Academic literature on the topic 'Cleavage furrow formation'

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.

Calcium signaling is known to be associated with cytokinesis; however, the detailed spatio-temporal pattern of calcium dynamics has remained unclear. We have studied changes of intracellular free calcium in cleavage-stage Xenopus embryos using fluorescent calcium indicator dyes, mainly Calcium Green-1. Cleavage formation was followed by calcium transients that localized to cleavage furrows and propagated along the furrows as calcium waves. The calcium transients at the cleavage furrows were observed at each cleavage furrow at least until blastula stage. The velocity of the calcium waves at the first cleavage furrow was approximately 3 microns/s, which was much slower than that associated with fertilization/egg activation. These calcium waves traveled only along the cleavage furrows and not in the direction orthogonal to the furrows. These observations imply that there exists an intracellular calcium-releasing activity specifically associated with cleavage furrows. The calcium waves occurred in the absence of extracellular calcium and were inhibited in embryos injected with heparin an inositol 1,4,5-trisphosphate (InsP3) receptor antagonist. These results suggest that InsP3 receptor-mediated calcium mobilization plays an essential role in calcium wave formation at the cleavage furrows.

During cytokinesis, cleavage furrow invagination requires an actomyosin-based contractile ring and addition of new membrane. Little is known about how this actin and membrane traffic to the cleavage furrow. We address this through live analysis of fluorescently tagged vesicles in postcellularized Drosophila melanogaster embryos. We find that during cytokinesis, F-actin and membrane are targeted as a unit to invaginating furrows through formation of F-actin–associated vesicles. F-actin puncta strongly colocalize with endosomal, but not Golgi-derived, vesicles. These vesicles are recruited to the cleavage furrow along the central spindle and a distinct population of microtubules (MTs) in contact with the leading furrow edge (furrow MTs). We find that Rho-specific guanine nucleotide exchange factor mutants, pebble (pbl), severely disrupt this F-actin–associated vesicle transport. These transport defects are a consequence of the pbl mutants' inability to properly form furrow MTs and the central spindle. Transport of F-actin–associated vesicles on furrow MTs and the central spindle is thus an important mechanism by which actin and membrane are delivered to the cleavage furrow.

Coordinated membrane and cytoskeletal remodeling activities are required for membrane extension in processes such as cytokinesis and syncytial nuclear division cycles in Drosophila. Pseudocleavage furrow membranes in the syncytial Drosophila blastoderm embryo show rapid extension and retraction regulated by actin-remodeling proteins. The F-BAR domain protein Syndapin (Synd) is involved in membrane tubulation, endocytosis, and, uniquely, in F-actin stability. Here we report a role for Synd in actin-regulated pseudocleavage furrow formation. Synd localized to these furrows, and its loss resulted in short, disorganized furrows. Synd presence was important for the recruitment of the septin Peanut and distribution of Diaphanous and F-actin at furrows. Synd and Peanut were both absent in furrow-initiation mutants of RhoGEF2 and Diaphanous and in furrow-progression mutants of Anillin. Synd overexpression in rhogef2 mutants reversed its furrow-extension phenotypes, Peanut and Diaphanous recruitment, and F-actin organization. We conclude that Synd plays an important role in pseudocleavage furrow extension, and this role is also likely to be crucial in cleavage furrow formation during cell division.

It has been proposed that a localized calcium (Ca) signal at the growing end of the cleavage furrow triggers cleavage furrow formation in large eggs. We have examined the possible role of a Ca signal in cleavage furrow formation in the Xenopus laevis egg during the first cleavage. We were able to detect two kinds of Ca waves along the cleavage furrow. However, the Ca waves appeared after cleavage furrow formation in late stages of the first cleavage. In addition, cleavage was not affected by injection of dibromoBAPTA or EGTA into the eggs at a concentration sufficient to suppress the Ca waves. Furthermore, even smaller classes of Ca release such as Ca puffs and Ca blips do not occur at the growing end of the cleavage furrow. These observations demonstrate that localized Ca signals in the cleavage furrow are not involved in cytokinesis. The two Ca waves have unique characteristics. The first wave propagates only in the region of newly inserted membrane along the cleavage furrow. On the other hand, the second wave propagates along the border of new and old membranes, suggesting that this wave might be involved in adhesion between two blastomeres.

After the separation of sister chromatids in anaphase, it is essential that the cell position a cleavage furrow so that it partitions the chromatids into two daughter cells of roughly equal size. The mechanism by which cells position this cleavage furrow remains unknown, although the best current model is that furrows always assemble midway between asters. We used micromanipulation of human cultured cells to produce mitotic heterokaryons with two spindles fused in a V conformation. The majority (15/19) of these cells cleaved along a single plane that transected the two arms of the V at the position where the metaphase plate had been, a result at odds with current views of furrow positioning. However, four cells did form an additional ectopic furrow between the spindle poles at the open end of the V, consistent with the established view. To begin to address the mechanism of furrow assembly, we have begun a detailed study of the properties of the chromosome passenger inner centromere protein (INCENP) in anaphase and telophase cells. We found that INCENP is a very early component of the cleavage furrow, accumulating at the equatorial cortex before any noticeable cortical shape change and before any local accumulation of myosin heavy chain. In mitotic heterokaryons, INCENP was detected in association with spindle midzone microtubules beneath sites of furrowing and was not detected when furrows were absent. A functional role for INCENP in cytokinesis was suggested in experiments where a nearly full-length INCENP was tethered to the centromere. Many cells expressing the chimeric INCENP failed to complete cytokinesis and entered the next cell cycle with daughter cells connected by a large intercellular bridge with a prominent midbody. Together, these results suggest that INCENP has a role in either the assembly or function of the cleavage furrow.

De novo formation of cells in the Drosophila embryo is achieved when each nucleus is surrounded by a furrow of plasma membrane. Remodeling of the plasma membrane during cleavage furrow ingression involves the exocytic and endocytic pathways, including endocytic tubules that form at cleavage furrow tips (CFT-tubules). The tubules are marked by amphiphysin but are otherwise poorly understood. Here we identify the septin family of GTPases as new tubule markers. Septins do not decorate CFT-tubules homogeneously: instead, novel septin complexes decorate different CFT-tubules or different domains of the same CFT-tubule. Using these new tubule markers, we determine that all CFT-tubule formation requires the BAR domain of amphiphysin. In contrast, dynamin activity is preferentially required for the formation of the subset of CFT-tubules containing the septin Peanut. The absence of tubules in amphiphysin-null embryos correlates with faster cleavage furrow ingression rates. In contrast, upon inhibition of dynamin, longer tubules formed, which correlated with slower cleavage furrow ingression rates. These data suggest that regulating the recycling of membrane within the embryo is important in supporting timely furrow ingression.

RhoA controls cleavage furrow formation during cell division, but whether RhoA suffices to orchestrate spatiotemporal dynamics of furrow formation is unknown. In this issue, Wagner and Glotzer (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201603025) show that RhoA activity can induce furrow formation in all cell cortex positions and cell cycle phases.

The Chytridiomycete Allomyces macrogynus generates new membranes for cleavage furrow and nuclear-cap formation during gametogenesis and zoosporogenesis. Transmission electron microscopy after impregnation with a mixture of zinc iodide and osmium tetroxide clearly demonstrated changes in the endoplasmic reticulum. Endoplasmic reticulum was intensely stained but did not appear to contribute to the formation of the unstained flagellar membranes or cleavage furrows. However, the relative cytoplasmic volume of endoplasmic reticulum decreased as positively stained nuclear-cap membrane formed. These observations are consistent with the hypothesis that flagellar membranes and cleavage furrows are derived from trans-Golgi equivalents, whereas the nuclear-cap membrane is derived from the endoplasmic reticulum. Key words: Allomyces macrogynus, Chytridiomycetes, endoplasmic reticulum, gametogenesis, zoosporogenesis.

Cytokinesis is the final stage of the cell cycle. It partitions sister genomes and separates the cytoplasm of nascent daughter cells. Cytokinesis is initiated by the formation of a cleavage furrow whose ingression is powered by an actomyosin network known as the contractile ring. Following furrow ingression, the process of cell separation is completed by a membrane scission reaction. For the accurate inheritance of genetic information, it is crucial that furrow formation is initiated at the cell equator between segregating chromosomes and that this occurs after chromatin has cleared the cleavage plane. In animal cells, the mitotic spindle plays a pivotal role in the formation and placement of the cleavage furrow. The coupling of cytokinesis and chromosome segregation to the mitotic spindle ensures that nuclear and cytoplasmic division are tightly coordinated. The spindle midzone, a structure that is formed at anaphase onset between segregating sister genomes, is thought to play an important instructive role during cleavage furrow formation. How the mitotic spindle controls cytokinetic events at the cell envelope is a key challenge in cell division research. Formation of the cytokinetic furrow in animal cells requires activation of the GTPase RhoA by the conserved guanine nucleotide exchange factor Ect2. How Ect2, which is associated with the spindle midzone, controls RhoA activity at the equatorial cell periphery during anaphase is not understood. Using a genetic complementation system, I have been able to replace the endogenous Ect2 protein with a fluorescently-tagged transgene to study its dynamic localization during cytokinesis. Using live-cell time-lapse microscopy, I found that Ect2 concentrates not only at the spindle midzone but also accumulates at equatorial plasma membrane during cytokinesis. The association of Ect2 with the plasma membrane in vivo involves a pleckstrin homology domain and a polybasic cluster that bind to phosphoinositide lipids in vitro. I further demonstrated that both guanine nucleotide exchange activity and the membrane targeting domains of Ect2 are essential for RhoA activation, contractile ring formation and cleavage furrow ingression in human cells. Membrane localization of Ect2 is spatially confined to the equator by centralspindlin, Ect2’s spindle midzone anchor complex, and is also temporally coordinated with chromosome segregation through the activation state of Cdk1. My results suggest that targeting of Ect2 to the equatorial membrane may represent a key step in the delivery of the cytokinetic signal to the cortex.

Reduced activity of Fragile X mental retardation protein (FMRP) in brain neurons results in the most common form of heritable mental retardation in humans, Fragile X Syndrome (FXS). FMRP is a selective RNA-binding protein that is implicated in the translational regulation of specific mRNAs in neurons. Although very few direct targets of FMRP have been identified and verified in vivo, FXS is thought to result from the aberrant regulation of potentially hundreds of mRNAs causing defects in neuron morphology and synapse function. Identifying additional targets will be important for elucidating the mechanism of FMRP regulation as well as the etiology of FXS. Drosophila melanogaster offers a unique and powerful system for studying the function of FMRP. Flies with loss of FMRP activity have neuronal and behavioral defects similar to those observed in humans with FXS. Importantly, FMRP regulates common target mRNAs in neurons in both mice and flies. Here, I will describe our discovery of a previously unknown requirement for Drosophila FMRP (dFMRP) during the cleavage stage of early embryonic development. First, we identified a requirement for dFMRP for proper cleavage furrow formation and found that dFMRP functions to regulate the expression of specific target mRNAs during the cleavage stage. Among these is trailer hitch (tral) mRNA, which encodes a translational regulator as well, and represents a new in vivo target of dFMRP translational regulation. In addition, I have identified twenty-eight proteins that change in expression in the absence of dFMRP using a comparative proteomics based screen for dFMRP targets. One of these is the Chaperonin containing tcp-1 complex (CCT), a previously unidentified target, which I found is itself also required for cleavage furrow formation. Finally, we have identified a new dFMRP protein-binding partner, Caprin, and found that together dFMRP and Caprin are required for the proper timing of the MBT. This set of work has led to a better understanding of the mechanism of dFMRP-dependent regulation of cellular morphogenesis in early embryos and has the potential to lead to a better understanding of the etiology of FXS.
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In animal cells, the duplicated genetic material is aligned on a microtubule-based structure known as the mitotic spindle during mitosis. At the mitotic exit, the mitotic spindle elongates, and the sister chromatids get separated. The separation of sister chromatids is followed by the cleavage furrow formation and its ingression, which eventually partition the cytoplasmic constituents and genetic material into newly formed daughter cells. How the chromosome separation is coordinated with cleavage furrow formation is incompletely understood. Also, when animal cells enter mitosis, the chromatin gets highly condensed, and the transcription is chiefly paused. However, when cells exit mitosis, the chromatin should get decondensed in a tightly regulated manner to ensure proper landscaping of chromosome territories, which makes it competent enough for DNA-based processes like replication and transcription. The accurate functioning of these processes is critical for the development and for stem cell divisions. In this study, we have linked the function of an evolutionarily conserved protein, nuclear mitotic apparatus (NuMA), in cleavage furrow formation and chromatin decondensation at the mitotic exit. In the first part of my thesis, we have tried to characterize the function of chromatin-localized NuMA in regulating chromatin decondensation. In the second part, we have attempted to provide insight into how spatial localization of NuMA at the plasma membrane coordinates chromosome separation with cleavage furrow formation. 1). NuMA regulates chromatin decondensation at the mitotic exit and nuclear shape in interphase cells NuMA is a highly abundant (~10^6 copies) protein of interphase nuclei. Few studies hint that nuclear NuMA may have a role in chromatin organization, and it is hypothesized to be a part of the nuclear structural framework. In this regard, the loss of NuMA's function based on antibody-based microinjections was associated with nuclear shape defects. However, since the depletion of NuMA is linked with multiple mitotic abnormalities, it remained unclear whether the nuclear shape defects seen upon NuMA depletion is an indirect effect due to impairment of NuMA's mitotic function or a direct outcome of the absence of NuMA in the nucleus. Further, whether NuMA is bound to chromatin in the nucleus was also unknown. Even if NuMA is bound to chromatin, what mechanisms ensure its release upon mitotic entry was unknown. In this work, by utilizing fluorescence recovery after photobleaching (FRAP) and biochemical analysis, we report that NuMA is transiently bound to chromatin in the nucleus. We show that NuMA, which is bound to DNA, is released in late prophase upon nuclear envelope breakdown (NEBD) by the action of Cdk1-CyclinB kinase. Importantly, we identify evolutionarily conserved sequences rich in basic amino acids, arginine, and lysine, at the C-terminus of NuMA that aid in its direct interaction with DNA. In the absence of such interaction, NuMA becomes significantly mobile in the nucleus. Notably, the expression of the DNA-binding deficient mutant of NuMA delays chromatin decondensation at the mitotic exit. Furthermore, we discovered that DNA binding deficient NuMA polymerizes into high-order structures such as fibrillar networks, which perturbs nuclear shape. The DNA-binding property of NuMA prevents the formation of these higher-order structures and thus helps in maintaining the proper nuclear architecture. Overall, this study links the chromatin binding ability of NuMA with the proper chromatin decondensation at mitotic exit and maintenance of nuclear shape in interphase, independent of its mitotic role. 2). Polarized membrane distribution of NuMA/dynein and Ect2/Cyk4/Mklp1 regulate cleavage furrow formation Animal cells partition their genetic material and cellular constituents through cytokinesis. The initiation of cytokinesis is regulated by the activation of small GTPase RhoA that helps in myosin II activation and actin polymerization at the equatorial membrane, resulting in cleavage furrow formation. RhoA is spatiotemporally regulated by a heterotetrameric complex known as centralspindlin consisting of a dimer of kinesin-6 member Mklp1 and a dimer of RhoGAP Cyk4. The centralspindlin complex localizes at the spindle midzone and promotes the localization of its downstream effectors RhoGEF Ect2 which directly activates RhoA and regulates cytokinesis. However, how a precise RhoA zone at the equatorial membrane is established and maintained remained unclear. In anaphase, the mitotic protein NuMA is enriched at the polar membrane via its direct interaction with membrane phospholipids, PtIns(4)P and PtIns(4,5)P2 and is vital for proper spindle elongation by cortically anchoring the dynein/dynactin complex. However, despite the presence of PtIns(4)P and PtIns(4,5)P2 throughout the membrane, the NuMA/dynein complexes are restricted to the polar membrane and are excluded from the equatorial membrane, which is mutually exclusively occupied by RhoA. The mechanism of equatorial membrane exclusion of NuMA/dynein complex and its biological relevance remained unknown. In this work, we uncovered that Ect2, Cyk4, and Mklp1 are critical in restricting NuMA/dynein to the polar cortical region. In the absence of Ect2, Cyk4, or Mklp1, NuMA/dynein complex occupies the equatorial cortex, which impacts proper spindle elongation. Further, we show that Ect2 is in complex with Cyk4 and Mklp1 in anaphase cells. We establish that the membrane localization, but not the spindle midzone localization of the Ect2/Cyk4/Mklp1 complex, is critical for NuMA/dynein exclusion and, thus, for proper spindle elongation. Conversely, we show that polar membrane localization of the NuMA/dynein complex confines RhoA to a narrow zone at the equatorial membrane, which ensures cleavage furrow formation and cytokinesis. Overall our work provides insight into the mechanism that restricts NuMA/dynein and Ect2/Cyk4/Mklp1 to mutually exclusive membrane surfaces, which ensures proper chromatin segregation and cleavage furrow formation in animal cells. This coordination is critical for an error-free cell division program.