Drosophila F-BAR protein Syndapin contributes to coupling the plasma membrane and contractile ring in cytokinesis

Cytokinesis is a highly ordered cellular process driven by interactions between central spindle microtubules and the actomyosin contractile ring linked to the dynamic remodelling of the plasma membrane. The mechanisms responsible for reorganizing the plasma membrane at the cell equator and its coupling to the contractile ring in cytokinesis are poorly understood. We report here that Syndapin, a protein containing an F-BAR domain required for membrane curvature, contributes to the remodelling of the plasma membrane around the contractile ring for cytokinesis. Syndapin colocalizes with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) at the cleavage furrow, where it directly interacts with a contractile ring component, Anillin. Accordingly, Anillin is mislocalized during cytokinesis in Syndapin mutants. Elevated or diminished expression of Syndapin leads to cytokinesis defects with abnormal cortical dynamics. The minimal segment of Syndapin, which is able to localize to the cleavage furrow and induce cytokinesis defects, is the F-BAR domain and its immediate C-terminal sequences. Phosphorylation of this region prevents this functional interaction, resulting in reduced ability of Syndapin to bind to and deform membranes. Thus, the dephosphorylated form of Syndapin mediates both remodelling of the plasma membrane and its proper coupling to the cytokinetic machinery.     

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.     

Anillin, a contractile ring protein that cycles from the nucleus to the cell cortex

We report the cDNA sequence and localization of a protein first identified by actin filament chromatography of Drosophila embryo extracts as ABP8 (Miller, K. G., C. M. Field, and B. M. Alberts. 1989. J. Cell Biol. 109:2963-2975). The cDNA encodes a 1201-amino acid protein which we name anillin. Anillin migrates at 190 kD on SDS-PAGE. Anillin is expressed throughout Drosophila development and in tissue culture cells. By immunofluorescence, anillin localizes to the nucleus of interphase cells, except in the syncytial embryo where it is always cytoplasmic. During metaphase, it is present in the cytoplasm and cortex, and during anaphase-telophase it becomes highly enriched in the cleavage furrow along with myosin II. In the syncytial embryo, anillin, along with myosin-II, is enriched in cortical areas undergoing cell cycle regulated invagination including metaphase furrows and the cellularization front. In contractile rings, metaphase furrows, and nascent ring canals, anillin remains bound to the invaginated cortex suggesting a stabilizing role. Anillin is not expressed in cells that have left the cell cycle. Anillin isolated from embryo extracts binds directly to actin filaments. The domain responsible for this binding has been mapped to a region of 244 amino acids by expression of protein fragments in bacteria. This domain, which is monomeric in solution, also bundles actin filaments. We speculate that anillin plays a role in organizing and/or stabilizing the cleavage furrow and other cell cycle regulated, contractile domains of the actin cytoskeleton.     

Functional analysis of the Drosophila diaphanous FH protein in early embryonic development

The Drosophila Formin Homology (FH) protein Diaphanous has an essential role during cytokinesis. To gain insight into the function of Diaphanous during cytokinesis and explore its role in other processes, we generated embryos deficient for Diaphanous and analyzed three cell-cycle-regulated actin-mediated events during embryogenesis: formation of the metaphase furrow, cellularization and formation of the pole cells. In dia embryos, all three processes are defective. Actin filaments do not organize properly to the metaphase and cellularization furrows and the actin ring is absent from the base of the presumptive pole cells. Furthermore, plasma membrane invaginations that initiate formation of the metaphase furrow and pole cells are missing. Immunolocalization studies of wild-type embryos reveal that Diaphanous localizes to the site where the metaphase furrow is anticipated to form, to the growing tip of cellularization furrows, and to contractile rings. In addition, the dia mutant phenotype reveals a role for Diaphanous in recruitment of myosin II, anillin and Peanut to the cortical region between actin caps. Our findings thus indicate that Diaphanous has a role in actin cytoskeleton organization and is essential for many, if not all, actin-mediated events involving membrane invagination. Based on known biochemical functions of FH proteins, we propose that Diaphanous serves as a mediator between signaling molecules and actin organizers at specific phases of the cell cycle.     

Microtuble organization in Xenopus eggs during the first cleavage and its role in cytokinesis

It has been suggested that the organization of microtubules during mitosis plays an important role in cytokinesis in animal cells. We studied the organization of microtubules during the first cleavage and its role in cytokinesis of Xenopus eggs. First, we examined the immunofluorescent localization of microtubules in Xenopus eggs at various stages during the first cleavage. The astral microtubules that extend from each of the two centrosomes towards the division plane meet and connect with each other at the division plane as cytokinesis proceeds. The microtubular connection thus advances from the animal pole to the vegetal pole, and its leading edge is located approximately beneath the leading edge of the cleavage furrow. Furthermore, an experiment using nocodazole suggests that microtubules have an essential role in advancement of the cleavage furrow, but neither in contraction nor maintenance of the already formed contractile ring which underlies the cleavage furrow membrane. These results suggest that the astral microtubules play an important role in controlling the formation of the contractile ring in Xenopus eggs.     

An essential role for a membrane lipid in cytokinesis. Regulation of contractile ring disassembly by redistribution of phosphatidylethanolamine

Phosphatidylethanolamine (PE) is a major membrane phospholipid that is mainly localized in the inner leaflet of the plasma membrane. We previously demonstrated that PE was exposed on the cell surface of the cleavage furrow during cytokinesis. Immobilization of cell surface PE by a PE-binding peptide inhibited disassembly of the contractile ring components, including myosin II and radixin, resulting in formation of a long cytoplasmic bridge between the daughter cells. This blockade of contractile ring disassembly was reversed by removal of the surface-bound peptide, suggesting that the PE exposure plays a crucial role in cytokinesis. To further examine the role of PE in cytokinesis, we established a mutant cell line with a specific decrease in the cellular PE level. On the culture condition in which the cell surface PE level was significantly reduced, the mutant ceased cell growth in cytokinesis, and the contractile ring remained in the cleavage furrow. Addition of PE or ethanolamine, a precursor of PE synthesis, restored the cell surface PE on the cleavage furrow and normal cytokinesis. These findings provide the first evidence that PE is required for completion of cytokinesis in mammalian cells, and suggest that redistribution of PE on the cleavage furrow may contribute to regulation of contractile ring disassembly.     

Anillin is a scaffold protein that links RhoA, actin, and myosin during cytokinesis

Cell division after mitosis is mediated by ingression of an actomyosin-based contractile ring. The active, GTP-bound form of the small GTPase RhoA is a key regulator of contractile-ring formation. RhoA concentrates at the equatorial cell cortex at the site of the nascent cleavage furrow. During cytokinesis, RhoA is activated by its RhoGEF, ECT2. Once activated, RhoA promotes nucleation, elongation, and sliding of actin filaments through the coordinated activation of both formin proteins and myosin II motors (reviewed in [1, 2]). Anillin is a 124 kDa protein that is highly concentrated in the cleavage furrow in numerous animal cells in a pattern that resembles that of RhoA [3-7]. Although anillin contains conserved N-terminal actin and myosin binding domains and a PH domain at the C terminus, its mechanism of action during cytokinesis remains unclear. Here, we show that human anillin contains a conserved C-terminal domain that is essential for its function and localization. This domain shares homology with the RhoA binding protein Rhotekin and directly interacts with RhoA. Further, anillin is required to maintain active myosin in the equatorial plane during cytokinesis, suggesting it functions as a scaffold protein to link RhoA with the ring components actin and myosin. Although furrows can form and initiate ingression in the absence of anillin, furrows cannot form in anillin-depleted cells in which the central spindle is also disrupted, revealing that anillin can also act at an early stage of cytokinesis.     

A secreted protein promotes cleavage furrow maturation during cytokinesis

Developmental modifications in cell shape depend on dynamic interactions between the extracellular matrix and cytoskeleton. In contrast, existing models of cytokinesis describe substantial cell surface remodeling that involves many intracellular regulatory and structural proteins but includes no contribution from the extracellular matrix [1-3]. Here, we show that extracellular hemicentins assemble at the cleavage furrow of dividing cells in the C.?elegans germline and in preimplantation mouse embryos. In the absence of hemicentin, cleavage furrows form but retract prior to completion, resulting in multinucleate cells. In addition to their role in tissue organization, the data indicate that hemicentins are the first secreted proteins required during mammalian development and the only known secreted proteins required for cytokinesis, with an evolutionarily conserved role in stabilizing and preventing retraction of nascent cleavage furrows. Together with studies showing that extracellular polysaccharides are required for cytokinesis in diverse species [4-9], our data suggest that assembly of a cell type-specific extracellular matrix may be a general requirement for cleavage furrow maturation and contractile ring function during cytokinesis.     

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.     

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.     

PtdIns(4,5)P2 functions at the cleavage furrow during cytokinesis

Phosphoinositides play important roles in regulating the cytoskeleton and vesicle trafficking, potentially important processes at the cleavage furrow. However, it remains unclear which, if any, of the phosphoinositides play a role during cytokinesis. A systematic analysis to determine if any of the phosphoinositides might be present or of functional importance at the cleavage furrow has not been published. Several studies hint at a possible role for one or more phosphoinositides at the cleavage furrow. The best of these are genetic data identifying mutations in phosphoinositide-modifying enzymes (a PtdIns(4)P-5-kinase in S. pombe and a PI-4-kinase in D. melanogaster) that interfere with cytokinesis. The genetic nature of these experiments leaves questions as to how direct may be their contribution to cytokinesis. Here we show that a single phosphoinositide, PtdIns(4,5)P2, specifically accumulates at the furrow. Interference with PtdIns(4,5)P2 interferes with adhesion of the plasma membrane to the contractile ring at the furrow. Finally, four distinct interventions to specifically interfere with PtdIns(4,5)P2 each impair cytokinesis. We conclude that PtdIns(4,5)P2 is present at the cleavage furrow and is required for normal cytokinesis at least in part because of a role in adhesion between the contractile ring and the plasma membrane.     

Polo-like kinase controls vertebrate spindle elongation and cytokinesis

During cell division, chromosome segregation must be coordinated with cell cleavage so that cytokinesis occurs after chromosomes have been safely distributed to each spindle pole. Polo-like kinase 1 (Plk1) is an essential kinase that regulates spindle assembly, mitotic entry and chromosome segregation, but because of its many mitotic roles it has been difficult to specifically study its post-anaphase functions. Here we use small molecule inhibitors to block Plk1 activity at anaphase onset, and demonstrate that Plk1 controls both spindle elongation and cytokinesis. Plk1 inhibition did not affect anaphase A chromosome to pole movement, but blocked anaphase B spindle elongation. Plk1-inhibited cells failed to assemble a contractile ring and contract the cleavage furrow due to a defect in Rho and Rho-GEF localization to the division site. Our results demonstrate that Plk1 coordinates chromosome segregation with cytokinesis through its dual control of anaphase B and contractile ring assembly.     

Interactions between myosin and actin crosslinkers control cytokinesis contractility dynamics and mechanics

INTRODUCTION: Contractile networks are fundamental to many cellular functions, particularly cytokinesis and cell motility. Contractile networks depend on myosin-II mechanochemistry to generate sliding force on the actin polymers. However, to be contractile, the networks must also be crosslinked by crosslinking proteins, and to change the shape of the cell, the network must be linked to the plasma membrane. Discerning how this integrated network operates is essential for understanding cytokinesis contractility and shape control. Here, we analyzed the cytoskeletal network that drives furrow ingression in Dictyostelium. RESULTS: We establish that the actin polymers are assembled into a meshwork and that myosin-II does not assemble into a discrete ring in the Dictyostelium cleavage furrow of adherent cells. We show that myosin-II generates regional mechanics by increasing cleavage furrow stiffness and slows furrow ingression during late cytokinesis as compared to myoII nulls. Actin crosslinkers dynacortin and fimbrin similarly slow furrow ingression and contribute to cell mechanics in a myosin-II-dependent manner. By using FRAP, we show that the actin crosslinkers have slower kinetics in the cleavage furrow cortex than in the pole, that their kinetics differ between wild-type and myoII null cells, and that the protein dynamics of each crosslinker correlate with its impact on cortical mechanics. CONCLUSIONS: These observations suggest that myosin-II along with actin crosslinkers establish local cortical tension and elasticity, allowing for contractility independent of a circumferential cytoskeletal array. Furthermore, myosin-II and actin crosslinkers may influence each other as they modulate the dynamics and mechanics of cell-shape change.     

Contractile ring-independent localization of DdINCENP, a protein important for spindle stability and cytokinesis

Dictyostelium DdINCENP is a chromosomal passenger protein associated with centromeres, the spindle midzone, and poles during mitosis and the cleavage furrow during cytokinesis. Disruption of the single DdINCENP gene revealed important roles for this protein in mitosis and cytokinesis. DdINCENP null cells lack a robust spindle midzone and are hypersensitive to microtubule-depolymerizing drugs, suggesting that their spindles may not be stable. Furthermore DdCP224, a protein homologous to the microtubule-stabilizing protein TOGp/XMAP215, was absent from the spindle midzone of DdINCENP null cells. Overexpression of DdCP224 rescued the weak spindle midzone defect of DdINCENP null cells. Although not required for the localization of the myosin II contractile ring and subsequent formation of a cleavage furrow, DdINCENP is important for the abscission of daughter cells at the end of cytokinesis. Finally, we show that the localization of DdINCENP at the cleavage furrow is modulated by myosin II but it occurs by a mechanism different from that controlling the formation of the contractile ring.     

Phospholipase C isoforms are localized at the cleavage furrow during cytokinesis

It has recently been demonstrated that phosphatidylinositol 4,5-bisphosphate (PIP2) is localized at the cleavage furrow in dividing cells and its hydrolysis is required for complete cytokinesis, suggesting a pivotal role of PIP2 in cytokinesis. Here, we report that at least three mammalian isoforms of phosphoinositide-specific phospholipase C (PLC), PLCdelta1, PLCdelta3 and PLCbeta1, are localized to the cleavage furrow during cytokinesis. Targeting of the delta1 isoform to the furrow depends on the specific interaction between the PH domain and PIP2 in the plasma membrane. The necessity of active PLC in animal cell cytokinesis was confirmed using the specific inhibitors for PIP2 hydrolysis. These results support the model that activation of selected PLC isoforms at the cleavage furrow controls progression of cytokinesis through regulation of PIP2 levels: induction of the cleavage furrow by a contractile ring consisting of actomyosin is regulated by PIP2-dependent actin-binding proteins and formation of specific lipid domains required for membrane separation is affected by alterations in the lipid composition of the furrow.     

Rho-dependent control of anillin behavior during cytokinesis

Anillin is a conserved protein required for cytokinesis but its molecular function is unclear. Anillin accumulation at the cleavage furrow is Rho guanine nucleotide exchange factor (GEF)(Pbl)-dependent but may also be mediated by known anillin interactions with F-actin and myosin II, which are under RhoGEF(Pbl)-dependent control themselves. Microscopy of Drosophila melanogaster S2 cells reveal here that although myosin II and F-actin do contribute, equatorial anillin localization persists in their absence. Using latrunculin A, the inhibitor of F-actin assembly, we uncovered a separate RhoGEF(Pbl)-dependent pathway that, at the normal time of furrowing, allows stable filamentous structures containing anillin, Rho1, and septins to form directly at the equatorial plasma membrane. These structures associate with microtubule (MT) ends and can still form after MT depolymerization, although they are delocalized under such conditions. Thus, a novel RhoGEF(Pbl)-dependent input promotes the simultaneous association of anillin with the plasma membrane, septins, and MTs, independently of F-actin. We propose that such interactions occur dynamically and transiently to promote furrow stability.     

A Novel Guanine Nucleotide Exchange Factor,MYOGEF, is Required for Cytokinesis

The cleavage furrow is created by an actomyosin contractile ring that isregulated by small GTPase proteins such as Rac1 and RhoA. Guanine nucleotideexchange factors (GEFs) are positive regulators of the small GTPase proteins andhave been implicated as important factors in regulating cytokinesis. However, it isstill unclear how GEFs regulate the contractile ring during cytokinesis inmammalian cells. Here we report that a novel GEF, which is termed MyoGEF(myosin-interacting GEF), interacts with nonmuscle myosin II and exhibits activitytoward RhoA. MyoGEF and nonmuscle myosin II colocalize to the cleavage furrowin early anaphase cells. Disruption of MyoGEF expression in U2OS cells by RNAinterference (RNAi) results in the formation of multinucleated cells. These resultssuggest that MyoGEF, RhoA, and nonmuscle myosin II act as a functional unit atthe cleavage furrow to advance furrow ingression during cytokinesis.     

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.     

The cytoskeletal involvement in cellularization of theDrosophila melanogaster embryo

Summary The distribution and arrangement of cytoskeletal components in the early embryo ofDrosophila melanogaster were examined by thin-section electron microscopy to elucidate their involvement in the formation of the cellular blastoderm, a process called cellularization. During the final nuclear division in the cortex of the syncytial blastoderm bundles of astral microtubules were closely associated with the surface plasma membrane along the midline where a new gutter was initiated. Thus the new gutter together with the pre-formed ones compartmentalized the embryo surface to reflect underlying individual daughter nuclei. Subsequently such gutters became deeper by further invagination of the plasma membrane between adjacent nuclei to form so-called cleavage furrows. Nuclei simultaneously elongated in the direction perpendicular to the embryo surface and numerous microtubules from the centrosomes ran longitudinally between the nucleus and the cleavage furrow. Microtubules often appeared to be in close association with the nuclear envelope and the cleavage furrow membrane. The plasma membrane at the advancing tip of the furrow was always undercoated with an electron-dense layer, which could be shown to be mainly composed of 5–6 nm microfilaments. These microfilaments were decorated with H-meromyosin to be identified as actin filaments. As cleavage proceeded, each nucleus with its perikaryon became demarcated by the furrow membrane, which then extended laterally to constrict the cytoplasmic connection between each newly forming cell and the central yolk region. The cytoplasmic strand thus formed possessed a prominent circular bundle of microfilaments which were also decorated with H-meromyosin and bidirectionally arranged, similar in structure to the contractile ring in cytokinesis. These observations strongly suggest that both microtubules and actin filaments play a crucial role in cellularization ofDrosophila embryos.     

Mutations in sticky lead to defective organization of the contractile ring during cytokinesis and are enhanced by Rho and suppressed by Rac

The contractile ring is a highly dynamic structure, but how this dynamism is accomplished remains unclear. Here, we report the identification and analysis of a novel Drosophila gene, sticky (sti), essential for cytokinesis in all fly proliferating tissues. sti encodes the Drosophila orthologue of the mammalian Citron kinase. RNA interference-mediated silencing of sti in cultured cells causes them to become multinucleate. Components of the contractile ring and central spindle are recruited normally in such STICKY-depleted cells that nevertheless display asymmetric furrowing and aberrant blebbing. Together with an unusual distribution of F-actin and Anillin, these phenotypes are consistent with defective organization of the contractile ring. sti shows opposite genetic interactions with Rho and Rac genes suggesting that these GTPases antagonistically regulate STICKY functions. Similar genetic evidence indicates that RacGAP50C inhibits Rac during cytokinesis. We discuss that antagonism between Rho and Rac pathways may control contractile ring dynamics during cytokinesis.