhite LL, Lohka MJ, Wilson KL, Scherson TY, Cisek LJ, Corden JL, Pollard TD: Phosphorylation of myosin-II regulatory light chain by cyclin-p35cdc2: a mechanism for the timing of cytokinesis. J Cell Biol 1992, 118:595-605. 101. Yamakita Y, Yamashiro S, Matsumura F: In vivo phosphorylation of regulatory light chain of myosin II during mitosis of cultured cells. J Cell Biol 1994, 124:129-137. 102. Shuster CB, Burgess DR: Parameters that specify the timing of • cytokinesis. J Cell Biol 1999, 146:981-992. Challenges the traditionally held view that phosphorylation by p34 cdc2 of myosin regulatory light chain (RMLC) on inhibitory sites (Ser1/2) is important during mitosis for delaying the onset of cytokinesis. The authors found no sign of Ser1/2 phosphorylation on cortical RMLC even in the presence of constitutively active p34cdc2. Along with [99•], this paper suggests that regulation of actomyosin contractility during mitosis and cytokinesis is primarily through the control of RMLC phosphorylation on activating sites.