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Microbiology and Molecular Biology Reviews, June 2002, p. 155-178, Vol. 66, No. 2
1092-2172/02/$04.00+0 DOI: 10.1128/MMBR.66.2.155-178.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts 01605
SUMMARY INTRODUCTION POSITIONING THE SITE OF CELL DIVISION Division Plane Specification in Plant Cells Division Plane Specification in Yeast Cells Division Plane Specification in Animal Cells ASSEMBLING DIVISION MACHINERY AND DIVIDING Plants: Constructing the Cell Plate Assembly of Actomyosin Ring in Yeast and Animal Cells Myosin II. Septins. Cdc15-like proteins. IQGAP. Small GTPases. Formins. Order of assembly. Furrow Formation and Spindle Midzone Signals in Animals Chromosomal passenger proteins. Microtubule motor proteins. Interplay between the spindle midzone and contractile ring. Finishing Cytokinesis in Yeast and Animal Cells Models for ring constriction. Membranes, vesicles, and cytokinesis. COORDINATION OF CYTOKINESIS WITH NUCLEAR CYCLE CDK Polo Kinase Signal Transduction Pathways and Timing Cytokinesis SIN. MEN. Signaling cytokinesis in higher eukaryotes. CYTOKINESIS AND DEVELOPMENT: VARIATIONS ON THE COMMON THEME Asymmetric Division Incomplete Cytokinesis Pseudocleavage CONCLUDING REMARKS REFERENCES
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| INTRODUCTION |
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In general, the goal of cytokinesis is common in all organisms: to physically separate a mother cell into two daughter cells. How different organisms conduct cytokinesis varies, but the major events are universal (Fig. 1). In animal cells, the division site is first chosen, generally at the cell equator, and subsequently the cleavage furrow is assembled at the division site (Fig. 1D). The furrow contains actin, myosin, and other proteins that are organized into a contractile ring called the actomyosin ring. The ring then ingresses or contracts, generating a membrane barrier between the cytoplasmic contents of each daughter cell. The ingressing furrow constricts components of the spindle midzone into a focused structure called the midbody. In the final cytokinetic event, called abscission, the furrow "seals," generating two completely separate cells. The overall approach to division is well conserved between the fission yeast Schizosaccharomyces pombe and animals. Fission yeast also positions an actomyosin ring at the cell equator (Fig. 1C). However, unlike animal cells, S. pombe cells synthesize a division septum behind the ring as it constricts, generating new cell wall material between daughter cells. The septum is ultimately degraded by digestive enzymes, physically separating the daughter cells. Unlike animal cells and fission yeast, the yeast Saccharomyces cerevisiae divides by budding (Fig. 1B). First the site to bud is marked, and then through polarized cell growth the bud grows outward from the mother cell cortex, gradually increasing in size. The contractile ring is assembled at the bud neck, and similar to fission yeast, a division septum is synthesized behind the constricting ring. Plants significantly differ from yeast and animals in that they do not use a contractile ring to divide (Fig. 1A). In contrast, plants interdigitate microtubules and actin to build a dense structure called the phragmoplast between divided nuclei. Microtubule-directed vesicles containing cell wall material are targeted to the phragmoplast, and fusion of these vesicles causes the phragmoplast to grow outward toward the cell cortex, forming a cell plate. The cell plate fuses to the parent cell wall, dividing the parent cell into two daughters.
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In the following sections, we review recent studies using several eukaryotic model organisms to study cytokinesis, focusing on mammalian cells, invertebrates, yeast, and higher plants. The major events contributing to cytokinesis—including determining the division site, building the division apparatus and mechanically dividing, and coordinating cytokinesis with the nuclear division cycle—will be discussed. We conclude with a brief description of variations on the common theme of cytokinesis important for development.
| POSITIONING THE SITE OF CELL DIVISION |
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Like most plant and animal cells, S. pombe divides by medial fission to generate two equal-size daughter cells (Fig. 2C). S. pombe cells do not grow equal amounts from each end; therefore, the position of the cell middle changes during cell cycle progression (172). Thus, the mechanism by which cells specify their middle must be dynamic. In S. pombe nuclear position is maintained in the cell middle through opposing pushing forces generated by microtubules (262), leading to a model in which the position of the nucleus determines the division site (38). Consistent with this, the nucleus is frequently mispositioned in tubulin mutants; however, the division site always correlates with the position of the nucleus (38).
How does nuclear position specify the division site? Insight into this has come from analysis of plo1 and mid1 mutants, which are defective in positioning the actomyosin ring (15, 38, 240). In interphase, Mid1p resides primarily in the nucleus and can be seen faintly at the medial cell cortex (15, 202, 240). Mid1p appears to shuttle in and out of the nucleus and is thought to continuously mark the cell cortex proximal to the nucleus (202). Upon entry into mitosis, Mid1p becomes phosphorylated and exits the nucleus in a Plo1p-dependent manner. After exiting the nucleus, Mid1p strongly localizes to the cortex overlying the nucleus, recruiting proteins involved in actomyosin ring assembly such as actin and thus marking the cell division site (15, 240). This pathway may be conserved, as Polo kinases and the Mid1p-related protein anillin are required for cytokinesis in Drosophila melanogaster and human cells (76, 196). Both Mid1p and anillin cycle from the nucleus in interphase to the contractile ring in mitosis. In addition, the two proteins have some structural similarities, including a proline-rich domain and a carboxy-terminal PH domain (76, 240; D. McCollum, unpublished observations). Other proteins involved in actomyosin ring positioning, such as the Pom1-kinase (16) and products of the pos genes (63), have been identified. The mechanism of their action in actomyosin ring positioning is not yet known.
In late mitosis, after chromosome segregation, the central or interzonal region of the mitotic spindle, which is the region between separated chromosomes, undergoes a reorganization in which microtubules bundle into antiparallel interdigitating arrays. For the purpose of this review we refer to this centralized microtubule bundle as the spindle midzone. During furrow contraction, the spindle midzone is compacted into an electron-dense structure called the midbody. After furrowing completes in animal cells, they remain connected through a structure called the cytoplasmic bridge, which contains the condensed spindle midzone microtubules. This structure persists until the bridge "cuts" during abscission.
The spindle midzone has been shown to directly contribute to signaling actin ring assembly and constriction during later stages of mitosis. In one report, placing an artificial barrier between the spindle midzone and cell cortex in cultured cells during metaphase caused cleavage furrow ingression to be inhibited; however, if the barrier was created in early anaphase, cytokinesis was successful (31). This suggested that transient signals from the spindle midzone were required for cleavage site formation. Wheatley and Wang then examined the relationship between microtubules and cleavage site formation in cultured cells that had been fluorescently labeled for tubulin (276). They confirmed that cleavage furrow formation was linked to microtubules of the spindle midzone as opposed to spindle poles and depolymerization of midzone microtubules with a drug resulted in failed cytokinesis. Consistent with these results, it was observed that during male meiosis in D. melanogaster KLP3A mutants (discussed below), mitotic spindles formed normally, but the central spindle midzone failed to form (280) and furrowing was not initiated. Thus, taken at face value, cleavage site positional information seems to be derived from different sources in marine invertebrate embryos compared to cultured cells; however, a closer examination of earlier experiments may reveal more similarities than differences. Since division site selection and furrow ingression occurs virtually simultaneously in animal cells, a more detailed description of the components of the spindle midzone is discussed in the next section.
| ASSEMBLING DIVISION MACHINERY AND DIVIDING |
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The cytoskeletal network of the phragmoplast and the cell plate then migrate outward towards the parent cell wall. The expansion of the phragmoplast is somehow directed towards the site in the parent cell wall that was previously marked by the PPB (see above). This directed process seems to be facilitated by the cytoskeletal network (238, 239).
Further clues to how the centrifugal expansion of the cell plate might be facilitated were found with the recent discovery of the mitogen-activated protein kinase (MAPK) kinase kinase NPK1 in tobacco. Expression of a kinase-dead mutant of NPK1 results in multinucleate cells, suggesting that this MAPK kinase kinase regulates cytokinesis but not the cell cycle. Furthermore, NPK1 localizes to the leading edge of the cell plate in mitosis, and biochemical experiments showed an increase in activity in late mitosis. This makes NPK1 a strong candidate for a part of a kinase pathway regulating cytokinesis in plants (188). In alfalfa, a MAPK MMK3 was observed to localize to the phragmoplast with increased activity in mitosis through anaphase. This together with its activity's being dependent on microtubules makes it likely to be involved in the regulation of cytokinesis (27).
This mechanism for cytokinesis is not rigidly adhered to by all cell types. In the Arabidopsis endosperm, phragmoplast formation is not dependent on a mitotic spindle remnant. The endosperm arises as a syncytium around the embryo early in development, containing many unseparated nuclei. In this case, to generate distinct cells, the phragmoplast needs to form at more locations than just between a pair of daughter nuclei. To accomplish this, microtubules originating from the nuclear periphery of neighboring nuclei interdigitate, forming several miniphragmoplasts, which then give rise to a tubular network that eventually generates a cell plate (201).
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Although actin and myosin probably function together to bring about cytokinesis, recent work suggests that their localization to the furrow is at least partly independent. In budding yeast, the myosin ring assembles at the bud neck early in the cell cycle, well before localization of actin polymers, which localize to the bud neck at the end of anaphase (23, 140). In S. pombe, myosin can localize to the cell middle in the absence of filamentous actin polymers but cannot organize into a contractile ring (178, 184). Moreover, in both S. cerevisiae and S. pombe, myosin ring localization is not affected by actin depolymerization once the actomyosin ring has formed (23, 184). Consistent with myosin localization being somewhat independent of actin, the actin binding motor domain of myosin is not essential for myosin localization to the ring (184, 290, 291). In D. discoideum, phosphorylation of the Myo2 tail is important for ring localization (56); however, tail binding proteins that mediate ring localization have not been identified.
Some controversy has existed over the years as to the role of the ELC and RLC for myosin function. In D. discoideum both myosin light chains are essential (42, 53); however, cells expressing Myo2 in which the RLC binding site has been deleted perform normal cytokinesis (266). One possibility is that the RLC is not important for myosin function but has a separate essential function in cytokinesis. However, recent work with S. pombe suggests this is not the case (183). S. pombe cells from which the S. pombe RLC, Rlc1p, has been deleted have severe cytokinesis defects (132, 183). Expression of a myosin heavy chain in which the Rlc1p binding domain has been deleted rescues the cytokinesis defects of rlc1 deletion mutants (183). These results suggest that RLCs function to relieve auto-inhibition of Myo2p. Phosphorylation of the RLC may be essential for regulating myosin activity coordinately with progression through mitosis (see below) (218, 254).
Septins. Septins were first discovered in budding yeast, where they localize to the bud neck and are required for cytokinesis, bud morphogenesis, and chitin deposition (3; for a review, see references 74 and 149). Septins contain a GTPase domain, the significance of which is unclear; can form filaments in vitro (75); and may associate with the plasma membrane through binding to phospholipids (292). The Drosophila septin peanut localizes to the cleavage furrow, and peanut mutants exhibit cytokinetic defects (185). Also, injection of antibodies against the mammalian septin Nedd5 into cultured cells blocks cytokinesis (114). Despite these results, septins do not appear to be absolutely required for cytokinesis. In Drosophila peanut mutants, some cells are able to complete cytokinesis. However, in these mutants, another Drosophila septin, Sep2, localized normally, suggesting possible redundancy among septins (2). In Caenorhabditis elegans there are only two septin genes, unc-59 and unc-61. Both unc-59 and unc-61 are required for normal postembryonic cytokinesis and morphogenesis but have no essential function in embryogenesis (186). Similarly, S. pombe septin mutants have only minor defects in cytokinesis (149).
What is the role of septins in cytokinesis? In S. cerevisiae, septins seem to function as scaffold proteins for assembly of other ring components (57). Septins have also been identified in complexes with proteins involved in secretion, including the exocyst complex (100) and SNARE proteins (21), suggesting a possible role in directing polarized secretion.
Cdc15-like proteins. Cdc15p was the founding member of a family of SH3-domain containing proteins first identified in S. pombe (71). Both Cdc15p and a related S. pombe protein, Imp2p, localize to the actomyosin contractile ring (58, 71). cdc15 mutants can form an actomyosin contractile ring, but the rings are unstable and fail to recruit actin polymers, resulting in cytokinesis failure (18). Two related proteins exist in mammalian cells, PSTPIP and PSTPIP2, both of which localize to the cleavage furrow (247, 281). It is not yet known if these proteins are required for cytokinesis. The Cdc15p-related protein Cyk2/Hof1p in S. cerevisiae is also involved in cytokinesis (111, 141). Cyk2p/Hof1p forms a double ring coincidently with septin ring formation early in the cell cycle, and this becomes a single ring colocalizing with the actin ring just prior to contracting (141, 267).
IQGAP. IQGAP family proteins contain a number of different functional domains, including an actin binding domain termed the calponin homology domain, IQ domains, and a GTPase activation (GTPase-activating protein [GAP]) domain (96, 126). The Dictyostelium IQGAP-related protein, GAPA, as well as the S. pombe and S. cerevisiae IQGAP proteins Rng2p and Cyk1p/Iqg1p are required for cytokinesis (1, 64, 65, 140, 199). In S. pombe, cells from which the IQGAP homologue rng2 has been deleted form a spot-like structure containing actin in the medial region of the cell but are unable to form actin rings (64). Rng2p localizes to the actomyosin ring and spindle pole body (SPB) of interphase and mitotic cells. Furthermore, Rng2p interacts with calmodulin, a component of the SPB and actomyosin ring. S. cerevisiae IQGAP Cyk1/Iqg1 also localizes to the actomyosin ring in late anaphase (65, 140, 199) and is required to recruit actin through its calponin homology domain (140, 226).
Both S. pombe and S. cerevisiae IQGAP proteins interact with calmodulin, presumably through the IQ motifs. In S. cerevisiae the IQ motifs are required for localization of Cyk1p/Iqg1p to the bud neck (64, 226) and bind a calmodulin-related protein, Mlc1p (28, 227). Mlc1p serves as a light chain for the type V myosin Myo2p (249), and the type II myosin Myo1p (28). Mlc1p localizes to the bud neck prior to Cyk1p/Iqg1p and is required to recruit Cyk1p/Iqg1p to the bud neck (28, 227). Mammalian myosin ELC has been shown to bind to IQGAP1, suggesting that light chain binding may be a conserved property of IQGAP proteins (274). Deletion of the carboxyl-terminal GAP-related domain of Cyk1p/Iqg1p does not affect localization or actin recruitment to the ring but prevents actomyosin ring contraction. In vitro binding experiments show that Cyk1p/Iqg1p binds to Tem1p GTPase (226). This result is quite interesting since Tem1p is a component of the mitotic exit network (MEN), which seems to be required for initiation of actin ring constriction at the end of anaphase (see below).
Small GTPases. Several small GTPases of the Rho, Rac, and Cdc42 families are required for cytokinesis in animal cells (207). GTPases are generally considered molecular switches that cycle between active (GTP-bound) and inactive (GDP-bound) states in response to specific cellular cues. GTP-bound GTPases are potent activators of intracellular signaling networks. GDP-GTP cycling is regulated by guanine nucleotide exchange factors (GEFs) and GAPs, which catalyze the activation and deactivation of GTPases, respectively.
Small GTPases of the Rho family are most prominent in carrying out essential functions of cytokinesis. Inactivation of Rho GTPase in animal cells inhibits cytokinesis by disrupting normal assembly of actin filaments and triggering disassembly of the contractile ring (8, 60, 115, 127, 154, 173, 193). Mutants in the Dictyostelium rac protein racE are also defective in cytokinesis (127). Rho GTPase is localized to the cleavage furrow and midbody during cytokinesis (60, 189, 253). Regulators of Rho GTPases have also been shown to be important for cytokinesis. Two RhoGEFs required for cytokinesis, human ECT2 and Drosophila pebble, have been identified (208, 257). Both localize to the spindle midzone during cytokinesis. A Rho GAP required for cytokinesis, CYK-4, has been identified in C. elegans (102). CYK-4 also localizes to the spindle midzone. CYK-4 and the kinesin-like protein ZEN-4 show a mutual dependence for localization, suggesting these two central spindle midzone proteins may cooperate in executing cytokinesis.
There are several known effectors of Rho GTPases, some of which have been implicated in regulating cytokinesis. Of those required for cytokinesis, three include protein kinases: the Dictyostelium p21-activated serine/threonine kinase a (46), bovine Rho-associated kinase (122, 285), and murine citron kinase (157), all of which are implicated in various aspects of assembling and contraction of the actomyosin ring. Rho appears to be crucial for localization of citron kinase to the cleavage furrow and midbody (62).
Formins. The most compelling evidence for a downstream effector of Rho GTPases is that for formin homology (FH) proteins. Formins comprise a large family of proteins—including diaphanous of Drosophila (34), Bni1p of S. cerevisiae (67, 120), Cdc12p of S. pombe (39), and sepA of Aspergillus spp. (95)—and are involved in cell polarity and cytokinesis. Formins bind the small GTPases Rho and Cdc42 but also numerous other proteins, including actin; the actin-associated proteins profilin, Bud6p (Aip3p), and Spa2p; and the Cdc15-like protein Hof1p (39, 67, 82, 101, 111, 120, 273). Because of their ability to bind numerous components of the actomyosin contractile ring, it has been proposed that formins serve as scaffolds to organize macromolecular protein complexes involved in actomyosin ring formation (39). Consistent with a role in cytokinesis, formins localize to the actomyosin contractile ring (7, 39, 111). Moreover, S. pombe Cdc12p is essential for actomyosin ring formation. In C. elegans, the cyk-1 gene encodes an FH protein required for a late step in cytokinesis, as cleavage furrows ingress normally in cyk-1 mutant embryos and regress once mitosis completes (252). CYK-1 localizes to the cleavage furrow subsequent to initiation of furrow ingression, supportive of a role in latter stages of cytokinesis. S. cerevisiae bni1 mutants have a mild cytokinesis defect (67, 120); however, Bni1p also appears to be involved in spindle orientation (83, 130, 169, 286), which helps ensure that cytokinesis is spatially coordinated with mitosis.
Order of assembly. The study of cleavage furrow formation has consisted largely of characterization of new proteins that either localize to the furrow and/or are required for assembly of the furrow. Because an enormous number of proteins localize to the cleavage furrow simultaneously, determining their order of function and interdependence has been a daunting task. A number of factors have made S. cerevisiae a useful organism for these types of studies. First, this yeast is useful because the bud neck forms early and assembly of actomyosin ring components is spread over much of the cell cycle. Second, unlike in other organisms, the actomyosin ring is not essential, allowing easier study of ring mutants. Recent studies indicate that myosin ring formation depends on septins (23, 140) and that the myosin ring can form without filamentous actin but does not constrict without actin (23). The multifunctional myosin light chain Mlc1p depends on septins for localization to the bud neck, and it in turn is required for recruitment of the IQGAP protein Iqg1p/Cyk1p (28, 227). Iqg1p/Cyk1p is essential for formation of the actin ring (140, 226). Although these types of studies are still at an early stage they appear quite promising. It will be of interest to see if the dependency relationships in ring formation identified in S. cerevisiae are conserved in other organisms.
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The first chromosomal passenger protein identified was INCENP (49). Because of its localization to metaphase centromeres and then to the central spindle and midbody, it was initially thought that such passenger proteins marked the cleavage furrow position so that it would bisect segregated chromosomes. A role for INCENP in cytokinesis eluded researchers for some time until dominant negative forms of INCENP which caused cells to fail cytokinesis were identified (61, 156). Additionally, both RNA interference experiments with flies and worms and knockout experiments with mice have revealed that INCENP is essential for cytokinesis (4, 110, 156).
A second chromosomal passenger protein involved in cytokinesis is the Aurora-B/Ipl1 kinase. Aurora-B/Ipl1-related kinases are part of a large family of serine/threonine kinases important for regulating chromosome segregation and cytokinesis (24). Aurora-B/Ipl1 proteins in C. elegans (AIR-2), Drosophila (Aurora-B), and humans (AIM-1) are all required for cytokinesis (88, 162, 221, 256, 265). Importantly, however, the target(s) of Aurora kinases at the spindle midzone is unknown.
A third family of chromosomal passenger proteins implicated in cytokinesis is the family of Survivin-like proteins (233). Survivin was originally identified in baculovirus as an inhibitor of apoptosis protein that associates with mitotic spindles (10, 135). Later work showed that a C. elegans Survivin-like protein, BIR-1, was required for cytokinesis (80). This defect could partially be rescued by transgenic expression of a mammalian homolog. In addition, mouse null embryos indicated that Survivin was required for mitosis during development, further suggesting that Survivin function is conserved (264). More recently, Survivin localization was shown to mirror that of chromosomal passenger proteins (237, 246, 264).
Based on the similar localization and mutant phenotypes of the chromosomal passenger proteins, it was hypothesized that these proteins may function as part of a complex (Fig. 4). Recent reports have now uncovered that chromosomal passengers do appear to function as a complex in cells. INCENP directly interacts with Aurora-related kinases from Xenopus laevis (XAIRK2), C. elegans (AIR-2), and humans (AIRK2) (5, 110). In addition, a dominant negative INCENP mutant disrupted AIRK2 localization during mitosis in HeLa cells, further suggesting that this interaction is conserved and essential in vivo (5). In C. elegans, inactivation of Survivin protein prevented the proper localization of the aurora kinase AIR-2 (246). In a recent report, human Survivin was shown to interact directly with both Aurora-B and INCENP by yeast two-hybrid and in vitro pull down assays, and loss of INCENP localization disrupted the ability of Survivin to properly localize (277). Thus, to date, there is biochemical evidence for at least a tripartite chromosomal passenger complex at the spindle midzone.
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Microtubule motor proteins. The major motor proteins associated with the spindle midzone are the microtubule motor protein kinesins, specifically, those of the CHO1/MKLP1 and KLP3A families. Both are required for mitotic progression, localize along spindles during metaphase, and concentrate in the spindle midzone region during anaphase (191, 192, 224, 280). Bacterially expressed CHO1 can bundle antiparallel microtubules in vitro and, in the presence of Mg-ATP, can cause sliding of antiparallel microtubules (192). Mutants in the Drosophila CHO1/MKLP1 homolog, pavarotti, or Klp3A resulted in normal anaphase but cytokinesis failure due to disruption in the structure of the spindle midzone and contractile ring assembly (6, 280). The furrow proteins peanut, actin, and anillin do not localize in pavarotti mutants. The failure in contractile ring formation when the spindle midzone is disrupted indicates that the midzone plays a key role in furrow assembly in Drosophila. This may result from a failure to transport furrow components along microtubules to the cleavage site. Consistent with this, it has been shown that in mammalian cells, preexisting actin filaments get transported to the furrow (32).
Cytokinesis is also defective in Drosophila polo mutants, where Polo is required for the correct localization of pavarotti (which is mutually required for Polo localization) and the septin peanut and for normal formation of the actomyosin ring (6, 33). Polo is a protein kinase localized to interzone microtubules during anaphase and to the midbody during late cytokinesis, where it colocalizes with the kinesin-like protein CHO1/MKLP1 (129). In S. pombe, disruption of the Polo homolog Plo1 results in a population of cells defective in actocyosin ring formation, suggesting that Polo kinase may play a direct role in this process (198).
In C. elegans embryos lacking the CHO1/MKLP1 homolog ZEN-4, cells continue through mitosis but fail to undergo normal cytokinesis, resulting in multinucleate embryos (206, 209). Close inspection of these cells revealed that furrow ingression initiates normally but fails and regresses prior to completion and is preceded by disruption of spindle midzone microtubules. Thus, in C. elegans, the spindle midzone is required not for furrow formation but for completion of cytokinesis, suggesting a second function for the spindle midzone.
What then is the function of spindle midzone motor proteins? One possibility is that they provide structural support for interdigitating microtubules at the midzone region, which may provide a platform to localize furrow components to the cell middle. Another alternative is that motors help deliver the necessary molecules to build the cleavage furrow. Along these lines, the spindle midzone may be necessary for vesicle delivery required to complete cytokinesis, much like the phragmoplast in plants. These models are probably not mutually exclusive; however, proof awaits future studies.
Interplay between the spindle midzone and contractile ring. Is there a mutual dependency between the spindle midzone and contractile ring formation? Two models have emerged from genetic experiments with C. elegans and Drosophila. Work with Drosophila suggests that a mutual dependent relationship may exist, while studies with C. elegans argue that they are genetically separable entities.
In Drosophila, a cooperative interdependence may exist in which if either the spindle midzone or contractile ring is disrupted, the other fails to form properly (85). The Drosophila mutants chickadee (chic), diaphanous (dia), spaghetti squash (sqh), KLP3A, and polo (33, 86, 97, 280) all have defects in formation of both the spindle midzone and contractile ring. The chic gene encodes the actin binding protein profilin, dia encodes a formin homology protein (discussed above), and squ encodes a myosin II RLC (34, 50, 86, 112). Since they are all furrow components, mutants in these genes probably lead to defects in the contractile ring. As described above, the products of the KLP3A and polo genes localize and appear to function at the spindle midzone. Therefore, mutants in these genes probably lead to defects in midzone formation. The fact that all cytokinetic mutants identified in Drosophila to date are either defective for both spindle midzone and contractile ring formation, or for neither, has led to a model in which a cooperative interdependence exists between these two structures. One speculative idea is that the spindle midzone may promote assembly of furrow components, and in turn, the furrow may stabilize bundled ends of midzone microtubules. In support of such a model, KLP3A was found to bind both actin and mitcrotubules (235).
Using C. elegans as a genetic model, Severson et al. observed that ZEN-4/MKLP localization was normal in CYK-1 mutant embryos (CYK-1 is a formin which localizes to the cleavage furrow and is required for cytokinesis [see above]), and CYK-1 localization to the leading edge of the furrow was normal in ZEN-4 mutant embryos (225). In addition, genetic experiments indicated that air-2 zen-4 double mutants were equally as defective as single mutants, in that cleavage furrows ingressed and then failed. In contrast, air-2 or zen-4 mutants in combination with a cyk-1 mutation were more defective, in that furrows even failed to ingress. These data suggest that the spindle midzone proteins AIR-2 and ZEN-4 are genetically separable from the ring protein CYK-1 and may function in separate pathways required for cytokinesis. Moreover, it appears that formation of the contractile ring in C. elegans does not require an intact central spindle and organization of the central spindle does not require an intact contractile ring. Functionally, one model is that CYK-1 contributes to contractile ring dynamics, while AIR-2 and ZEN-4 may be involved in membrane secretion.
Recent experiments have begun to reveal the mechanics behind cytokinesis (214). Using atomic force microscopy, Matzke et al. were able to record mechanical measurements of the cell cortex during the cell cycle and cytokinesis in mammalian cell cultures (163). It was observed that global "stiffness" of the cell cortex was higher in metaphase compared to interphase. Global stiffness increased until anaphase onset, and then the equatorial region where the cleavage furrow was to form began to show a higher degree of stiffness than background global stiffness. During furrowing, equatorial stiffness reached up to 10-fold higher than background stiffness, probably as a result of cytoskeletal and associated components concentrating and contracting at the cleavage site. Such an observation indicates that forces generated by cytokinesis are not distributed across the entire cell cortex, perhaps ensuring that furrowing remains concentrated at the cell middle.
In another approach to understanding how cortical regions of cells contribute to cytokinesis, O'Connell et al. applied localized cytochalasin D treatment to disrupt actin-based cellular functions in mammalian cells (194). When applied to the equatorial region, cytokinesis seemed to be facilitated, probably as a result of weakening the equatorial stiffness. In contrast, when applied between the spindle pole and distal end of cells, cytokinesis was inhibited, indicating that polar cortices somehow "communicate" with the equatorial cortex during cytokinesis. In a reciprocal experiment, cells were treated with the drug jasplakinolide, which stabilizes actin filaments. When this drug was applied to the equatorial region, cytokinesis was inhibited; however, cytokinesis was unaffected when it was applied to polar regions. While Matzke et al. indicate that the cortex is not elastic and focuses cortical stiffness to the cleavage furrow, O'Connell et al. emphasize that the equatorial region alone is not sufficient to complete cytokinesis, but rather division involves coordination of the entire cell cortex. Future studies comparing such high-resolution mechanical measuring with genetic and biochemical studies will be crucial in fully characterizing the interface between the mechanical forces of cleavage and the molecules that generate them.
Yeast cell and cells of the filamentous fungus Aspergillus nidulans are different from animal cells in that a septum forms centripetally through the deposition of cell wall material behind the constricting actomyosin ring. Studies with S. pombe suggest that the actomyosin ring requires septum formation for its ability to constrict (107, 145). One explanation for the difference between yeast and animal cells is that in yeast, the contractile force of the ring may not be sufficient to counteract the turgor pressure of the cell without cell wall formation. Perhaps the primary purpose of the ring is to localize and guide septum formation. This is consistent with the fact that the actomyosin ring is not essential in S. cerevisiae, possibly because the bud neck is quite narrow and an actomyosin ring is not essential to guide septum formation.
Membranes, vesicles, and cytokinesis. Based on early morphological studies of plants, it seemed clear that vesicle fusion and secretion might play a role in plant cytokinesis; however, the idea that cytokinesis in animal cells might also require secretion has only recently gained wide acceptance. It was thought that actomyosin-based constriction of the ring is sufficient to pinch a cell in two and there is no need to add new membrane to get the process to work. Nonetheless, it seems reasonable to suggest that membrane addition could contribute to cytokinesis. First, addition of new membrane to the furrow may facilitate constriction of the ring by reducing the force necessary for ring constriction. The ring would be required not simply to pull in the plasma membrane but to guide the direction of furrowing, as in yeast cells. Second, secretion to the furrow could be important for delivery of membrane proteins specifically to the furrow to facilitate cytokinesis. Third, vesicle delivery could play a role in the final stage of membrane fusion at the end of cytokinesis, as in plant cells.
Early evidence of a role for secretion in animal cell cytokinesis came from pioneering work using cleaving Xenopus embryos. These early studies suggested that the cleavage furrow is formed by fusion of new Golgi-derived membrane near the leading edge of the furrow (26, 30, 234). Later studies suggested that this vesicle delivery depended on a specialized array of microtubules at the cleavage furrow termed the furrow microtubule array (FMA) (Fig. 3B). Using confocal microscopy on dividing Xenopus eggs, Danilchik et al. observed the FMA as a V-shaped array of microtubules emanating from a point just ahead of the advancing furrow that seems to form from spindle midzone microtubules (54). Appearance of this array is coincident with furrow ingression and new membrane insertion and is suggestive of an active role for microtubules in membrane insertion during furrowing. In fact, using depolymerizing drugs, it was shown that disrupting the microtubule cytoskeleton blocked new membrane insertion and cytokinesis while disrupting the actin cytoskeleton neither blocked new membrane assembly nor displaced the FMA but did block furrow ingression (54, 219). It also should be noted that once these embryos reached the midblastula stage, FMAs disappeared and were replaced by midbody-like structures similar to those found in somatic cells, suggesting this specialized structure for membrane delivery may only be needed for cytokinesis in large cells.
FMAs are also found associated with furrows in dividing blastomere stage zebra fish embryos (105). These studies indicated that secretion to the furrow may be important for delivery of membrane proteins specifically to the furrow (after cytokinesis in early embryonic cells, daughter cells do not move apart but rather tightly adhere in a cadherin-dependent process called cohesion). These data suggest that during furrowing, FMAs deliver specific cargoes to the cleavage site, probably as membrane vesicles. In support of this, labeling of membranes in dividing blastomeres indicated that new membrane accumulates near furrows in a microtubule-dependent manner (105). Genetic evidence is also consistent with this hypothesis. In a mutant screen for zebra fish maternal effect mutations, the gene nebel was identified as having cell adhesion defects (203). nebel mutants have a defective FMA and do not effectively localize ß-catenin or concanavalin A (another cleavage plane marker) to the cleavage site; however, other microtubule structures appear normal. Initiation of furrowing occurs in nebel mutants, but blastomeres fail to adhere, and in some cases the furrows regress. This suggests that target delivery of cell adhesion molecules may allow for dividing cells to be adhered behind the constricting furrow, lessening the force required for constriction and thereby promoting cytokinesis. This mechanism seems similar to the targeted delivery of cell wall-synthesizing enzymes in yeast, without which the actomyosin ring is unable to constrict (145).
Several motor proteins, both plus and minus end directed, localize to the spindle midzone region (discussed above). It will be interesting in future studies to determine what motors use these specialized FMAs and what cargoes they transport. It is also not clear whether FMAs are a universal feature of dividing cells or specific to early embryos. Recently Skop et al. inhibited secretion in early C. elegans embryos with a drug and found that the spindle midzone appeared intact and furrow ingression initiated normally, but furrows soon regressed, resulting in failed cytokinesis (236). Using a membrane probe, it was also observed that membrane accumulation occurred at the apices of late cleavage furrows, which form the intracellular bridge (236). The authors propose that the final stage of cell separation in animal cells may be quite similar to cytokinesis in plants, with the spindle midzone or midbody serving a function similar to that of the phragmoplast in plants.
One thing is clear: microtubule networks are important for targeted vesicle delivery to the furrow. Once there, vesicles need to be directed to fuse to appropriate membranes. The first definitive hint that targeted vesicle fusion machinery contributed to cytokinesis came from the identification and characterization of the SNARE family member KNOLLE (syntaxin) and KEULE (s-1) genes in plants (discussed above). Recently, genetic studies have suggested a role for vesicle fusion proteins in cytokinesis in animal cells. In Drosophila, syntaxin1 is required for a specialized form of cell division in the early embryo called cellularization (29). In the Drosophila early blastoderm, a syncytium of about 6,000 nuclei is located in the peripheral cytoplasm. During interphase of cycle 14, membrane vesicles that were concentrated in the periplasm are redistributed to align between the nuclei (148). These vesicles fuse to form double membranes, which continue to recruit additional vesicles, ultimately building a membrane that encircles each nucleus. During cellularization, syntaxin1 protein is localized to newly forming cell surface and cleavage furrows, and lack of syntaxin1 causes cellularization to fail, generating large acellular patches (29). Also in C. elegans it was discovered that disruption of the syntaxin syn-4 (103) or rab-11 (236) caused defects in early embryonic cleavage divisions.
Another family of proteins that may be important for vesicle fusion during cytokinesis is the septin family (74, 113). As described earlier, septins are conserved in most eukaryotic organisms with the exception of plants and function in regulating cell morphological changes. How might septins function in vesicle fusion during cytokinesis? Two reports of septin-interacting proteins have shed light on this question. Using coimmunoprecipitation experiments, Hsu et al. found that septins associated with the exocyst complex, which is required in yeast for transport of Golgi-derived vesicles (77, 94, 100). The exocyst is a large complex that functions in vesicle fusion and is thought to help target vesicles to specific sites at the plasma membrane (100). A second septin-interacting protein was identified as the SNARE protein syntaxin. Two septins, CDCrel-1 and Nedd5, were found to physically associate with syntaxin in a complex (21). Overexpression of CDCrel-1 inhibits exocytosis, while overexpression of a dominant negative mutant can enhance exocytosis, an effect that can be alleviated by coincidently preventing SNARE complex formation. These reports suggest that septins may function in both targeting of membrane vesicles through interactions with the exocyst complex and mediating membrane fusion in conjunction with SNARE proteins.
| COORDINATION OF CYTOKINESIS WITH NUCLEAR CYCLE |
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Another potential role for CDK activity is to inhibit myosin through inhibitory phosphorylation of the myosin RLC (171, 217, 259). Myosin RLC can be phosphorylated by Cdk1, which inhibits its actin-activated ATPase activity in vitro. Phosphorylation of the RLC at a different site by myosin light chain kinase promotes actin-activated ATPase activity in vitro (223). It has been shown that inhibitory phosphorylation increases early in mitosis and subsequently decreases, coincidently with Cdk1 inactivation in anaphase. Simultaneously, activating phosphorylation of RLC increases (283). It is not entirely clear which kinases perform these phosphorylation events in vivo. For example, although myosin light chain kinase is thought to be responsible for activating phosphorylation of RLC, AIM-1 (182) and Rho-kinase (122) are also capable of this phosphorylation. Moreover, activating phosphorylation of RLC seems to be important for cytokinesis in vivo (109, 121), but it is not clear how significant Cdk1 phosphorylation of RLC is for regulating cytokinesis (232). It seems likely that CDK phosphorylation inhibits cytokinesis through numerous mechanisms.
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Inactivation of Cdc16p or Byr4p in G1 phase, when CDK activity is normally low, or in cells blocked in G2 phase by inactivation of Cdc2p, leads to inappropriate septation. However, inactivation of Cdc16p in G2 phase when CDK activity has not been inhibited does not lead to uncontrolled septation (36). Thus, Cdc16p-Byr4p seems to function to ensure that cells do not initiate cytokinesis during interphase when CDK activity is low (36, 134). These results indicated that, in S. pombe, CDK inhibition of cytokinesis seems to function through inhibition of the SIN. This effect appears to be mediated, at least in part, through the Sid1p kinase, whose localization is inhibited by Cdc2p kinase (41, 93). Interestingly, cells expressing a nondestructible cyclin arrest in anaphase B with Cdc7p at both poles, indicating that CDK inactivation is also important for asymmetric activation of Spg1p; however, the functional significance of this is unclear (41).
Since Cdc2p inactivation occurs coincidently with chromosome segregation, coupling initiation of cytokinesis to Cdc2p inactivation ensures that cell division does not initiate before chromosomes have been segregated. However, this mechanism renders cytokinesis sensitive to Cdc2p activity, which begins to rise as the next cell cycle initiates around the time of septation (160). If cytokinesis is delayed, the rising Cdc2p activity could inhibit the SIN and cytokinesis unless the cell has a way to inhibit CDK activity until cytokinesis is complete. Evidence for a cytokinesis checkpoint to ensure that cytokinesis is complete before initiation of the next nuclear division cycle was revealed through characterization of cytokinesis mutants defective for either actomyosin ring formation or septum synthesis (Fig. 5B) (133, 144, 145, 263). These mutants complete mitosis and then arrest the cell cycle after failing cytokinesis with two interphase nuclei. The block in nuclear division is relieved by inactivation of either the CDK inhibitory kinase Wee1p; the SIN (133, 144, 145); or Clp1p/Flp1p, the S. pombe homolog of the S. cerevisiae Cdc14p phosphatase (51, 263). Clp1p/Flp1p localizes to the nucleolus and SPB in interphase, and then in mitosis it exits the nucleolus and localizes to the mitotic spindle and actomyosin ring. Once cytokinesis is complete Clp1p/Flp1p relocalizes to the nucleolus. It appears that Clp1p/Flp1p functions to keep CDK activity low by promoting inhibitory tyrosine phosphorylation of the S. pombe CDK, Cdc2p (263). This lets the SIN stay on until cytokinesis is complete. When cytokinesis fails or is delayed, the SIN keeps Clp1p out of the nucleolus until cytokinesis can be completed. This complex feedback loop helps keep cells from becoming multinucleate and thus polyploid when cytokinesis is delayed.
MEN. In S. cerevisiae the pathway analogous to the SIN is termed the MEN (see Table 3 for a comparison of SIN and MEN genes). Like the SIN, the MEN appears to be a GAP kinase cascade, whose components localize to both the SPB and the bud neck (167). At present, the MEN consists of four protein kinases (Cdc5p, Cdc15p, Dbf2p, and Dbf20p), a GTPase (Tem1p), an exchange factor (Lte1p), a protein phosphatase (Cdc14p), and a Dbf2p binding protein (Mob1p) (108, 116, 151, 220, 229, 230, 260, 271). Additionally the SPB component Nud1p seems to function to anchor MEN proteins at the cytoplasmic face of the SPB (92). Lte1p is presumed to activate Tem1p, which then recruits Cdc15p to the SPB(s) (19, 204). Cdc15p is thought to activate Dbf2-Mob1 (131, 159). The MEN functions in anaphase to inhibit CDK activity and cause mitotic exit by promoting release of the Cdc14p phosphatase from the nucleolus by an unknown mechanism (231, 268). Cdc14p
