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An anillin-Ect2 complex stabilizes central spindle microtubules at the cortex during cytokinesis.

ABSTRACT: Cytokinesis occurs due to the RhoA-dependent ingression of an actomyosin ring. During anaphase, the Rho GEF (guanine nucleotide exchange factor) Ect2 is recruited to the central spindle via its interaction with MgcRacGAP/Cyk-4, and activates RhoA in the central plane of the cell. Ect2 also localizes to the cortex, where it has access to RhoA. The N-terminus of Ect2 binds to Cyk-4, and the C-terminus contains conserved DH (Dbl homologous) and PH (Pleckstrin Homology) domains with GEF activity. The PH domain is required for Ect2's cortical localization, but its molecular function is not known. In cultured human cells, we found that the PH domain interacts with anillin, a contractile ring protein that scaffolds actin and myosin and interacts with RhoA. The anillin-Ect2 interaction may require Ect2's association with lipids, since a novel mutation in the PH domain, which disrupts phospholipid association, weakens their interaction. An anillin-RacGAP50C (homologue of Cyk-4) complex was previously described in Drosophila, which may crosslink the central spindle to the cortex to stabilize the position of the contractile ring. Our data supports an analogous function for the anillin-Ect2 complex in human cells and one hypothesis is that this complex has functionally replaced the Drosophila anillin-RacGAP50C complex. Complexes between central spindle proteins and cortical proteins could regulate the position of the contractile ring by stabilizing microtubule-cortical interactions at the division plane to ensure the generation of active RhoA in a discrete zone.

SUBMITTER: Frenette P

PROVIDER: S-EPMC3325936 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Active Ran regulates anillin function during cytokinesis.

Project description:Cytokinesis cleaves a cell into two daughters at the end of mitosis, and must be spatially coordinated with chromosome segregation to prevent aneuploidy. The dogma is that the mitotic spindle governs the assembly and constriction of an actomyosin ring. Here, we reveal a function for active Ran in spatially restricting the ring. Our model is that during anaphase, "free" importins, whose gradient inversely correlates with active Ran and chromatin position, function as a molecular ruler for the recruitment and localization of anillin, a contractile protein and a crucial regulator of cytokinesis. We found that decreasing Ran-GTP levels or tethering active Ran to the equatorial membrane affects anillin's localization and causes cytokinesis phenotypes. Anillin contains a conserved nuclear localization signal (NLS) at its C-terminus that binds to importin-? and is required for cortical polarity and cytokinesis. Mutating the NLS decreases anillin's cortical affinity, causing it to be more dominantly regulated by microtubules. Anillin contains a RhoA-GTP binding domain, which autoinhibits the NLS and the neighboring microtubule-binding domain, and RhoA-GTP binding may relieve this inhibition during mitosis. Retention of the C-terminal NLS in anillin homologues suggests that this is a conserved mechanism for controlling anillin function.

| S-EPMC5683762 | biostudies-literature

Rho-dependent control of anillin behavior during cytokinesis.

Project description: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.

| S-EPMC2213597 | biostudies-literature

Importin binding mediates the intramolecular regulation of anillin during cytokinesis.

Project description:Cytokinesis occurs by the ingression of an actomyosin ring that cleaves a cell into two daughters. This process is tightly controlled to avoid aneuploidy, and we previously showed that active Ran coordinates ring positioning with chromatin. Active Ran is high around chromatin, and forms an inverse gradient to cargo-bound importins. We found that the ring component anillin contains a nuclear localization signal (NLS) that binds to importin and is required for its function during cytokinesis. Here we reveal the mechanism whereby importin binding favors a conformation required for anillin's recruitment to the equatorial cortex. Active RhoA binds to the RhoA-binding domain causing an increase in accessibility of the nearby C2 domain containing the NLS. Importin binding subsequently stabilizes a conformation that favors interactions for cortical recruitment. In addition to revealing a novel mechanism for the importin-mediated regulation of a cortical protein, we also show how importin binding positively regulates protein function.

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Anillin acts as a bifunctional linker coordinating midbody ring biogenesis during cytokinesis.

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| S-EPMC3698874 | biostudies-literature

Central-spindle microtubules are strongly coupled to chromosomes during both anaphase A and anaphase B.

Project description:Spindle microtubules, whose dynamics vary over time and at different locations, cooperatively drive chromosome segregation. Measurements of microtubule dynamics and spindle ultrastructure can provide insight into the behaviors of microtubules, helping elucidate the mechanism of chromosome segregation. Much work has focused on the dynamics and organization of kinetochore microtubules, that is, on the region between chromosomes and poles. In comparison, microtubules in the central-spindle region, between segregating chromosomes, have been less thoroughly characterized. Here, we report measurements of the movement of central-spindle microtubules during chromosome segregation in human mitotic spindles and Caenorhabditis elegans mitotic and female meiotic spindles. We found that these central-spindle microtubules slide apart at the same speed as chromosomes, even as chromosomes move toward spindle poles. In these systems, damaging central-spindle microtubules by laser ablation caused an immediate and complete cessation of chromosome motion, suggesting a strong coupling between central-spindle microtubules and chromosomes. Electron tomographic reconstruction revealed that the analyzed anaphase spindles all contain microtubules with both ends between segregating chromosomes. Our results provide new dynamical, functional, and ultrastructural characterizations of central-spindle microtubules during chromosome segregation in diverse spindles and suggest that central-spindle microtubules and chromosomes are strongly coupled in anaphase.

| S-EPMC6743361 | biostudies-literature

The 3Ms of central spindle assembly: microtubules, motors and MAPs.

Project description:During metaphase, sister chromatids are positioned at the midpoint of the microtubule-based mitotic spindle in preparation for their segregation. The onset of anaphase triggers inactivation of the key mitotic kinase cyclin-dependent kinase 1 (CDK1) and the polewards movement of sister chromatids. During anaphase, the mitotic spindle reorganizes in preparation for cytokinesis. Kinesin motor proteins and microtubule-associated proteins bundle the plus ends of interpolar microtubules and generate the central spindle, which regulates cleavage furrow initiation and the completion of cytokinesis. Complementary approaches, including cell biology, genetics and computational modelling, have provided new insights into the mechanism and regulation of central spindle assembly.

| S-EPMC2789570 | biostudies-literature

Critical Roles of a RhoGEF-Anillin Module in Septin Architectural Remodeling during Cytokinesis.

Project description:How septin architecture is remodeled from an hourglass to a double ring during cytokinesis in fungal and animal cells remains unknown. Here, we show that during the hourglass-to-double-ring transition in budding yeast, septins acquire a "zonal architecture" in which paired septin filaments that are organized along the mother-bud axis associate with circumferential single septin filaments, the Rho guanine-nucleotide-exchange factor (RhoGEF) Bud3, and the anillin-like protein Bud4 exclusively at the outer zones and with myosin-II filaments in the middle zone. Deletion of Bud3 or its Bud4-interacting domain, but not its RhoGEF domain, leads to a complete loss of the single filaments, whereas deletion of Bud4 or its Bud3-interacting domain destabilizes the transitional hourglass, especially at the mother side, with partial loss of both filament types. Deletion of Bud3 and Bud4 together further weakens the transitional structure and abolishes the double ring formation while causing no obvious defect in actomyosin ring constriction. This and further analyses suggest that Bud3 stabilizes the single filaments, whereas Bud4 strengthens the interaction between the paired and single filaments at the outer zones of the transitional hourglass, as well as in the double ring. This study reveals a striking zonal architecture for the transitional hourglass that pre-patterns two cytokinetic structures-a septin double ring and an actomyosin ring-and also defines the essential roles of a RhoGEF-anillin module in septin architectural remodeling during cytokinesis at the filament level.

| S-EPMC7176533 | biostudies-literature

KIF5B modulates central spindle organization in late-stage cytokinesis in chondrocytes.

Project description:BackgroundThe growth plate is a special region of the cartilage that drives longitudinal growth of long bones. Proliferating chondrocytes in the growth plate, arranged in columns, divide perpendicular to the long axis of the growth plate then intercalate to re-align with parental columns. Which molecular partners maintain growth plate columnar structures and chondrocyte cytokinesis has not been fully revealed. It is reported that kinesin family member 3A (KIF3A), a subunit of kinesin-2, plays an important role in maintaining columnar organization in growth plates via controlling primary cilia formation and cell proliferation.ResultHere we identify kinesin family member 5B (KIF5B), the heavy chain of kinesin-1, a ubiquitously expressed motor protein for anterograde intracellular transport along the microtubule network, as a key modulator of cytokinesis in chondrocytes via maintenance of central spindle organization. We show that KIF5B is concentrated in the central spindle during cytokinesis in both primary chondrocytes and chondrogenic ATDC5 cells.ConclusionThe failure of cytokinesis in KIF5B null chondrocytes leads to incomplete cell rotation, disrupting proliferation and differentiation, and results in a disorganized growth plate.

| S-EPMC6794761 | biostudies-literature

Recruitment of Polo kinase to the spindle midzone during cytokinesis requires the Feo/Klp3A complex.

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| S-EPMC1894651 | biostudies-literature

KIFC3 promotes mitotic progression and integrity of the central spindle in cytokinesis.

Project description:Kinesin-14 motor proteins play a variety of roles during metaphase and anaphase. However, it is not known whether members of this family of motors also participate in the dramatic changes in mitotic spindle organization during the transition from telophase to cytokinesis. We have identified the minus-end-directed motor, KIFC3, as an important contributor to central bridge morphology at this stage. KIFC3's unique motor-dependent localization at the central bridge allows it to congress microtubules, promoting efficient progress through cytokinesis. Conversely, when KIFC3 function is perturbed, abscission is delayed, and the central bridge is both widened and extended. Examination of KIFC3 on growing microtubules in interphase indicates that it caps microtubules released from the centrosome, both in the region of the centrosome and in the cell periphery. In line with other kinesin-14 family members, KIFC3 may guide free microtubules to their destination at the bridge and/or may slide and crosslink central bridge microtubules in order to stage the cells for abscission.

| S-EPMC3956538 | biostudies-literature

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