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.     

Nir2, a human homolog of Drosophila melanogaster retinal degeneration B protein,is essential for cytokinesis

Cytokinesis, the final stage of eukaryotic cell division, ensures the production of two daughter cells. It requires fine coordination between the plasma membrane and cytoskeletal networks, and it is known to be regulated by several intracellular proteins, including the small GTPase Rho and its effectors. In this study we provide evidence that the protein Nir2 is essential for cytokinesis. Microinjection of anti-Nir2 antibodies into interphase cells blocks cytokinesis, as it results in the production of multinucleate cells. Immunolocalization studies revealed that Nir2 is mainly localized in the Golgi apparatus in interphase cells, but it is recruited to the cleavage furrow and the midbody during cytokinesis. Nir2 colocalizes with the small GTPase RhoA in the cleavage furrow and the midbody, and it associates with RhoA in mitotic cells. Its N-terminal region, which contains a phosphatidylinositol transfer domain and a novel Rho-inhibitory domain (Rid), is required for normal cytokinesis, as overexpression of an N-terminal-truncated mutant blocks cytokinesis completion. Time-lapse videomicroscopy revealed that this mutant normally initiates cytokinesis but fails to complete it, due to cleavage furrow regression, while Rid markedly affects cytokinesis due to abnormal contractility. Rid-expressing cells exhibit aberrant ingression and ectopic cleavage sites; the cells fail to segregate into daughter cells and they form a long unseparated bridge-like cytoplasmic structure. These results provide new insight into the cellular functions of Nir2 and introduce it as a novel regulator of cytokinesis.     

ARF6 GTPase protects the post-mitotic midbody from 14-3-3-mediated disintegration

In cytokinesis, there is a lengthy interval between cleavage furrow ingression and abscission, during which the midbody microtubule bundle provides both structural support for a narrow intercellular bridge and a platform that orchestrates the biochemical preparations for abscission. It is currently unclear how the midbody structure is stably maintained during this period. Here, we report a novel role for the ADP-ribosylation factor 6 (ARF6) GTPase in the post-mitotic stabilisation of midbody. Centralspindlin kinesin-6/RhoGAP complex, a midbody component critical for both the formation and function of the midbody, assembles in a sharp band at the centre of the structure in a manner antagonised by 14-3-3 protein. We show that ARF6 competes with 14-3-3 for binding to centralspindlin such that midbodies formed by centralspindlin mutants that can bind 14-3-3 but not ARF6 frequently collapse before abscission. These data indicate a novel mechanism for the regulation of midbody dynamics in which ARF6 protects the compacted centralspindlin assembly from dissipation by 14-3-3.     

Mip1 associates with both the Mps1 kinase and actin,and is required for cell cortex stability and anaphase spindle positioning

The Mps1 family of protein kinases contributes to cell cycle control by regulating multiple microtubule cytoskeleton activities. We have uncovered a new Mps1 substrate that provides a novel link between Mps1 and the actin cytoskeleton. We have identified a conserved human Mps1 (hMps1) interacting protein we have termed Mps1 interacting protein-1 (Mip1). Mip1 defines an uncharacterized family of conserved proteins that contain coiled-coil and calponin homology domains. We demonstrate that Mip1 is a phosphoprotein that interacts with hMps1 in vitro and in vivo and is a hMps1 substrate. Mip1 exhibits dynamic localization during the cell cycle; Mip1 localizes to the actin cytoskeleton during interphase, the spindle in early mitosis, and the cleavage furrow during cytokinesis. Mip1 function is required to ensure proper spindle positioning at the onset of anaphase after cells begin furrow ingression. Cells depleted of Mip1 exhibit aberrant mitotic actin filament organization, excessive membrane blebbing, dramatic spindle rocking, and chromosome distribution errors during early cytokinesis producing high numbers of binucleate cells. Our data indicate that Mip1 is a newly recognized component of the actin cytoskeleton that interacts with hMps1 and that it is essential to ensure proper segregation of the genome during cell cleavage.     

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.     

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.     

Mip1 associates with both the Mps1 kinase and actin,and is required for cell cortex stability and anaphase spindle positioning

The Mps1 family of protein kinases contributes to cell cycle control by regulating multiple microtubule cytoskeleton activities. We have uncovered a new Mps1 substrate that provides a novel link between Mps1 and the actin cytoskeleton. We have identified a conserved human Mps1 (hMps1) interacting protein and have termed Mps1 interacting protein-1 (Mip1). Mip1 defines an uncharacterized family of conserved proteins that contain coiled-coil and calponin homology domains. We demonstrate that Mip1 is a phosphoprotein that interacts with hMps1 in vitro and in vivo and is a hMps1 substrate. Mip1 exhibits dynamic localization during the cell cycle; Mip1 localizes to the actin cytoskeleton during interphase, the spindle in early mitosis and the cleavage furrow during cytokinesis. Mip1 function is required to ensure proper spindle positioning at the onset of anaphase after cells begin furrow ingression. Cells depleted of Mip1 exhibit aberrant mitotic actin filament organization, excessive membrane blebbing, dramatic spindle rocking and chromosome distribution errors during early cytokinesis producing high numbers of binucleate cells. Our data indicate that Mip1 is a newly recognized component of the actin cytoskeleton that interacts with hMps1 and that it is essential to ensure proper segregation of the genome during cell cleavage.Key words: Mps1 kinase, actin, Mip1, cytokinesis     

The large GTPase dynamin associates with the spindle midzone and is required for cytokinesis

Cytokinesis involves the concerted efforts of the microtubule and actin cytoskeletons as well as vesicle trafficking and membrane remodeling to form the cleavage furrow and complete daughter cell separation. The exact mechanisms that support membrane remodeling during cytokinesis remain largely undefined. In this study, we report that the large GTPase dynamin, a protein involved in membrane tubulation and vesiculation, is essential for successful cytokinesis. Using biochemical and morphological methods, we demonstrate that dynamin localizes to the spindle midzone and the subsequent intercellular bridge in mammalian cells and is also enriched in spindle midbody extracts. In Caenorhabditis elegans, dynamin localized to newly formed cleavage furrow membranes and accumulated at the midbody of dividing embryos in a manner similar to dynamin localization in mammalian cells. Further, dynamin function appears necessary for cytokinesis, as C. elegans embryos from a dyn-1 ts strain, as well as dynamin RNAi-treated embryos, showed a marked defect in the late stages of cytokinesis. These findings indicate that, during mitosis, conventional dynamin is recruited to the spindle midzone and the subsequent intercellular bridge, where it plays an essential role in the final separation of dividing cells.     

Endogenous ARF6 interacts with Rac1 upon angiotensin II stimulation to regulate membrane ruffling and cell migration

ARF6 and Rac1 are small GTPases known to regulate remodelling of the actin cytoskeleton. Here, we demonstrate that these monomeric G proteins are sequentially activated when HEK 293 cells expressing the angiotensin type 1 receptor (AT(1)R) are stimulated with angiotensin II (Ang II). After receptor activation, ARF6 and Rac1 transiently form a complex. Their association is, at least in part, direct and dependent on the nature of the nucleotide bound to both small G proteins. ARF6-GTP preferentially interacts with Rac1-GDP. AT(1)R expressing HEK293 cells ruffle, form membrane protrusions, and migrate in response to agonist treatment. ARF6, but not ARF1, depletion using small interfering RNAs recapitulates the ruffling and migratory phenotype observed after Ang II treatment. These results suggest that ARF6 depletion or Ang II treatment are functionally equivalent and point to a role for endogenous ARF6 as an inhibitor of Rac1 activity. Taken together, our findings reveal a novel function of endogenously expressed ARF6 and demonstrate that by interacting with Rac1, this small GTPase is a central regulator of the signaling pathways leading to actin remodeling.     

Nuf, a Rab11 effector, maintains cytokinetic furrow integrity by promoting local actin polymerization

Plasma membrane ingression during cytokinesis involves both actin remodeling and vesicle-mediated membrane addition. Vesicle-based membrane delivery from the recycling endosome (RE) has an essential but ill-defined involvement in cytokinesis. In the Drosophila melanogaster early embryo, Nuf (Nuclear fallout), a Rab11 effector which is essential for RE function, is required for F-actin and membrane integrity during furrow ingression. We find that in nuf mutant embryos, an initial loss of F-actin at the furrow is followed by loss of the associated furrow membrane. Wild-type embryos treated with Latrunculin A or Rho inhibitor display similar defects. Drug- or Rho-GTP-induced increase of actin polymerization or genetically mediated decrease of actin depolymerization suppresses the nuf mutant F-actin and membrane defects. We also find that RhoGEF2 does not properly localize at the furrow in nuf mutant embryos and that RhoGEF2-Rho1 pathway components show strong specific genetic interactions with Nuf. We propose a model in which RE-derived vesicles promote furrow integrity by regulating the rate of actin polymerization through the RhoGEF2-Rho1 pathway.     

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.     

Calcium sensitivity of α-actinin is required for equatorial actin assembly during cytokinesis

The actin cross-linking protein, α-actinin, plays a crucial role in mediating furrow ingression during cytokinesis. However, the mechanism by which its dynamics are regulated during this process is poorly understood. Here we have investigated the role of calcium sensitivity of α-actinin in the regulation of its dynamics by generating a functional calcium-insensitive mutant (EFM). GFP-tagged EFM (EFM-GFP) localized to the equatorial regions during cell division. However, the maximal equatorial accumulation of EFM-GFP was significantly smaller in comparison to α-actinin-GFP when it was expressed in normal cells and cells depleted of endogenous α-actinin. No apparent defects in cytokinesis were observed in these cells. However, F-actin levels at the equator were significantly reduced in cells expressing EFM-GFP as compared with α-actinin-GFP at furrow initiation but were recovered during furrow ingression. These results suggest that calcium sensitivity of α-actinin is required for its equatorial accumulation that is crucial for the initial equatorial actin assembly but is dispensable for cytokinesis. Equatorial RhoA localization was not affected by EFM-GFP overexpression, suggesting that equatorial actin assembly is predominantly driven by the RhoA-dependent mechanism. Our observations shed new light on the role and regulation of the accumulation of pre-existing actin filaments in equatorial actin assembly during cytokinesis.     

A role for sphingomyelin-rich lipid domains in the accumulation of phosphatidylinositol-4,5-bisphosphate to the cleavage furrow during cytokinesis

Cytokinesis is a crucial step in the creation of two daughter cells by the formation and ingression of the cleavage furrow. Here, we show that sphingomyelin (SM), one of the major sphingolipids in mammalian cells, is required for the localization of phosphatidylinositol-4,5-bisphosphate (PIP(2)) to the cleavage furrow during cytokinesis. Real-time observation with a labeled SM-specific protein, lysenin, revealed that SM is concentrated in the outer leaflet of the furrow at the time of cytokinesis. Superresolution fluorescence microscopy analysis indicates a transbilayer colocalization between the SM-rich domains in the outer leaflet and PIP(2)-rich domains in the inner leaflet of the plasma membrane. The depletion of SM disperses PIP(2) and inhibits the recruitment of the small GTPase RhoA to the cleavage furrow, leading to abnormal cytokinesis. These results suggest that the formation of SM-rich domains is required for the accumulation of PIP(2) to the cleavage furrow, which is a prerequisite for the proper translocation of RhoA and the progression of cytokinesis.     

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 requirement for ARF6 during the completion of cytokinesis

During cancer development, coordinated changes in cell motility and cell cycle progression are required for the gradual transformation of normal cells into cancer cells. Previous studies have shown that ARF6 is a critical regulator of epithelial cell integrity and motility via its role in membrane movement and actin-based cytoskeletal remodeling. Recently, we have found that ARF6 also plays a role during cell division. It localizes to the cleavage furrow and midbody of cells during mitosis, and its activity is regulated during cytokinesis. Here, we investigate the requirement for ARF6 during mitosis and find that depletion of ARF6 using RNA interference disrupts the completion of cytokinesis. This finding demonstrates that ARF6 is essential during the final stages of cytokinesis. In addition, we have identified Ku70, a DNA-binding protein that is required for DNA damage repair, as a new ARF6-interacting protein and found that it is part of a complex with ARF6, especially during mitosis. These results clarify the importance of ARF6 activity during cytokinesis and begin to reveal other molecules that may contribute to the function of ARF6.     

Endocytosis resumes during late mitosis and is required for cytokinesis

Recent work has underscored the importance of membrane trafficking events during cytokinesis. For example, targeted membrane secretion occurs at the cleavage furrow in animal cells, and proteins that regulate endocytosis also influence the process of cytokinesis. Nonetheless, the prevailing dogma is that endosomal membrane trafficking ceases during mitosis and resumes after cell division is complete. In this study, we have characterized endocytic membrane trafficking events that occur during mammalian cell cytokinesis. We have found that, although endocytosis ceases during the early stages of mitosis, it resumes during late mitosis in a temporally and spatially regulated pattern as cells progress from anaphase to cytokinesis. Using fixed and live cell imaging, we have found that, during cleavage furrow ingression, vesicles are internalized from the polar region and subsequently trafficked to the midbody area during later stages of cytokinesis. In addition, we have demonstrated that cytokinesis is inhibited when clathrin-mediated endocytosis is blocked using a series of dominant negative mutants. In contrast to previous thought, we conclude that endocytosis resumes during the later stages of mitosis, before cytokinesis is completed. Furthermore, based on our findings, we propose that the proper regulation of endosomal membrane traffic is necessary for the successful completion of cytokinesis.     

Membrane phospholipid dynamics during cytokinesis: regulation of actin filament assembly by redistribution of membrane surface phospholipid

To study molecular motion and function of membrane phospholipids, we have developed various probes which bind specifically to certain phospholipids. Using a novel peptide probe, RoO9-0198, which binds specifically to phosphatidylethanolamine (PE) in biological membranes, we have analyzed the cell surface movement of PE in dividing CHO cells. We found that PE was exposed on the cell surface specifically at the cleavage furrow during the late telophase of cytokinesis. PE was exposed on the cell surface only during the late telophase and no alteration in the distribution of the plasma membranebound peptide was observed during the cytokinesis, suggesting that the surface exposure of PE reflects the enhanced transbilayer movement of PE at the cleavage furrow. Furthermore, cell surface immobilization of PE induced by adding of the cyclic peptide coupled with streptavidin to prometaphase cells effectively blocked the cytokinesis at late telophase. The peptide-streptavidin complex bound specifically to cleavage furrow and inhibited both actin filament disassembly at cleavage furrow and subsequent plasma membrane fusion. Binding of the peptide complex to interphase cells also induced immediate disassembly of stress fibers followed by assembly of cortical actin filaments to the local area of plasma membrane where the peptide complex bound. The cytoskeletal reorganizations caused by the peptide complex were fully reversible; removal of the surface-bound peptide complex by incubating with PE-containing liposome caused gradual disassembly of the cortical actin filaments and subsequent formation of stress fibers. These observations suggest that the redistribution of plasma membrane phospholipids act as a regulator of actin cytoskeleton organization and may play a crucial role in mediating a coordinate movement between plasma membrane and actin cytoskeleton to achieve successful cell division.     

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.     

On the mechanism of cleavage furrow ingression in Dictyostelium

The ability of Dictyostelium cells to divide without myosin II in a cell cycle-coupled manner has opened two questions about the mechanism of cleavage furrow ingression. First, are there other possible functions for myosin II in this process except for generating contraction of the furrow by a sliding filament mechanism? Second, what could be an alternative mechanical basis for the furrowing? Using aberrant changes of the cell shape and anomalous localization of the actin-binding protein cortexillin I during asymmetric cytokinesis in myosin II-deficient cells as clues, it is proposed that myosin II filaments act as a mechanical lens in cytokinesis. The mechanical lens serves to focus the forces that induce the furrowing to the center of the midzone, a cortical region where cortexillins are enriched in dividing cells. Additionally, continual disassembly of a filamentous actin meshwork at the midzone is a prerequisite for normal ingression of the cleavage furrow and a successful cytokinesis. If this process is interrupted, as it occurs in cells that lack cortexillins, an overassembly of filamentous actin at the midzone obstructs the normal cleavage. Disassembly of the crosslinked actin network can generate entropic contractile forces in the cortex, and may be considered as an alternative mechanism for driving ingression of the cleavage furrow. Instead of invoking different types of cytokinesis that operate under attached and unattached conditions in Dictyostelium, it is anticipated that these cells use a universal multifaceted mechanism to divide, which is only moderately sensitive to elimination of its constituent mechanical processes.