Inn1 couples contraction of the actomyosin ring to membrane ingression during cytokinesis in budding yeast

By rapidly depleting each of the essential budding yeast proteins of unknown function, we identified a novel factor that we call Inn1, which associates with the contractile actomyosin ring at the end of mitosis and is needed for cytokinesis. We show that Inn1 has a C2 domain at the amino terminus of the protein that is required for ingression of the plasma membrane, whereas the remainder of the protein recruits Inn1 to the actomyosin ring. The lethal effects of deleting the INN1 gene can be suppressed by artificial fusion of the C2 domain to other components of the actomyosin ring, restoring membrane ingression on contraction of the actomyosin ring. Our data indicate that recruitment of the C2 domain of Inn1 to the contractile actomyosin ring is crucial for ingression of the plasma membrane during cytokinesis in budding yeast.     

The actin-binding and bundling protein,EPLIN, is required for cytokinesis

Cytokinesis involves two phases: 1) membrane ingression followed by 2) membrane abscission. The ingression phase generates a cleavage furrow and this requires co-operative function of the actin-myosin II contractile ring and septin filaments. We demonstrate that the actin-binding protein, EPLIN, locates to the cleavage furrow during cytokinesis and this is possibly via association with the contractile ring components, myosin II, and the septin, Sept2. Depletion of EPLIN results in formation of multinucleated cells and this is associated with inefficient accumulation of active myosin II (MRLCS19) and Sept2 and their regulatory small GTPases, RhoA and Cdc42, respectively, to the cleavage furrow during the final stages of cytokinesis. We suggest that EPLIN may function during cytokinesis to maintain local accumulation of key cytokinesis proteins at the furrow.     

Augmin shapes the anaphase spindle for efficient cytokinetic furrow ingression and abscission

During anaphase, distinct populations of microtubules (MTs) form by either centrosome-dependent or augmin-dependent nucleation. It remains largely unknown whether these different MT populations contribute distinct functions to cytokinesis. Here we show that augmin-dependent MTs are required for the progression of both furrow ingression and abscission. Augmin depletion reduced the accumulation of anillin, a contractile ring regulator at the cell equator, yet centrosomal MTs were sufficient to mediate RhoA activation at the furrow. This defect in contractile ring organization, combined with incomplete spindle pole separation during anaphase, led to impaired furrow ingression. During the late stages of cytokinesis, astral MTs formed bundles in the intercellular bridge, but these failed to assemble a focused midbody structure and did not establish tight linkage to the plasma membrane, resulting in furrow regression. Thus augmin-dependent acentrosomal MTs and centrosomal MTs contribute to nonredundant targeting mechanisms of different cytokinesis factors, which are required for the formation of a functional contractile ring and midbody.     

A role for very-long-chain fatty acids in furrow ingression during cytokinesis in Drosophila spermatocytes

Cell shape and membrane remodeling rely on regulated interactions between the lipid bilayer and cytoskeletal arrays at the cell cortex. During cytokinesis, animal cells build an actomyosin ring anchored to the plasma membrane at the equatorial cortex. Ring constriction coupled to plasma-membrane ingression separates the two daughter cells. Plasma-membrane lipids influence membrane biophysical properties such as membrane curvature and elasticity and play an active role in cell function, and specialized membrane domains are emerging as important factors in regulating assembly and rearrangement of the cytoskeleton. Here, we show that mutations in the gene bond, which encodes a Drosophila member of the family of Elovl proteins that mediate elongation of very-long-chain fatty acids, block or dramatically slow cleavage-furrow ingression during early telophase in dividing spermatocytes. In bond mutant cells at late stages of division, the contractile ring frequently detaches from the cortex and constricts or collapses to one side of the cell, and the cleavage furrow regresses. Our findings implicate very-long-chain fatty acids or their derivative complex lipids in allowing supple membrane deformation and the stable connection of cortical contractile components to the plasma membrane during cell division.     

Initial diameter of the polar body contractile ring is minimized by the centralspindlin complex

Polar body formation is an essential step in forming haploid eggs from diploid oocytes. This process involves completion of a highly asymmetric cytokinesis that results in a large egg and two small polar bodies. Unlike mitotic contractile rings, polar body contractile rings assemble over one spindle pole so that the spindle must move through the contractile ring before cytokinesis. During time-lapse imaging of C. elegans meiosis, the contractile ring moved downward along the length of the spindle and completed scission at the midpoint of the spindle, even when spindle length or rate of ring movement was increased. Patches of myosin heavy chain and dynamic furrowing of the plasma membrane over the entire embryo suggested that global cortical contraction forces the meiotic spindle and overlying membrane out through the contractile ring center. Consistent with this model, depletion of myosin phosphatase increased the velocity of ring movement along the length of the spindle. Global dynamic furrowing, which was restricted to anaphase I and II, was dependent on myosin II, the anaphase promoting complex and separase, but did not require cortical contact by the spindle. Large cortical patches of myosin during metaphase I and II indicated that myosin was already in the active form before activation of separase. To identify the signal at the midpoint of the anaphase spindle that induces scission, we depleted two proteins that mark the exact midpoint of the spindle during late anaphase, CYK-4 and ZEN-4. Depletion of either protein resulted in the unexpected phenotype of initial ingression of a polar body ring with twice the diameter of wild type. This phenotype revealed a novel mechanism for minimizing polar body size. Proteins at the spindle midpoint are required for initial ring ingression to occur close to the membrane-proximal spindle pole.     

Anillin and the septins promote asymmetric ingression of the cytokinetic furrow

During cytokinesis, constriction of a cortical contractile ring generates a furrow that partitions one cell into two. The contractile ring contains three filament systems: actin, bipolar myosin II filaments, and septins, GTP-binding hetero-oligomers that polymerize to form a membrane-associated lattice. The contractile ring also contains a potential filament crosslinker, Anillin, that binds all three filament types. Here, we show that the contractile ring possesses an intrinsic symmetry-breaking mechanism that promotes asymmetric furrowing. Asymmetric ingression requires Anillin and the septins, which promote the coalescence of components on one side of the contractile ring, but is insensitive to a 10-fold reduction in myosin II levels. When asymmetry is disrupted, cytokinesis becomes sensitive to partial inhibition of contractility. Thus, asymmetric furrow ingression, a prevalent but previously unexplored feature of cell division in metazoans, is generated by the action of two conserved furrow components and serves a mechanical function that makes cytokinesis robust.     

Polo-like kinase 1 triggers the initiation of cytokinesis in human cells by promoting recruitment of the RhoGEF Ect2 to the central spindle

Cytokinesis of animal cells requires ingression of the actomyosin-based contractile ring between segregated sister genomes. Localization of the RhoGEF Ect2 to the central spindle at anaphase promotes local activation of the RhoA GTPase, which induces assembly and ingression of the contractile ring. Here we have used BI 2536, an inhibitor of the mitotic kinase Plk1, to analyze the functions of this enzyme during late mitosis in human cells. We show that Plk1 acts after Cdk1 inactivation and independently from Aurora B to promote RhoA accumulation at the equator, contractile ring formation, and cleavage furrow ingression. Inhibition of Plk1 abolishes the interaction of Ect2 with its activator and midzone anchor, HsCyk-4, thereby preventing localization of Ect2 to the central spindle. We propose that late mitotic Plk1 activity promotes recruitment of Ect2 to the central spindle, triggering the initiation of cytokinesis and contributing to cleavage plane specification in human cells.     

Spermatocyte cytokinesis requires rapid membrane addition mediated by ARF6 on central spindle recycling endosomes

The dramatic cell shape changes during cytokinesis require the interplay between microtubules and the actomyosin contractile ring, and addition of membrane to the plasma membrane. Numerous membrane-trafficking components localize to the central spindle during cytokinesis, but it is still unclear how this machinery is targeted there and how membrane trafficking is coordinated with cleavage furrow ingression. Here we use an arf6 null mutant to show that the endosomal GTPase ARF6 is required for cytokinesis in Drosophila spermatocytes. ARF6 is enriched on recycling endosomes at the central spindle, but it is required neither for central spindle nor actomyosin contractile ring assembly, nor for targeting of recycling endosomes to the central spindle. However, in arf6 mutants the cleavage furrow regresses because of a failure in rapid membrane addition to the plasma membrane. We propose that ARF6 promotes rapid recycling of endosomal membrane stores during cytokinesis, which is critical for rapid cleavage furrow ingression.     

Microtubules, membranes and cytokinesis

Proper division of the cell requires coordination between chromosome segregation by the mitotic spindle and cleavage of the cell by the cytokinetic apparatus. Interactions between the mitotic spindle, the contractile ring and the plasma membrane ensure that the cleavage furrow is properly placed between the segregating chromosomes and that new membrane compartments are formed to produce two daughter cells. The microtubule midzone is able to stimulate the cortex of the cell to ensure proper ingression and completion of the cleavage furrow. Specialized microtubule structures are responsible for directing membrane vesicles to the site of cell cleavage, and vesicle fusion is required for the proper completion of cytokinesis.     

The contractile ring coordinates curvature-dependent septum assembly during fission yeast cytokinesis

The functions of the actin-myosin–based contractile ring in cytokinesis remain to be elucidated. Recent findings show that in the fission yeast Schizosaccharomyces pombe, cleavage furrow ingression is driven by polymerization of cell wall fibers outside the plasma membrane, not by the contractile ring. Here we show that one function of the ring is to spatially coordinate septum cell wall assembly. We develop an improved method for live-cell imaging of the division apparatus by orienting the rod-shaped cells vertically using microfabricated wells. We observe that the septum hole and ring are circular and centered in wild-type cells and that in the absence of a functional ring, the septum continues to ingress but in a disorganized and asymmetric manner. By manipulating the cleavage furrow into different shapes, we show that the ring promotes local septum growth in a curvature-dependent manner, allowing even a misshapen septum to grow into a more regular shape. This curvature-dependent growth suggests a model in which contractile forces of the ring shape the septum cell wall by stimulating the cell wall machinery in a mechanosensitive manner. Mechanical regulation of the cell wall assembly may have general relevance to the morphogenesis of walled cells.     

The mechanism of cortical ingression during early cytokinesis: thinking beyond the contractile ring hypothesis

Owing to the rapid advances in genomic, proteomic and imaging technologies, the field of cytokinesis has seen rapid advances during the past decade. However, the basic model for the early stage of ingression, known as the contractile ring hypothesis, remains largely unchanged. From recent observations, it is becoming clear that early cytokinesis of animal cells involves a more extensive set of events, both temporally and spatially, than what is encompassed by the original contractile ring hypothesis. Activities relevant to cytokinesis, such as cortical contraction, can initiate well before onset of anaphase. Furthermore, equatorial ingression can involve multiple events in different regions of the cortex, including the establishment of anterior-posterior polarity, the modulation of cortical deformability, the expansion and compression of the cell cortex, and forces directed towards the interior of the cell or away from the equator. In this article (which is part of the Cytokinesis series), I evaluate critically key observations on when, where and how early ingression of animal cells takes place.     

Dissecting the role of Rho-mediated signaling in contractile ring formation

In anaphase, microtubules provide a specification signal for positioning of the contractile ring. However, the nature of the signal remains unknown. The small GTPase Rho is a potent regulator of cytokinesis, but the involvement of Rho in contractile ring formation is disputed. Here, we show that Rho serves as a microtubule-dependent signal that specifies the position of the contractile ring. We found that Rho translocates to the equatorial region before furrow ingression. The Rho-specific inhibitor C3 exoenzyme and small interfering RNA to the Rho GDP/GTP exchange factor ECT2 prevent this translocation and disrupt contractile ring formation, indicating that active Rho is required for contractile ring formation. ECT2 forms a complex with the GTPase-activating protein MgcRacGAP and the kinesinlike protein MKLP1 at the central spindle, and the localization of ECT2 at the central spindle depends on MgcRacGAP and MKLP1. In addition, we show that the bundled microtubules direct Rho-mediated signaling molecules to the furrowing site and regulate furrow formation. Our study provides strong evidence for the requirement of Rho-mediated signaling in contractile ring formation.     

Characterization of anillin mutants reveals essential roles in septin localization and plasma membrane integrity

Anillin is a conserved component of the contractile ring that is essential for cytokinesis, and physically interacts with three conserved cleavage furrow proteins, F-actin, myosin II and septins in biochemical assays. We demonstrate that the Drosophila scraps gene, identified as a gene involved in cellularization, encodes Anillin. We characterize defects in cellularization, pole cell formation and cytokinesis in a series of maternal effect and zygotic anillin alleles. Mutations that result in amino acid changes in the C-terminal PH domain of Anillin cause defects in septin recruitment to the furrow canal and contractile ring. These mutations also strongly perturb cellularization, altering the timing and rate of furrow ingression. They cause dramatic vesiculation of new plasma membranes, and destabilize the stalk of cytoplasm that normally connects gastrulating cells to the yolk mass. A mutation closer to the N terminus blocks separation of pole cells with less effect on cellularization, highlighting mechanistic differences between contractile processes. Cumulatively, our data point to an important role for Anillin in scaffolding cleavage furrow components, directly stabilizing intracellular bridges, and indirectly stabilizing newly deposited plasma membrane during cellularization.     

Inhibition of cytokinesis by wiskostatin does not rely on N-WASP/Arp2/3 complex pathway

Background  

Cytokinesis is the final step of cell division taking place at the end of mitosis during which the cytoplasmic content and replicated chromosomes of a cell are equally partitioned between the two daughter cells. This process is achieved by the formation and the ingression of an actomyosin contractile ring under the control of equatorial microtubules. The mechanisms of contractile ring formation are not fully understood but involve recruitment of preexisting actin filaments and de novo actin polymerisation.     

Distinct roles of Rho1, Cdc42, and Cyk3 in septum formation and abscission during yeast cytokinesis

In yeast and animal cytokinesis, the small guanosine triphosphatase (GTPase) Rho1/RhoA has an established role in formation of the contractile actomyosin ring, but its role, if any, during cleavage-furrow ingression and abscission is poorly understood. Through genetic screens in yeast, we found that either activation of Rho1 or inactivation of another small GTPase, Cdc42, promoted secondary septum (SS) formation, which appeared to be responsible for abscission. Consistent with this hypothesis, a dominant-negative Rho1 inhibited SS formation but not cleavage-furrow ingression or the concomitant actomyosin ring constriction. Moreover, Rho1 is temporarily inactivated during cleavage-furrow ingression; this inactivation requires the protein Cyk3, which binds Rho1-guanosine diphosphate via its catalytically inactive transglutaminase-like domain. Thus, unlike the active transglutaminases that activate RhoA, the multidomain protein Cyk3 appears to inhibit activation of Rho1 (and thus SS formation), while simultaneously promoting cleavage-furrow ingression through primary septum formation. This work suggests a general role for the catalytically inactive transglutaminases of fungi and animals, some of which have previously been implicated in cytokinesis.     

A high-resolution shape fitting and simulation demonstrated equatorial cell surface softening during cytokinesis and its promotive role in cytokinesis

Different models for animal cell cytokinesis posit that the stiffness of the equatorial cortex is either increased or decreased relative to the stiffness of the polar cortex. A recent work has suggested that the critical cytokinesis signaling complex centralspindlin may reduce the stiffness of the equatorial cortex by inactivating the small GTPase Rac. To determine if such a reduction occurs and if it depends on centralspindlin, we devised a method to estimate cortical bending stiffness with high spatio-temporal resolution from in vivo cell shapes. Using the early Caenorhabditis elegans embryo as a model, we show that the stiffness of the equatorial cell surface is reduced during cytokinesis, whereas the stiffness of the polar cell surface remains stiff. The equatorial reduction of stiffness was compromised in cells with a mutation in the gene encoding the ZEN-4/kinesin-6 subunit of centralspindlin. Theoretical modeling showed that the absence of the equatorial reduction of stiffness could explain the arrest of furrow ingression in the mutant. By contrast, the equatorial reduction of stiffness was sufficient to generate a cleavage furrow even without the constriction force of the contractile ring. In this regime, the contractile ring had a supportive contribution to furrow ingression. We conclude that stiffness is reduced around the equator in a centralspindlin-dependent manner. In addition, computational modeling suggests that proper regulation of stiffness could be sufficient for cleavage furrow ingression.     

Extracellular cell wall β(1,3)glucan is required to couple septation to actomyosin ring contraction

Cytokinesis has been extensively studied in different models, but the role of the extracellular cell wall is less understood. Here we studied this process in fission yeast. The essential protein Bgs4 synthesizes the main cell wall β(1,3)glucan. We show that Bgs4-derived β(1,3)glucan is required for correct and stable actomyosin ring positioning in the cell middle, before the start of septum formation and anchorage to the cell wall. Consequently, β(1,3)glucan loss generated ring sliding, oblique positioned rings and septa, misdirected septum synthesis indicative of relaxed rings, and uncoupling between a fast ring and membrane ingression and slow septum synthesis, suggesting that cytokinesis can progress with defective septum pushing and/or ring pulling forces. Moreover, Bgs4-derived β(1,3)glucan is essential for secondary septum formation and correct primary septum completion. Therefore, our results show that extracellular β(1,3)glucan is required for cytokinesis to connect the cell wall with the plasma membrane and for contractile ring function, as proposed for the equivalent extracellular matrix in animal cells.     

Contributions of turgor pressure, the contractile ring, and septum assembly to forces in cytokinesis in fission yeast

A paradigm of cytokinesis in animal cells is that the actomyosin contractile ring provides the primary force to divide the cell [1]. In the fission yeast Schizosaccharomyces pombe, cytokinesis also involves a conserved cytokinetic ring, which has been generally assumed to provide the force for cleavage [2-4] (see also [5]). However, in contrast to animal cells, cytokinesis in yeast cells also requires the assembly of a cell wall septum [6], which grows centripetally inward as the ring closes. Fission yeast, like other walled cells, also possess high (MPa) turgor pressure [7-9]. Here, we show that turgor pressure is an important factor in the mechanics of cytokinesis. Decreasing effective turgor pressure leads to an increase in cleavage rate, suggesting that the inward force generated by the division apparatus opposes turgor pressure. The contractile ring, which is predicted to provide only a tiny fraction of the mechanical stress required to overcome turgor, is largely dispensable for ingression; once septation has started, cleavage can continue in the absence of the contractile ring. Scaling arguments and modeling suggest that the large forces for cytokinesis are not produced by the contractile ring but are driven by the assembly of cell wall polymers in the growing septum.     

An ECT2-centralspindlin complex regulates the localization and function of RhoA

In anaphase, the spindle dictates the site of contractile ring assembly. Assembly and ingression of the contractile ring involves activation of myosin-II and actin polymerization, which are triggered by the GTPase RhoA. In many cells, the central spindle affects division plane positioning via unknown molecular mechanisms. Here, we dissect furrow formation in human cells and show that the RhoGEF ECT2 is required for cortical localization of RhoA and contractile ring assembly. ECT2 concentrates on the central spindle by binding to centralspindlin. Depletion of the centralspindlin component MKLP1 prevents central spindle localization of ECT2; however, RhoA, F-actin, and myosin still accumulate on the equatorial cell cortex. Depletion of the other centralspindlin component, CYK-4/MgcRacGAP, prevents cortical accumulation of RhoA, F-actin, and myosin. CYK-4 and ECT2 interact, and this interaction is cell cycle regulated via ECT2 phosphorylation. Thus, central spindle localization of ECT2 assists division plane positioning and the CYK-4 subunit of centralspindlin acts upstream of RhoA to promote furrow assembly.     

Cytokinesis and the Spindle Midzone

At the end of the cell cycle a cell physically divides into two daughter cells in a process called cytokinesis. Cytokinesis consists of at least four steps: 1. The position of the presumptive cytokinesis furrow is specified. 2. A contractile ring is formed. 3. The contractile ring contracts, resulting in furrow ingression. 4. Cytokinesis completes with sealing of the membranes. The mitotic spindle positions the cytokinesis furrow at the cell cortex midway along the longitudinal axis of the spindle, which is both the mid-point between the two asters and the location of the spindle midzone. The mitotic spindle emits two consecutive signals that position the furrow: Microtubule asters provide a first signal; the spindle midzone provides a second signal. Our results support the view that the spindle midzone is dispensable for completion of cytokinesis. However, the spindle midzone can negatively affect aster-positioned cytokinesis, possibly because the aster- and midzone-positioned furrows compete for contractile elements.