Wnt11 family dependent morphogenesis during frog gastrulation is marked by the cleavage furrow protein anillin

Wnt11 family proteins are ligands that activate a type of Dishevelled-mediated, non-canonical Wnt signaling pathway. Loss of function causes defects in gastrulation and/or anterior-posterior axis extension in all vertebrates. Non-mammalian vertebrate genomes encode two Wnt11 family proteins whose distinct functions have been unclear. We knocked down zygotic Wnt11b and Wnt11, separately and together, in Xenopus laevis. Single morphants exhibited very similar phenotypes of delayed blastopore closure, but they had different phenotypes at the tailbud stage. In response to their very similar gastrulation phenotypes, we chose to characterize dual morphants. Using dark field illuminated time-lapse imaging and kymograph analysis, we identified a failure of dorsal blastopore lip maturation that correlated with slower blastopore closure and failure to internalize the endoderm at the dorsal blastopore lip. We connected these externally visible phenotypes to cellular events in the internal tissues – including the archenteron – by imaging intact embryos stained for anillin and microtubules. The cleavage furrow protein anillin provided an exceptional cytological marker for blastopore lip and archenteron morphogenesis and the consequent disruption through loss of Wnt11 signaling. These cytological changes suggest a novel role for the regulation of contractility and stiffness of the epithelial cells that result in dramatic shape changes and are important in gastrulation.

Septin Remodeling During Mammalian Cytokinesis

Cytokinesis mediates the final separation of a mother cell into two daughter cells. Septins are recruited to the cleavage furrow at an early stage. During cytokinetic progression the septin cytoskeleton is constantly rearranged, ultimately leading to a concentration of septins within the intercellular bridge (ICB), and to the formation of two rings adjacent to the midbody that aid ESCRT-dependent abscission. The molecular mechanisms underlying this behavior are poorly understood. Based on observations that septins can associate with actin, microtubules and associated motors, we review here established roles of septins in mammalian cytokinesis, and discuss, how septins may support cytokinetic progression by exerting their functions at particular sites. Finally, we discuss how this might be assisted by phosphoinositide-metabolizing enzymes.

Endosome mediated delivery of ceramide phosphoethanolamine (CPE) with unique acyl chain anchors to the cleavage furrow is essential for male meiosis cytokinesis

Division of one cell into two daughter cells is fundamental in all living organisms. Cytokinesis, the final step of cell division, begins with the formation of an actomyosin contractile ring, positioned midway between the segregated chromosomes. Constriction of the ring with concomitant membrane deposition in a spatiotemporal precision generates a cleavage furrow that physically divides the cytoplasm. Unique lipids with specific biophysical properties have been shown to localize to midbodies however, their delivery mechanisms and biological roles were largely unknown. In this study, we show that Ceramide phosphoethanolamine (CPE), the structural analog of sphingomyelin, has unique acyl chain anchors in spermatocytes and is essential for meiosis cytokinesis. We found that disengagement of the central spindle from the contractile ring but not localization of phosphatidyl inositols (PIPs) at the plasma membrane was responsible for the male meiosis cytokinesis defect in CPE deficient animals. Further, we demonstrate that enrichment of CPE in Rab7 and Rab11 positive endosomes which in turn translocate to the cleavage furrows to promote cytokinesis. Our results implicate endosomal delivery of CPE to ingressing membranes is crucial for meiotic cytokinesis.

Expansion microscopy facilitates quantitative super-resolution studies of cytoskeletal structures in kinetoplastid parasites

Expansion microscopy (ExM) has become a powerful super-resolution method in cell biology. It is a simple, yet robust approach, which does not require any instrumentation or reagents beyond those present in a standard microscopy facility. In this study, we used kinetoplastid parasites Trypanosoma brucei and Leishmania major, which possess a complex, yet well-defined microtubule-based cytoskeleton, to demonstrate that this method recapitulates faithfully morphology of structures as previously revealed by a combination of sophisticated electron microscopy (EM) approaches. Importantly, we also show that due to rapidness of image acquisition and 3D reconstruction of cellular volumes ExM is capable of complementing EM approaches by providing more quantitative data. This is demonstrated on examples of less well-appreciated microtubule structures, such as the neck microtubule of T. brucei or the pocket, cytosolic, and multivesicular tubule-associated microtubules of L. major. We further demonstrate that ExM enables identifying cell types rare in a population, such as cells in mitosis and cytokinesis. 3D reconstruction of an entire volume of these cells provided details on morphology of the mitotic spindle and the cleavage furrow. Finally, we show that established antibody markers of major cytoskeletal structures function well in ExM, which together with the ability to visualize proteins tagged with small epitope tags will facilitate studies of the kinetoplastid cytoskeleton.

Spatiotemporal recruitment of RhoGTPase protein GRAF inhibits actomyosin ring constriction in Drosophila cellularization

Actomyosin contractility is regulated by Rho-GTP in cell migration, cytokinesis and morphogenesis in embryo development. Whereas Rho activation by Rho-GTP exchange factor (GEF), RhoGEF2 is well known in actomyosin contractility during cytokinesis at the base of invaginating membranes in Drosophila cellularization, Rho inhibition by RhoGTPase activating proteins (GAP) remains to be studied. We have found that the RhoGAP, GRAF inhibits actomyosin contractility during cellularization. GRAF is enriched at the cleavage furrow tip during actomyosin assembly and initiation of ring constriction. Graf depletion shows increased Rho-GTP, increased Myosin II and ring hyper constriction dependent upon the loss of the RhoGTPase domain. GRAF and RhoGEF2 are present in a balance for appropriate activation of actomyosin ring constriction. RhoGEF2 depletion and abrogation of Myosin II activation in Rho Kinase mutants suppresses the Graf hyper constriction defect. Therefore, GRAF recruitment restricts Rho-GTP levels in a spatiotemporal manner for inhibiting actomyosin contractility during cellularization.

Rab14/MACF2 Complex Regulates Endosomal Targeting During Cytokinesis

Abscission is a complex cellular process that is required for mitotic division. It is well-established that coordinated and localized changes in actin and microtubule dynamics are vital for cytokinetic ring formation, as well as establishment of the abscission site. Actin cytoskeleton reorganization during abscission would not be possible without the interplay between Rab11- and Rab35-containing endosomes and their effector proteins, whose roles in regulating endocytic pathways at the cleavage furrow have now been studied extensively. Here, we identified Rab14 as a novel regulator of cytokinesis. We demonstrate that depletion of Rab14 causes either cytokinesis failure or significantly prolongs division time. We show that Rab14 contributes to the efficiency of recruiting Rab11-endosomes to the ICB microtubules and that Rab14 knockout leads to inhibition of actin clearance at the abscission site. Finally, we demonstrate that Rab14 binds to microtubule minus-end interacting MACF2/CAMSAP3 complex and that this binding affects targeting of endosomes to the ICB microtubules. Collectively, our data identified Rab14 and MACF2/CAMSAP3 as proteins that regulate actin depolymerization and endosome targeting during cytokinesis. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Calcium spikes accompany cleavage furrow ingression and cell separation during fission yeast cytokinesis

Calcium rises transiently at the division plane during cytokinesis of embryonic cells, but the conservation and function of such calcium transients remain unclear. We discovered similar calcium spikes during fission yeast cytokinesis, and demonstrated that calcium promotes contractile ring constriction and daughter cell integrity.

Peripheral microtubules ensure asymmetric furrow positioning in neural stem cells

Neuroblast (NB) cell division is characterized by a basal positioning of the cleavage furrow resulting in a large difference in size between the future daughter cells. In animal cells, furrow placement and assembly is governed by centralspindlin, a highly conserved complex that accumulates at the equatorial cell cortex of the future cleavage site and at the spindle midzone. In contrast to model systems studied so far, these two centralspindlin populations are spatially and temporally separated in NBs. A cortical leading pool is located at the basal cleavage furrow site and a second pool accumulates at the midzone before travelling to the site of the basal cleavage furrow during cytokinesis completion. By manipulating microtubule (MT) dynamics, we show that the cortical centralspindlin population requires peripheral astral microtubules and the Chromosome Passenger Complex (CPC) for efficient recruitment. Loss of this pool does not prevent cytokinesis but enhances centralspindlin levels at the midzone leading to furrow repositioning towards the equator and decreased size asymmetry between daughter cells. Together these data reveal that the asymmetrical furrow placement characteristic of NBs results from a competition between spatially and functionally separate centralspindlin pools in which the cortical pool is dominant and requires peripheral astral microtubules.

Cleavage-furrow formation without F-actin inChlamydomonas

It is widely believed that cleavage-furrow formation during cytokinesis is driven by the contraction of a ring containing F-actin and type-II myosin. However, even in cells that have such rings, they are not always essential for furrow formation. Moreover, many taxonomically diverse eukaryotic cells divide by furrowing but have no type-II myosin, making it unlikely that an actomyosin ring drives furrowing. To explore this issue further, we have used one such organism, the green algaChlamydomonas reinhardtii. We found that although F-actin is associated with the furrow region, none of the three myosins (of types VIII and XI) is localized there. Moreover, when F-actin was eliminated through a combination of a mutation and a drug, furrows still formed and the cells divided, although somewhat less efficiently than normal. Unexpectedly, division of the largeChlamydomonaschloroplast was delayed in the cells lacking F-actin; as this organelle lies directly in the path of the cleavage furrow, this delay may explain, at least in part, the delay in cytokinesis itself. Earlier studies had shown an association of microtubules with the cleavage furrow, and we used a fluorescently tagged EB1 protein to show that microtubules are still associated with the furrows in the absence of F-actin, consistent with the possibility that the microtubules are important for furrow formation. We suggest that the actomyosin ring evolved as one way to improve the efficiency of a core process for furrow formation that was already present in ancestral eukaryotes.