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.     

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.     

Annexin A2 is required for the early steps of cytokinesis

Cytokinesis requires the formation of an actomyosin contractile ring between the two sets of sister chromatids. Annexin A2 is a calcium- and phospholipid-binding protein implicated in cortical actin remodeling. We report that annexin A2 accumulates at the equatorial cortex at the onset of cytokinesis and depletion of annexin A2 results in cytokinetic failure, due to a defective cleavage furrow assembly. In the absence of annexin A2, the small GTPase RhoA—which regulates cortical cytoskeletal rearrangement—fails to form a compact ring at the equatorial plane. Furthermore, annexin A2 is required for cortical localization of the RhoGEF Ect2 and to maintain the association between the equatorial cortex and the central spindle. Our results demonstrate that annexin A2 is necessary in the early phase of cytokinesis. We propose that annexin A2 participates in central spindle–equatorial plasma membrane communication.     

Astral signals spatially bias cortical myosin recruitment to break symmetry and promote cytokinesis

BACKGROUND: After anaphase, the segregated chromosomes are sequestered by cytokinesis into two separate daughter cells by a cleavage furrow formed by the actomyosin-based contractile ring. The failure to properly position the contractile ring between the segregated chromosomes can result in aneuploidy. In both C. elegans embryos and human cells, the central spindle regulates division-plane positioning in parallel with a second pathway that involves astral microtubules. RESULTS: We combined genetic and pharmacological manipulations with live cell imaging to spatially separate the two division cues in a single cell. We demonstrate that the two pathways for furrow formation are mechanistically and genetically distinct. By following the distribution of green fluorescent protein (GFP)-tagged nonmuscle myosin, we have found that the astral pathway for furrow formation involves the negative regulation of cortical myosin recruitment. An asymmetrically positioned spindle induces the asymmetric cortical accumulation of myosin. This cortical myosin behaves as a coherent contractile network. If the cortical network is nonuniform over the cell, the cortical contractile elements coalesce into a single furrow. This coalescence requires interconnections among contractile elements. CONCLUSIONS: We conclude that the two pathways of cleavage-furrow formation are mechanistically distinct. In particular, we conclude that the astral pathway for cleavage-furrow formation involves the negative regulation of myosin distribution by astral cues.     

Cortical PAR polarity proteins promote robust cytokinesis during asymmetric cell division

Cytokinesis, the physical division of one cell into two, is thought to be fundamentally similar in most animal cell divisions and driven by the constriction of a contractile ring positioned and controlled solely by the mitotic spindle. During asymmetric cell divisions, the core polarity machinery (partitioning defective [PAR] proteins) controls the unequal inheritance of key cell fate determinants. Here, we show that in asymmetrically dividing Caenorhabditis elegans embryos, the cortical PAR proteins (including the small guanosine triphosphatase CDC-42) have an active role in regulating recruitment of a critical component of the contractile ring, filamentous actin (F-actin). We found that the cortical PAR proteins are required for the retention of anillin and septin in the anterior pole, which are cytokinesis proteins that our genetic data suggest act as inhibitors of F-actin at the contractile ring. Collectively, our results suggest that the cortical PAR proteins coordinate the establishment of cell polarity with the physical process of cytokinesis during asymmetric cell division to ensure the fidelity of daughter cell formation.     

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.     

A global, myosin light chain kinase-dependent increase in myosin II contractility accompanies the metaphase-anaphase transition in sea urchin eggs

Myosin II is the force-generating motor for cytokinesis, and although it is accepted that myosin contractility is greatest at the cell equator, the temporal and spatial cues that direct equatorial contractility are not known. Dividing sea urchin eggs were placed under compression to study myosin II-based contractile dynamics, and cells manipulated in this manner underwent an abrupt, global increase in cortical contractility concomitant with the metaphase-anaphase transition, followed by a brief relaxation and the onset of furrowing. Prefurrow cortical contractility both preceded and was independent of astral microtubule elongation, suggesting that the initial activation of myosin II preceded cleavage plane specification. The initial rise in contractility required myosin light chain kinase but not Rho-kinase, but both signaling pathways were required for successful cytokinesis. Last, mobilization of intracellular calcium during metaphase induced a contractile response, suggesting that calcium transients may be partially responsible for the timing of this initial contractile event. Together, these findings suggest that myosin II-based contractility is initiated at the metaphase-anaphase transition by Ca2+-dependent myosin light chain kinase (MLCK) activity and is maintained through cytokinesis by both MLCK- and Rho-dependent signaling. Moreover, the signals that initiate myosin II contractility respond to specific cell cycle transitions independently of the microtubule-dependent cleavage stimulus.     

Two phases of astral microtubule activity during cytokinesis in C. elegans embryos

Microtubules of the mitotic spindle are believed to provide positional cues for the assembly of the actin-based contractile ring and the formation of the subsequent cleavage furrow during cytokinesis. In Caenorhabditis elegans, astral microtubules have been thought to inhibit cortical contraction outside the cleavage furrow. Here, we demonstrate by live imaging and RNA interference (RNAi) that astral microtubules play two distinct roles in initiating cleavage furrow formation. In early anaphase, microtubules are required for contractile ring assembly; in late anaphase, microtubules show different cortical behavior and seem to suppress cortical contraction at the poles, as suggested in previous studies. These two distinct phases of microtubule behavior depend on distinct regulatory pathways, one involving the gamma-tubulin complex and the other requiring aurora-A kinase. We propose that temporal and spatial regulation of two distinct phases of astral microtubule behavior is crucial in specifying the position and timing of furrowing.     

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.     

The terminal phase of cytokinesis in the Caenorhabditis elegans early embryo requires protein glycosylation

RNA interference (RNAi) was used to characterize the requirement of protein glycosylation for cell membrane stability during cytokinesis in the early embryo. This screen targeted 13 enzymes or components of polypeptide sugar transferases that initiate either N-glycosylation or three different pathways of O-glycosylation. RNAi of genes in the mucin-type and epidermal growth factor-fringe glycosylation pathways did not affect cytokinesis. However, embryos deficient in N-glycosylation exhibited a variable inability to complete cytokinesis. The most potent block in early embryonic cell division was obtained by RNAi of the polypeptide xylose transferase (ppXyl-T), which is required to initiate the proteoglycan modification pathway. Two generations of ppXyl-T RNAi-feeding treatment reduced the body size, mobility, brood size, and life span of adult animals. Embryos escaping ppXyl-T and Gal-T2 RNAi lethality develop to adulthood but have cytokinesis-deficient offspring, suggesting that glycosyltransferases in the proteoglycan pathway are maternal proteins in the early embryo. Gal-T2::GFP fusions and anti-Gal-T2 antibodies revealed a perinuclear staining pattern, consistent with the localization of the Golgi apparatus. RNAi in green fluorescent protein (GFP)-tagged strains to follow tubulin, PIE-1, and chromatin showed that deficient proteoglycan biosynthesis uncouples the stability of newly formed cell membranes from cytokinesis, whereas cleavage furrow initiation, mitotic spindle function, karyokinesis, and partitioning of intrinsic components are intact.     

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.     

The role of microtubules in contractile ring function.

During cytokinesis, a cortical contractile ring forms around a cell, constricts to a stable tight neck and terminates in separation of the daughter cells. At first cleavage, Ilyanassa obsoleta embryos form two contractile rings simultaneously. The cleavage furrow (CF), in the animal hemisphere between the spindle poles, constricts to a stable tight neck and separates the daughter cells. The third polar lobe constriction (PLC-3), in the vegetal hemisphere below the spindle, constricts to a transient tight neck, but then relaxes, allowing the polar lobe cytoplasm to merge with one daughter cell. Eggs exposed to taxol, a drug that stabilizes microtubules, before the CF or the PLC-3 develop, fail to form CFs, but form stabilized tight PLCs. Eggs exposed to taxol at the time of PLC-3 formation develop varied numbers of constriction rings in their animal hemispheres and one PLC in their vegetal hemisphere, none of which relax. Eggs exposed to taxol after PLC-3 initiation form stabilized tight CFs and PLCs. At maximum constriction, control embryos display immunolocalization of nonextractable alpha-tubulin in their CFs, but not in their PLCs, and reveal, via electron microscopy, many microtubules extending through their CFs, but not through their PLCs. Embryos which form stabilized tightly constricted CFs and PLCs in the presence of taxol display immunolocalization of nonextractable alpha-tubulin in both constrictions and show many polymerized microtubules extending through both CFs and PLCs. These results suggest that the extension of microtubules through a tight contractile ring may be important for stabilizing that constriction and facilitating subsequent cytokinesis.     

Terminal cytokinesis events uncovered after an RNAi screen

Much of our understanding of animal cell cytokinesis centers on the regulation of the equatorial acto-myosin contractile ring that drives the rapid ingression of a deep cleavage furrow. However, the central part of the mitotic spindle collapses to a dense structure that impedes the furrow and keeps the daughter cells connected via an intercellular bridge. Factors involved in the formation, maintenance, and resolution of this bridge are largely unknown. Using a library of 7,216 double-stranded RNAs (dsRNAs) representing the conserved genes of Drosophila, we performed an RNA interference (RNAi) screen for cytokinesis genes in Schneider's S2 cells. We identified both familiar and novel genes whose inactivation induced a multi-nucleate phenotype. Using live video microscopy, we show that three genes: anillin, citron-kinase (CG10522), and soluble N-ethylmaleimide sensitive factor (NSF) attachment protein (alpha-SNAP), are essential for the terminal (post-furrowing) events of cytokinesis. anillin RNAi caused gradual disruption of the intercellular bridge after furrowing; citron-kinase RNAi destabilized the bridge at a later stage; alpha-SNAP RNAi caused sister cells to fuse many hours later and by a different mechanism. We have shown that the stability of the intercellular bridge is essential for successful cytokinesis and have defined genes contributing to this stability.     

The aurora-related kinase AIR-2 recruits ZEN-4/CeMKLP1 to the mitotic spindle at metaphase and is required for cytokinesis

BACKGROUND: The Aurora/Ipl1p-related kinase AIR-2 is required for mitotic chromosome segregation and cytokinesis in early Caenorhabditis elegans embryos. Previous studies have relied on non-conditional mutations or RNA-mediated interference (RNAi) to inactivate AIR-2. It has therefore not been possible to determine whether AIR-2 functions directly in cytokinesis or if the cleavage defect results indirectly from the failure to segregate DNA. One intriguing hypothesis is that AIR-2 acts to localize the mitotic kinesin-like protein ZEN-4 (also known as CeMKLP1), which later functions in cytokinesis. RESULTS: Using conditional alleles, we established that AIR-2 is required at metaphase or early anaphase for normal segregation of chromosomes, localization of ZEN-4, and cytokinesis. ZEN-4 is first required late in cytokinesis, and also functions to maintain cell separation through much of the subsequent interphase. DNA segregation defects alone were not sufficient to disrupt cytokinesis in other mutants, suggesting that AIR-2 acts specifically during cytokinesis through ZEN-4. AIR-2 and ZEN-4 shared similar genetic interactions with the formin homology (FH) protein CYK-1, suggesting that AIR-2 and ZEN-4 function in a single pathway, in parallel to a contractile ring pathway that includes CYK-1. Using in vitro co-immunoprecipitation experiments, we found that AIR-2 and ZEN-4 interact directly. CONCLUSIONS: AIR-2 has two functions during mitosis: one in chromosome segregation, and a second, independent function in cytokinesis through ZEN-4. AIR-2 and ZEN-4 may act in parallel to a second pathway that includes CYK-1.     

Dynamic rearrangement of surface proteins is essential for cytokinesis

Cytokinesis is a complex process that involves dynamic cortical rearrangement. Our recent time-lapse recordings of the mouse egg unexpectedly revealed a high motility of the second polar body (2pb). Experiments to address its underlying mechanism show that neither mechanical compression by the zona pellucida nor the connection via the mid-body is required for the 2pb movement. Time-lapse recordings establish that the 2pb moves together with the cell membrane. These recordings, in which cell surface proteins are labeled with fluorescent latex-microbeads or monovalent antibodies against whole mouse proteins, indicate that the majority of the surface proteins dynamically accumulate in the cleavage furrow at every cell division. Comparable dynamics of the cell surface proteins, and specifically of E-cadherin, are also observed in cultured epithelial cells. The surface protein dynamics are closely correlated with, and dependent on, those of the underlying cortical actin. The cortical actin network may form a scaffold for membrane proteins and thereby transfer them during contractile ring formation toward the cleavage furrow. Immobilization of surface proteins by tetravalent lectin-mediated crosslinking results in the failure of cleavage, demonstrating that the observed protein dynamics are essential for cytokinesis. We propose that dynamic rearrangement of the cell surface proteins is a common feature of cytokinesis, playing a key role in modifying the mechanical properties of the cell membrane during cortical ingression.     

Cytokinesis in the C. elegans embryo: regulating contractile forces and a late role for the central spindle

Genetic and molecular studies in the nematode Caenorhabditis elegans have identified multiple essential pathways that regulate and execute cytokinesis in early embryonic cells. These pathways influence both the microfilament cytoskeleton and the microtubule cytoskeleton. Microfilaments are enriched throughout the cell cortex at all times during the cell cycle in embryonic cells. Cortical microfilaments are required for multiple processes in embryonic cells, including polar body extrusion during meiosis, anterior-posterior axis specification by the sperm-donated microtubule-organizing center, and cytokinesis during mitosis. In addition to contractile apparatus proteins that are required positively for cleavage furrow ingression, the Nedd8 ubiquitin-like protein modification pathway negatively regulates contractile forces outside the cleavage furrow during cytokinesis. Another pathway that acts positively during cytokinesis involves the mitotic spindle. The central spindle, where anti-parallel non-kinetochore microtubules overlap and are cross-linked, is required for a late step in cytokinesis, and other pathway(s) involved in membrane addition during cytokinesis may also require the central spindle. The amenability of C. elegans to classical genetics, the ease of reducing gene function with RNA interference, the completion of the genome sequence, and the availability of transgenic GFP fusion proteins that render the cytoskeleton fluorescent, all serve to make the early worm embryo an especially promising system for further advances in the identification of cytokinesis pathways, and in defining their interactions.     

The astral relaxation theory of cytokinesis revisited

Cytokinesis in animal cells is accomplished by the active constriction of the equatorial regions of a cell by an actomyosin-containing contractile ring. The mitotic apparatus specifies the position and orientation of the furrow such that the mitotic spindle is always bisected. Global cortical contractions occur in the cortex of a cell prior to cytokinesis that are independent of the presence of the mitotic apparatus. It was proposed some years ago that the asters of the mitotic apparatus could act to relax the preformed cortical tension in their vicinity. This would produce a differential in tension between the equatorial regions and the adjacent regions of the cortex so that the equatorial regions would contract, forming a cleavage furrow. It can be shown that, as it stands, this theory cannot explain cleavage. However, if cortical contractile elements are assumed to be laterally mobile in the plane of the cortex, then the astral relaxation theory can account for many of the aspects of cleavage, including the formation of the contractile ring. Similar schemes may account for the behaviour of the lamellapodia of motile cells.     

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.     

Relationship between p62 and p56, two proteins of the mammalian cortical granule envelope, and hyalin, the major component of the echinoderm hyaline layer, in hamsters

Mammalian cortical granules contain two polypeptides (p62 and p56) that are incorporated into the cortical granule envelope after fertilization and function in cleavage of the zygote and the preimplantation blastomeres. Since the echinoderm hyaline layer and mammalian cortical granule envelope are analogous, and since the hyaline layer protein, hyalin, functions in early echinoderm embryogenesis, this study was done to determine whether p62 and p56 and/or other components of the mammalian cortical granule envelope are related to hyalin. A polyclonal antibody (IL2) against purified S. purpuratus hyalin was shown by confocal scanning laser microscopy to bind to hamster cortical granules and to the cortical granule envelope of fertilized hamster oocytes and preimplantation embryos up to the blastocyst stage. In immunoblots, IL2 bound only to 62- and 56-kDa cortical granule proteins that were incorporated into the cortical granule envelope after fertilization. IL2 binding antigens appeared to be resynthesized by preimplantation embryos starting at the 2-cell stage of development. In vivo treatment of 2-cell-stage hamster embryos with IL2 inhibited blastomere cleavage, but treatment of morulae did not inhibit blastocyst implantation. These results support the idea that the mammalian cortical granule envelope proteins, p62/p56, share a common antigenic epitope(s) with echinoderm hyalin, and that p62/p56, like hyalin, play a role in early embryogenesis.     

Mechanics and regulation of cytokinesis

Recent advances are revealing quantitative aspects of cytokinesis. Further, genetic analyses and cell imaging are providing insights into the molecular dynamics of cleavage furrow ingression as well as further refining our knowledge of the zones of the mitotic spindle that regulate the contractile properties of the overlying cortex. Ultimately, however, cortical mechanics are the result of signals that emanate from the mitotic spindle. A genuine quantitative understanding of cytokinesis must include a thorough analysis of the mechanical properties of the cortex and how signals modify these properties to dictate a well-controlled, error-free cytokinesis.