CIF-1, a shared subunit of the COP9/signalosome and eukaryotic initiation factor 3 complexes, regulates MEL-26 levels in the Caenorhabditis elegans embryo

The COP9/signalosome (CSN) is an evolutionarily conserved macromolecular complex that regulates the cullin-RING ligase (CRL) class of E3 ubiquitin ligases, primarily by removing the ubiquitin-like protein Nedd8 from the cullin subunit. In the Caenorhabditis elegans embryo, the CSN controls the degradation of the microtubule-severing protein MEI-1 through CUL-3 deneddylation. However, the molecular mechanisms of CSN function and its subunit composition remain to be elucidated. Here, using a proteomic approach, we have characterized the CSN and CUL-3 complexes from C. elegans embryos. We show that the CSN physically interacts with the CUL-3-based CRL and regulates its activity by counteracting the autocatalytic instability of the substrate-specific adaptor MEL-26. Importantly, we identified the uncharacterized protein K08F11.3/CIF-1 (for CSN-eukaryotic initiation factor 3 [eIF3]) as a stoichiometric and functionally important subunit of the CSN complex. CIF-1 appears to be the only ortholog of Csn7 encoded by the C. elegans genome, but it also exhibits extensive sequence similarity to eIF3m family members, which are required for the initiation of protein translation. Indeed, CIF-1 binds eIF-3.F and inactivation of cif-1 impairs translation in vivo. Taken together, our results indicate that CIF-1 is a shared subunit of the CSN and eIF3 complexes and may therefore link protein translation and degradation.     

Levels of the ubiquitin ligase substrate adaptor MEL-26 are inversely correlated with MEI-1/katanin microtubule-severing activity during both meiosis and mitosis

The MEI-1/MEI-2 microtubule-severing complex, katanin, is required for oocyte meiotic spindle formation and function in C. elegans, but the microtubule-severing activity must be quickly downregulated so that it does not interfere with formation of the first mitotic spindle. Post-meiotic MEI-1 inactivation is accomplished by two parallel protein degradation pathways, one of which requires MEL-26, the substrate-specific adaptor that recruits MEI-1 to a CUL-3 based ubiquitin ligase. Here we address the question of how MEL-26 mediated MEI-1 degradation is triggered only after the completion of MEI-1's meiotic function. We find that MEL-26 is present only at low levels until the completion of meiosis, after which protein levels increase substantially, likely increasing the post-meiotic degradation of MEI-1. During meiosis, MEL-26 levels are kept low by the action of another type of ubiquitin ligase, which contains CUL-2. However, we find that the low levels of meiotic MEL-26 have a subtle function, acting to moderate MEI-1 activity during meiosis. We also show that MEI-1 is the only essential target for MEL-26, and possibly for the E3 ubiquitin ligase CUL-3, but the upstream ubiquitin ligase activating enzyme RFL-1 has additional essential targets.     

UNC-89 (obscurin) binds to MEL-26, a BTB-domain protein, and affects the function of MEI-1 (katanin) in striated muscle of Caenorhabditis elegans

The ubiquitin proteasome system is involved in degradation of old or damaged sarcomeric proteins. Most E3 ubiquitin ligases are associated with cullins, which function as scaffolds for assembly of the protein degradation machinery. Cullin 3 uses an adaptor to link to substrates; in Caenorhabditis elegans, one of these adaptors is the BTB-domain protein MEL-26 (maternal effect lethal). Here we show that MEL-26 interacts with the giant sarcomeric protein UNC-89 (obscurin). MEL-26 and UNC-89 partially colocalize at sarcomeric M-lines. Loss of function or gain of function of mel-26 results in disorganization of myosin thick filaments similar to that found in unc-89 mutants. It had been reported that in early C. elegans embryos, a target of the CUL-3/MEL-26 ubiquitylation complex is the microtubule-severing enzyme katanin (MEI-1). Loss of function or gain of function of mei-1 also results in disorganization of thick filaments similar to unc-89 mutants. Genetic data indicate that at least some of the mel-26 loss-of-function phenotype in muscle can be attributed to increased microtubule-severing activity of MEI-1. The level of MEI-1 protein is reduced in an unc-89 mutant, suggesting that the normal role of UNC-89 is to inhibit the CUL-3/MEL-26 complex toward MEI-1.     

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.     

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.     

Targeting Aurora B to the Equatorial Cortex by MKlp2 Is Required for Cytokinesis

Although Aurora B is important in cleavage furrow ingression and completion during cytokinesis, the mechanism by which kinase activity is targeted to the cleavage furrow and the molecule(s) responsible for this process have remained elusive. Here, we demonstrate that an essential mitotic kinesin MKlp2 requires myosin-II for its localization to the equatorial cortex, and this event is required to recruit Aurora B to the equatorial cortex in mammalian cells. This recruitment event is also required to promote the highly focused accumulation of active RhoA at the equatorial cortex and stable ingression of the cleavage furrow in bipolar cytokinesis. Specifically, in drug-induced monopolar cytokinesis, targeting Aurora B to the cell cortex by MKlp2 is essential for cell polarization and furrow formation. Once the furrow has formed, MKlp2 further recruits Aurora B to the growing furrow. This process together with continuous Aurora B kinase activity at the growing furrow is essential for stable furrow propagation and completion. In contrast, a MKlp2 mutant defective in binding myosin-II does not recruit Aurora B to the cell cortex and does not promote furrow formation during monopolar cytokinesis. This mutant is also defective in maintaining the ingressing furrow during bipolar cytokinesis. Together, these findings reveal that targeting Aurora B to the cell cortex (or the equatorial cortex) by MKlp2 is essential for the maintenance of the ingressing furrow for successful cytokinesis.     

The C. elegans anaphase promoting complex and MBK-2/DYRK kinase act redundantly with CUL-3/MEL-26 ubiquitin ligase to degrade MEI-1 microtubule-severing activity after meiosis

The C. elegans embryo supports both meiotic and mitotic spindles, requiring careful regulation of components specific to each spindle type. The MEI-1/katanin microtubule-severing complex is required for meiosis but must be inactivated prior to mitosis. Downregulation of MEI-1 depends on MEL-26, which binds MEI-1, targeting it for degradation by the CUL-3 E3 ubiquitin ligase complex. Here we report that other protein degradation pathways, involving the anaphase promoting complex (APC) and the MBK-2/DYRK kinase, act in parallel to MEL-26 to inactivate MEI-1. At 25 degrees all mel-26(null) embryos die due to persistence of MEI-1 into mitosis, but at 15 degrees a significant portion of embryos hatch due to lower levels of ectopic MEI-1, suggesting that a redundant pathway also regulates MEI-1 degradation at 15 degrees. Previously the MBK-2/DYRK kinase was suggested to trigger MEL-26 mediated MEI-1 degradation. However, mbk-2 enhances the incomplete lethality of mel-26(null) at 15 degrees, arguing that MEL-26 acts in parallel to MBK-2. APC mutants behave similarly. In mel-26 embryos, ectopic MEI-1 remains until the onset of gastrulation, but in mbk-2; apc embryos, MEI-1 only persists through the first mitosis. We propose that mbk-2 and apc couple the initial phase of MEI-1 degradation to meiotic exit, after which MEL-26 completes MEI-1 degradation.     

Endomitotic Megakaryocytes that Form a Bipolar Spindle Exhibit Cleavage Furrow Ingression Followed by Furrow Regression

Megakaryocyte (MK) differentiation is marked by the development of progressive polyploidy, due to repeated incomplete cell cycles in which mitosis is aborted during anaphase, a process termed endomitosis. We have postulated that anaphase in endomitotic MKs diverges from diploid mitosis at a point distal to the assembly of the midzone, possibly involving impaired cleavage furrow progression. To define the extent of furrow initiation and ingression in endomitosis, we performed time-lapse imaging of MKs expressing yellow fluorescent protein (YFP)-tubulin and monitored shape change as they progressed through anaphase. We found that in early endomitotic cells that have a bipolar spindle, cleavage furrows form that can undergo significant ingression, but furrows regress to produce polyploid cells. Compared to cells that divide, cells that exhibit furrow regression have a slower rate of furrow ingression and do not furrow as deeply. More highly polyploid MKs undergoing additional endomitotic cycles also show measurable furrowing that is followed by regression, but the magnitude of the shape change is less than seen in the early MKs. This suggests that in the earliest endomitotic cycles when there is formation of a bipolar spindle, the failure of cytokinesis occurs late, following assembly and initial constriction of the actin/myosin ring, whereas in endomitotic MKs that are already polyploid there is secondary inhibition of furrow progression. This behavior of furrow ingression followed by regression may explain why midbody remnants are occasionally observed in polyploid MKs. This finding has important implications for the potential mechanisms for cytokinesis failure in endomitosis.     

Cytokinesis failure in clathrin-minus cells is caused by cleavage furrow instability

The role of membrane traffic during cell division has only recently begun to be investigated. A growing number of trafficking proteins seem to be involved in the successful completion of cytokinesis. Clathrin was the first trafficking protein to be shown to be essential for cytokinesis in Dictyostelium. Here we investigate the nature of the cytokinesis defect of Dictyostelium clathrin null cells. We found that adherent clathrin null cells do form cleavage furrows but cannot maintain a consistent rate of furrow ingression. Clathrin null cells are completely defective in cytokinesis when placed in suspension. In these conditions, the cells develop an abnormal division morphology that consists of two lateral "furrows" on either side of a bulging equatorial region. Cells expressing GFP-myosin II were examined at various stages of cytokinesis. Clathrin null cells show multiple defects in myosin organization and localization that parallel the striking failure in furrow morphology. We postulate that this morphology is the result of contraction at the rear of the presumptive daughter cells in concert with incomplete furrow 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.     

Neddylation and deneddylation of CUL-3 is required to target MEI-1/Katanin for degradation at the meiosis-to-mitosis transition in C. elegans

BACKGROUND: SCF (Skp1-Cullin-F-box) complexes are a major class of E3 ligases that are required to selectively target substrates for ubiquitin-dependent degradation by the 26S proteasome. Conjugation of the ubiquitin-like protein Nedd8 to the cullin subunit (neddylation) positively regulates activity of SCF complexes, most likely by increasing their affinity for the E2 conjugated to ubiquitin. The Nedd8 conjugation pathway is required in C. elegans embryos for the ubiquitin-mediated degradation of the microtubule-severing protein MEI-1/Katanin at the meiosis-to-mitosis transition. Genetic experiments suggest that this pathway controls the activity of a CUL-3-based E3 ligase. Counteracting the Nedd8 pathway, the COP9/signalosome has been shown to promote deneddylation of the cullin subunit. However, little is known about the role of neddylation and deneddylation for E3 ligase activity in vivo. RESULTS: Here, we identified and characterized the COP9/signalosome in C. elegans and showed that it promotes deneddylation of CUL-3, a critical target of the Nedd8 conjugation pathway. As in other species, the C. elegans signalosome is a macromolecular complex containing at least six subunits that localizes in the nucleus and the cytoplasm. Reducing COP9/signalosome function by RNAi results in a failure to degrade MEI-1, leading to severe defects in microtubule-dependent processes during the first mitotic division. Intriguingly, reducing COP9/signalosome function suppresses a partial defect in the neddylation pathway; this suppression suggests that deneddylation and neddylation antagonize each other. CONCLUSIONS: We conclude that both neddylation and deneddylation of CUL-3 is required for MEI-1 degradation and propose that cycles of CUL-3 neddylation and deneddylation are necessary for its ligase activity in vivo.     

Furrow Constriction in Animal Cell Cytokinesis

Cytokinesis is the process of physical cleavage at the end of cell division; it proceeds by ingression of an acto-myosin furrow at the equator of the cell. Its failure leads to multinucleated cells and is a possible cause of tumorigenesis. Here, we calculate the full dynamics of furrow ingression and predict cytokinesis completion above a well-defined threshold of equatorial contractility. The cortical acto-myosin is identified as the main source of mechanical dissipation and active forces. Thereupon, we propose a viscous active nonlinear membrane theory of the cortex that explicitly includes actin turnover and where the active RhoA signal leads to an equatorial band of myosin overactivity. The resulting cortex deformation is calculated numerically, and reproduces well the features of cytokinesis such as cell shape and cortical flows toward the equator. Our theory gives a physical explanation of the independence of cytokinesis duration on cell size in embryos. It also predicts a critical role of turnover on the rate and success of furrow constriction. Scaling arguments allow for a simple interpretation of the numerical results and unveil the key mechanism that generates the threshold for cytokinesis completion: cytoplasmic incompressibility results in a competition between the furrow line tension and the cell poles’ surface tension.     

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.     

Completion of cytokinesis in C. elegans requires a brefeldin A-sensitive membrane accumulation at the cleavage furrow apex

BACKGROUND: The terminal phase of cytokinesis in eukaryotic cells involves breakage of the intercellular canal containing the spindle midzone and resealing of the daughter cells. Recent observations suggest that the spindle midzone is required for this process. In this study, we investigated the possibility that targeted secretion in the vicinity of the spindle midzone is required for the execution of the terminal phase of cytokinesis. RESULTS: We inhibited secretion in early C. elegans embryos by treatment with brefeldin A (BFA). Using 4D recordings of dividing cells, we showed that BFA induced stereotyped failures in the terminal phase of cytokinesis; although the furrow ingressed normally, after a few minutes the furrow completely regressed, even though spindle midzone and midbody microtubules appeared normal. In addition, using an FM1-43 membrane probe, we found that membrane accumulated locally at the apices of the late cleavage furrows that form the persisting intercellular canals between daughter cells. However, in BFA-treated embryos this membrane accumulation did not occur, which possibly accounts for the observed cleavage failures. CONCLUSIONS: We have shown that BFA disrupts the terminal phase of cytokinesis in the embryonic blastomeres of C. elegans. We observed that membrane accumulates at the apices of the late cleavage furrow by means of a BFA-sensitive mechanism. We suggest that this local membrane accumulation is necessary for the completion of cytokinesis and speculate that the spindle midzone region of animal cells is functionally equivalent to the phragmoplast of plants and acts to target secretion to the equatorial plane of a cleaving cell.     

Furrow Constriction in Animal Cell Cytokinesis

Cytokinesis is the process of physical cleavage at the end of cell division; it proceeds by ingression of an acto-myosin furrow at the equator of the cell. Its failure leads to multinucleated cells and is a possible cause of tumorigenesis. Here, we calculate the full dynamics of furrow ingression and predict cytokinesis completion above a well-defined threshold of equatorial contractility. The cortical acto-myosin is identified as the main source of mechanical dissipation and active forces. Thereupon, we propose a viscous active nonlinear membrane theory of the cortex that explicitly includes actin turnover and where the active RhoA signal leads to an equatorial band of myosin overactivity. The resulting cortex deformation is calculated numerically, and reproduces well the features of cytokinesis such as cell shape and cortical flows toward the equator. Our theory gives a physical explanation of the independence of cytokinesis duration on cell size in embryos. It also predicts a critical role of turnover on the rate and success of furrow constriction. Scaling arguments allow for a simple interpretation of the numerical results and unveil the key mechanism that generates the threshold for cytokinesis completion: cytoplasmic incompressibility results in a competition between the furrow line tension and the cell poles’ surface tension.     

A Highlights from MBoC Selection: Calcium spikes accompany cleavage furrow ingression and cell separation during fission yeast cytokinesis

The role of calcium signaling in cytokinesis has long remained ambiguous. Past studies of embryonic cell division discovered that calcium concentration increases transiently at the division plane just before cleavage furrow ingression, suggesting that these calcium transients could trigger contractile ring constriction. However, such calcium transients have only been found in animal embryos and their function remains controversial. We explored cytokinetic calcium transients in the fission yeast Schizosaccharomyces pombe by adopting GCaMP, a genetically encoded calcium indicator, to determine the intracellular calcium level of this model organism. We validated GCaMP as a highly sensitive calcium reporter in fission yeast, allowing us to capture calcium transients triggered by osmotic shocks. We identified a correlation between the intracellular calcium level and cell division, consistent with the existence of calcium transients during cytokinesis. Using time-lapse microscopy and quantitative image analysis, we discovered calcium spikes both at the start of cleavage furrow ingression and the end of cell separation. Inhibition of these calcium spikes slowed the furrow ingression and led to frequent lysis of daughter cells. We conclude that like the larger animal embryos, fission yeast triggers calcium transients that may play an important role in cytokinesis (197).     

PAR-4 and anillin regulate myosin to coordinate spindle and furrow position during asymmetric division

During asymmetric cell division, the mitotic spindle and polarized myosin can both determine the position of the cytokinetic furrow. However, how cells coordinate signals from the spindle and myosin to ensure that cleavage occurs through the spindle midzone is unknown. Here, we identify a novel pathway that is essential to inhibit myosin and coordinate furrow and spindle positions during asymmetric division. In Caenorhabditis elegans one-cell embryos, myosin localizes at the anterior cortex whereas the mitotic spindle localizes toward the posterior. We find that PAR-4/LKB1 impinges on myosin via two pathways, an anillin-dependent pathway that also responds to the cullin CUL-5 and an anillin-independent pathway involving the kinase PIG-1/MELK. In the absence of both PIG-1/MELK and the anillin ANI-1, myosin accumulates at the anterior cortex and induces a strong displacement of the furrow toward the anterior, which can lead to DNA segregation defects. Regulation of asymmetrically localized myosin is thus critical to ensure that furrow and spindle midzone positions coincide throughout cytokinesis.     

Reassessing the role and dynamics of nonmuscle myosin II during furrow formation in early Drosophila embryos

The early Drosophila embryo undergoes two distinct membrane invagination events believed to be mechanistically related to cytokinesis: metaphase furrow formation and cellularization. Both involve actin cytoskeleton rearrangements, and both have myosin II at or near the forming furrow. Actin and myosin are thought to provide the force driving membrane invagination; however, membrane addition is also important. We have examined the role of myosin during these events in living embryos, with a fully functional myosin regulatory light-chain-GFP chimera. We find that furrow invagination during metaphase and cellularization occurs even when myosin activity has been experimentally perturbed. In contrast, the basal closure of the cellularization furrows and the first cytokinesis after cellularization are highly dependent on myosin. Strikingly, when ingression of the cellularization furrow is experimentally inhibited by colchicine treatment, basal closure still occurs at the appropriate time, suggesting that it is regulated independently of earlier cellularization events. We have also identified a previously unrecognized reservoir of particulate myosin that is recruited basally into the invaginating furrow in a microfilament-independent and microtubule-dependent manner. We suggest that cellularization can be divided into two distinct processes: furrow ingression, driven by microtubule mediated vesicle delivery, and basal closure, which is mediated by actin/myosin based constriction.     

CAR-1, a protein that localizes with the mRNA decapping component DCAP-1, is required for cytokinesis and ER organization in Caenorhabditis elegans embryos

The division of one cell into two requires the coordination of multiple components. We describe a gene, car-1, whose product may provide a link between disparate cellular processes. Inhibition of car-1 expression in Caenorhabditis elegans embryos causes late cytokinesis failures: cleavage furrows ingress but subsequently regress and the spindle midzone fails to form, even though midzone components are present. The localized accumulation of membrane that normally develops at the apex of the cleavage furrow during the final phase of cytokinesis does not occur and organization of the endoplasmic reticulum is aberrant, indicative of a disruption in membrane trafficking. The car-1 gene has homologues in a number of species, including proteins that associate with RNA binding proteins. CAR-1 localizes to P-granules (germ-line specific ribonucleoprotein particles) and discrete, developmentally regulated cytoplasmic foci. These foci also contain DCAP-1, a protein involved in decapping mRNAs. Thus, CAR-1, a protein likely to be associated with RNA metabolism, plays an essential role in the late stage of cytokinesis, suggesting a novel link between RNA, membrane trafficking and cytokinesis in the C. elegans embryo.