End Of Cell Division Research Articles

What Drosophila spermatocytes tell us about the mechanisms underlying cytokinesis

Cytokinesis separates the genomic material and organelles of a dividing cell equitably into two physically distinct daughter cells at the end of cell division. This highly choreographed process involves coordinated reorganization and regulated action of the actin and microtubule cytoskeletal systems, an assortment of motor proteins, and membrane trafficking components. Due to their large size, the ease with which exquisite cytological analysis may be performed on them, and the availability of numerous mutants and other genetic tools, Drosophila spermatocytes have provided an excellent system for exploring the mechanistic basis for the temporally programmed and precise spatially localized events of cytokinesis. Mutants defective in male meiotic cytokinesis can be easily identified in forward genetic screens by the production of multinucleate spermatids. In addition, the weak spindle assembly checkpoint in spermatocytes, which causes only a small delay of anaphase onset in the presence of unattached chromosomes, allows investigation of whether gene products required for spindle assembly and chromosome segregation are also involved in cytokinesis. Perhaps due to the large size of spermatocytes and the requirement for two rapid-fire rounds of division without intervening S or growth phases during meiosis, male meiotic mutants have also revealed much about molecular mechanisms underlying new membrane addition during cytokinesis. Finally, cell type-specific differences in the events that set up and complete cytokinesis are emerging from comparison of spermatocytes with cells undergoing mitosis either elsewhere in the organism or in tissue culture.

Cytokinesis inDrosophila melanogaster

Cytokinesis is the process that physically separates two sibling cells, ensuring the proper partitioning of the nuclear and cytoplasmic contents at the end of cell division. Cytokinesis requires a fine-tuned molecular machinery that has to be assembled with high spatiotemporal precision. Drosophila melanogaster is an ideal model system to investigate this cellular process. Genetic screens performed in spermatocytes, neuroblasts, and Schneider cells revealed numerous evolutionary conserved components. These genetically amenable systems have proven to be very useful to further elucidate the cellular and molecular mechanism of cytokinesis, significantly contributing to our current understanding of this important cellular process. As in other organisms, cytokinesis is largely dependent on the mitotic spindle, providing positional cues for cleavage furrow placement and progression. However, spindle-independent mechanisms could also be important during special cases of cytokinesis, such as asymmetric cell division. Thus, powerful fly genetics combined with single-cell analysis, live imaging, and biochemical assays will continue to provide important insights into the mechanism of cytokinesis.

Cytokinesis in Drosophila male meiosis

Cytokinesis separates the cytoplasm and the duplicated genome into two daughter cells at the end of cell division. This process must be finely regulated to maintain ploidy and prevent tumor formation. Drosophila male meiosis provides an excellent cell system for investigating cytokinesis. Mutants affecting this process can be easily identified and spermatocytes are large cells particularly suitable for cytological analysis of cytokinetic structures. Over the past decade, the powerful tools of Drosophila genetics and the unique characteristics of this cell system have led researchers to identify molecular players of the cell cleavage machinery and to address important open questions. Although spermatocyte cytokinesis is incomplete, resulting in formation of stable intercellular bridges, the molecular mechanisms are largely conserved in somatic cells. Thus, studies of Drosophila male meiosis will shed new light on the complex cell circuits regulating furrow ingression and substantially further our knowledge of cancer and other human diseases.

Abscission checkpoint control: stuck in the middle with Aurora B

At the end of cell division, the cytoplasmic bridge joining the daughter cells is severed through a process that involves scission of the plasma membrane. The presence of chromatin bridges ‘stuck’ in the division plane is sensed by the chromosomal passenger complex (CPC) component Aurora B kinase, triggering a checkpoint that delays abscission until the chromatin bridges have been resolved. Recent work has started to shed some light on the molecular mechanism by which the CPC controls the timing of abscission.

The chromosomal passenger complex controls the function of endosomal sorting complex required for transport-III Snf7 proteins during cytokinesis

SummaryCytokinesis controls the proper segregation of nuclear and cytoplasmic materials at the end of cell division. The chromosomal passenger complex (CPC) has been proposed to monitor the final separation of the two daughter cells at the end of cytokinesis in order to prevent cell abscission in the presence of DNA at the cleavage site, but the precise molecular basis for this is unclear. Recent studies indicate that abscission could be mediated by the assembly of filaments comprising components of the endosomal sorting complex required for transport-III (ESCRT-III). Here, we show that the CPC subunit Borealin interacts directly with the Snf7 components of ESCRT-III in both Drosophila and human cells. Moreover, we find that the CPC's catalytic subunit, Aurora B kinase, phosphorylates one of the three human Snf7 paralogues—CHMP4C—in its C-terminal tail, a region known to regulate its ability to form polymers and associate with membranes. Phosphorylation at these sites appears essential for CHMP4C function because their mutation leads to cytokinesis defects. We propose that CPC controls abscission timing through inhibition of ESCRT-III Snf7 polymerization and membrane association using two concurrent mechanisms: interaction of its Borealin component with Snf7 proteins and phosphorylation of CHMP4C by Aurora B.

Class-1 Myosins at the Interphase between Spindles and Membranes

Class-1 myosins play key roles in the dynamic organization of the cytoskeleton and membrane shape as they act as divalent cross-linkers interconnecting and generating force between both actin and membrane systems. Previously we have shown that the long-tailed Dictyostelium myosin-1C not only is involved in membrane remodeling during endocytic processes, but also plays a role in spindle dynamics during mitosis through direct microtubule interactions. Here, we characterized myosin-1D as the second myosin-1 member with roles in mitosis. Our studies reveal that myosin-1D binds spindle pole microtubules and associates with the nuclear envelope of dividing cells. Myosin-1D targets microtubules and nuclear structures early during mitosis and decorates the membrane embedded chromatin masses until the end of cell division. This suggests a role of the motor in nuclear membrane fission by mediating microtubule-membrane interactions. Since the nuclear envelope in Dictyostelium remains intact throughout mitosis, except for regions of perforations that are found near the spindle poles, myosin-1D appears to act as a dynamic linker connecting spindle pole microtubules to the nuclear membrane important to hold the envelope together as it gets stretched upon spindle elongation. To investigate the mechanisms that govern myosin-1s to distinct mitotic structures, we have generated a set of motor and tail domain constructs and characterized binding affinities and kinetics of intermolecular interactions with lipid membranes, actin, and microtubules. Our results show that differences in strength and specificity of lipid and microtubule binding and regulatory roles of the tails account for the specialized functions of the motors in mitosis.

Integrin β1 Is Necessary for the Maintenance of Corneal Structural Integrity

The precise role of a normal keratocyte in maintaining corneal structural integrity is unclear; it is generally considered to remain quiescent at the end of cell division. Given that integrins are essential for cell/extracellular matrix interactions, the authors tested the hypothesis that integrin expression by keratocytes is essential for corneal structure and function. Using a tamoxifen-dependent cre recombinase expressed under the control of a fibroblast-specific promoter/enhancer, the authors conditionally deleted the integrin β1 (Itgb1) gene in mouse keratocytes during the postnatal matrix maturation phase of the cornea. The effects of this deletion were monitored histologically and by macroscopic observation of the cornea. The resultant cornea shows an initial thinning of the stroma, reduced space between collagen fibrils, loss of epithelial layers and subsequent edema, thickening of Descemet's membrane, and degenerative changes in the endothelial cell layer, with eventual scarring. These pathologic changes have some similarities to human corneal disease keratoconus. The phenotype did not develop when Itgb1 was deleted after complete corneal maturation. Loss of integrin β1 expression in keratocytes during the phase of stromal maturation results in corneal thinning and edema. Keratocyte-ECM interaction is essential for matrix maturation and thus in the maintenance of corneal structural integrity. This model has relevance in understanding corneal diseases such as keratoconus.

Proliferating versus differentiating stem and cancer cells exhibit distinct midbody-release behaviour

The central portion of the midbody, a cytoplasmic bridge between nascent daughter cells at the end of cell division, has generally been thought to be retained by one of the daughter cells, but has, recently, also been shown to be released into the extracellular space. The significance of midbody-retention versus -release is unknown. Here we show, by quantitatively analysing midbody-fate in various cell lines under different growth conditions, that the extent of midbody-release is significantly greater in stem cells than cancer-derived cells. Induction of cell differentiation is accompanied by an increase in midbody-release. Knockdown of the endosomal sorting complex required for transport family members, Alix and tumour-suppressor gene 101, or of their interaction partner, centrosomal protein 55, impairs midbody-release, suggesting mechanistic similarities to abscission. Cells with such impaired midbody-release exhibit enhanced responsiveness to a differentiation stimulus. Taken together, midbody-release emerges as a characteristic feature of cells capable of differentiation.

Hydrodynamics of Cellular Cortical Flows and the Formation of Contractile Rings

We propose a mechanism for the formation of contractile rings and the apparition of a flow in the cortical layer of cells undergoing cytokinesis at the end of cell division or during the healing of a wound in the cortex of Xenopus eggs. We generalize the hydrodynamic active gel theory along the lines of thin shell theory of continuum elasticity to describe the cell cortex. As in liquid crystal physics, the flow couples to the orientation of the actin filaments. The cortical flow is driven by an increased density of myosin motors in the cortex, and orients the filaments to form the ring.

Evidence for a dual role of PBP1 in the cell division and cell separation of Staphylococcus aureus

Penicillin-binding proteins (PBPs) catalyse the synthesis of cell wall peptidoglycan. PBP1 of Staphylococcus aureus is a high-molecular-weight monofunctional transpeptidase (TPase) and previous studies with a conditional mutant showed that this protein was essential for bacterial growth and survival: cells in which PBP1 was depleted stopped dividing but continued to enlarge in size, accompanied by rapid loss of viability. Also, cell walls produced under PBP1 depletion appeared to have normal composition. We describe here construction of a second PBP1 mutant in which the active site of the TPase domain was inactivated. Cells in which the wild-type PBP1 was replaced by the mutant protein were able to initiate and complete septa and undergo at least one or two cell divisions after which growth stopped accompanied by inhibition of cell separation, downregulation in the transcription of the autolytic system and production of cell walls with increased proportion of monomeric and dimeric muropeptides and decrease in oligomeric muropeptides. PBP1 seems to perform a dual role in the cell cycle of S. aureus: as a protein required for septation and also as a transpeptidase that generates a critical signal for cell separation at the end of cell division.

How to scaffold the contractile ring for a safe cytokinesis – lessons from Anillin-related proteins

The ingression of a cleavage furrow separates the two daughter cells at the end of cell division. In many organisms this furrow ingression is driven by the assembly and contraction of actomyosin filaments, forming a contractile ring. To achieve a successful cytokinesis, these actomyosin filaments need to be assembled in an organized manner. For this purpose, a network of cytoskeletal proteins is built at the cleavage site to act as a scaffold for actomyosin filaments and to connect them to the plasma membrane. The Drosophila melanogaster protein Anillin, and its related proteins in other organisms, has a pivotal role in the organization of this scaffold in many species, ranging from yeast to humans. Recent studies indicate that Anillin-related proteins interact not only with the structural components of the contractile ring, but also with the signalling factors that control their dynamics. In addition, Drosophila Anillin connects the actomyosin ring to the spindle microtubules through its interaction with the RacGAP component of the centralspindlin complex. Here I review the structures and functions of Anillin and Anillin-related proteins in various model systems, and aim to highlight both the common and distinctive features of these essential organizers of the molecular machinery that drives furrow ingression.

Drought‐induced changes in anthesis‐silking interval are related to silk expansion: a spatio‐temporal growth analysis in maize plants subjected to soil water deficit

The growth and emergence of maize silks has a considerable importance in yield determination under drought conditions. Spatial and temporal patterns of the rates of tissue expansion and of cell division were characterized in silks of plants subjected to different soil water potentials. In all cases, silk development consisted of four phases: (1) cell division and tissue expansion occurred together uniformly all along the silk; (2) cell division progressively ceased from tip to base, while expansion remained spatially uniform including during the phase (3) after the cessation of cell division; and (4) as the silk emerged from the husks, expansion ceased in the emerged portion, probably because of direct evaporative demand, while the relative growth rate progressively decreased in the enclosed part. The rates of tissue expansion and cell division were reduced with water deficit, resulting in delayed silk emergence. The duration of cell division was not affected, and in all cases, the end of cell division in the silk coincided with anther dehiscence. The duration of phase 3, between the end of cell division and the arrest of cell growth in silk apex, considerably increased with water deficit. It corresponded to the anthesis-silking interval used by breeders to characterize the response of cultivars to stress.

Mutations in a small region of the exportin Crm1p disrupt the daughter cell‐specific nuclear localization of the transcription factor Ace2p in Saccharomyces cerevisiae

The CBK1 gene of Saccharomyces cerevisiae encodes a protein kinase that is a member of the NDR (nuclear Dbf2-related) family of protein kinases, which are involved in morphogenesis and cell proliferation. Previous studies have shown that deletion of CBK1 leads to a loss of polarity and the formation of large aggregates of cells. This aggregation phenotype is due to the loss of the daughter cell-specific accumulation of the transcription factor Ace2p, which is responsible for the transcription of genes whose products are necessary for the final separation of the mother and the daughter at the end of cell division. We show that the daughter cell-specific localization of Ace2p does not occur via a specific localization of the ACE2 mRNA and that, in vivo, the transcription of CTS1, one of the principal targets of Ace2p, is daughter cell-specific. We have shown that extragenic suppressors of the Deltacbk1 aggregation phenotype are located in the nuclear exportin CRM1 and ACE2. These mutations disrupt the interaction of Ace2p and Crm1p, thus impairing Ace2p export and resulting in the accumulation of the protein in both mother and daughter cell nuclei. We propose that in the daughter cell nucleus Cbk1p phosphorylates the Ace2p nuclear export signal, and that this phosphorylation blocks the export of Ace2p via Crm1p, thus promoting the daughter cell-specific nuclear accumulation of Ace2p.

RacGAP50C is sufficient to signal cleavage furrow formation during cytokinesis

Several studies indicate that spindle microtubules determine the position of the cleavage plane at the end of cell division, but their exact role in triggering the formation and ingression of the cleavage furrow is still unclear. Here we show that in Drosophila depletion of either the GAP (GTPase-activating protein) or the kinesin-like subunit of the evolutionary conserved centralspindlin complex prevents furrowing without affecting the association of astral microtubules with the cell cortex. Moreover, time-lapse imaging indicates that astral microtubules serve to deliver the centralspindlin complex to the equatorial cortex just before furrow formation. However, when the GAP-signaling component was mislocalized around the entire cortex using a membrane-tethering motif, this caused ectopic furrowing even in the absence of its motor partner. Thus, the GAP component of centralspindlin is both necessary and sufficient for furrow formation and ingression and astral microtubules provide a route for its delivery to the cleavage site.

Local Change in Phospholipid Composition at the Cleavage Furrow Is Essential for Completion of Cytokinesis

Cell division ends up with the membrane separation of two daughter cells, presumably by a membrane fusion that requires dynamic changes of the distribution and the composition of membrane lipids. We have previously shown that a membrane lipid phosphatidylethanolamine (PE) is exposed on the cell surface of the cleavage furrow during late cytokinesis and that this PE movement is involved in regulation of the contractile ring disassembly. Here we show that immobilization of cell surface PE by a PE-binding peptide blocks the RhoA inactivation in the late stage of cytokinesis. Phosphatidylinositol 4-phosphate 5-kinase (PIP5K), but not other RhoA effectors, is co-localized with RhoA in the peptide-treated cells. Indeed, PIP5K and its product phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) are localized to the cleavage furrow of normally dividing cells. Both overexpression of a kinase-deficient PIP5K mutant and microinjection of anti-PI(4,5)P(2) antibodies compromise cytokinesis by preventing local accumulation of PI(4,5)P(2) in the cleavage furrow. These findings demonstrate that the localized production of PI(4,5)P(2) is required for the proper completion of cytokinesis and that the possible formation of a unique lipid domain in the cleavage furrow membrane may play a crucial role in coordinating the contractile rearrangement with the membrane remodeling during late cytokinesis.

Cleavage furrow formation and ingression during animal cytokinesis: a microtubule legacy

Cytokinesis ensures the proper partitioning of the nuclear and cytoplasmic contents into independent daughter cells at the end of cell division. Although the metazoan mitotic spindle has been implicated in the placement and advancement of the cleavage furrow, the molecules responsible for these processes have remained elusive. Recent studies have provided insights into the role of different microtubule structures and associated proteins in cleavage furrow positioning and ingression together with the signalling events that regulate the dynamics of the equatorial cell cortex during cytokinesis. We try to unify these findings into a general model of cytokinesis in which both astral and central spindle microtubules have the ability to induce furrowing. We further propose that the evolutionarily conserved centralspindlin complex serves as a master controller of cell cleavage in Drosophila by promoting both furrow formation and ingression. The same mechanism might be conserved in other organisms.

Cytokinesis: New Roles for Myosin

Myosin II is the motor for cytokinesis, an event at the end of cell division during which the animal cell uses a contractile ring to pinch itself in half. New and surprising research shows that myosin, either through light chain phosphorylation or through its ATPase activity, also plays an important role in both the assembly and disassembly of the actin contractile ring.

Molecular characterization of human ninein protein: two distinct subdomains required for centrosomal targeting and regulating signals in cell cycle

The centrosomal protein ninein has been identified as a microtubules minus end capping, centriole position, and anchoring protein, but the true physiological function remains to be determined. In this report, using immunofluorescence analysis and GFP-fusions we show that coiled-coil II domain (CCII domain, 1303–2096) co-localized with γ-tubulin and centrin at the centrosome. We further narrow down within 83 amino acids and classify a new centrosomal targeting signal. Interestingly, antibodies raised against CCII domain reveal that ninein protein declines from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Moreover, the data also suggest that fragment 1783–1866 may be attributed to declined signal of ninein. In kinase assay, we show that CCII domain could readily be phosphorylated by AIK and PKA. Taken together, our results suggest that ninein protein contains two distinct subdomains which are required for targeting and regulating asymmetry centrosomes. Importantly, the decline of ninein during mitosis implies that this centrosomal protein may play a role to regulate the process of chromosome segregation without discrimination. The model we propose here will foster a clearer picture of how two asymmetric centrosomes could direct and ensure the correct segregation of chromosomes during the mitotic stage.

A fruit-specific phospho enolpyruvate carboxylase is related to rapid growth of tomato fruit.

Malic and citric acids accumulate in cherry tomato (Lycopersicon esculentum Mill.) fruit during the period of rapid growth, from the end of cell division to the onset of ripening. The involvement of phospho enolpyruvate carboxylase (PEPCase, EC 4.1.1.31) in organic acid accumulation and tomato fruit development was investigated. Two PEPCases, named LYCes;Ppc1 and LYCes;Ppc2 and mapped to chromosomes 12 and 7, respectively, were shown to be differentially expressed during tomato fruit development. LYCes;Ppc1 mRNA was present in all fruit tissues and in all other plant organs examined. In contrast, LYCes;Ppc2 was strongly and specifically expressed in fruit from the end of cell division to ripening. No LYCes;Ppc2 expression was detected by northern blot in other plant tissues. In fruit, the increase in LYCes;Ppc2 mRNA was closely followed by an increase in fruit PEPCase protein and activity, and was coincident with the increased accumulation of malate and citrate during the initial period of rapid growth rate, from 8 to 20 days post anthesis. Localization of LYCes;Ppc2 mRNA in young tomato fruit by in situ hybridization revealed that LYCes;Ppc2 is preferentially expressed in large cells of the pericarp and in enlarging cells of the gel surrounding the seeds. Examination of the kinetic and regulatory properties of the PEPCases of growing and ripening fruit further showed that PEPCase in growing fruit is less sensitive to low pH and malate inhibition, indicating a high phosphorylation state and/or the presence of a PEPCase isoform with these characteristics. Taken together, these results indicate that in developing tomato fruit PEPCase is probably important in permitting the synthesis of organic acids to provide the turgor pressure necessary for cell expansion.

Effect of Pulp Cell Number and Assimilate Availability on Dry Matter Accumulation Rate in a Banana Fruit [ Musa sp. AAA group ‘Grande Naine’ (Cavendish subgroup)

Fruit position on the bunch (inflorescence) is an important part of variability in banana fruit weight at harvest, as fruits at the bottom of the bunch (distal fruits) are approx. 40% smaller than those at the top (proximal fruits). In this study, the respective roles of cell number and cell filling rate in the development of pulp dry weight are estimated. To this end, the source/sink ratio in the plant was altered at different stages of fruit development. Leaf shading (reducing resource availability), bunch bagging (increasing sink activity by increasing fruit temperature), and bunch trimming (decreasing sink size by fruit pruning), applied once cell division had finished, showed that the pulp filling rate depends on resource availability. Bunch bagging and bunch trimming were also carried out before the end of cell division to estimate the role of pulp cell number in the development of pulp dry weight. A sampling method was calibrated to evaluate pulp cell number from the digestion of a fixed portion of the pulp in a solution of chromic and nitric acids. A relationship was found between pulp cell number and fruit length at the end of cell division. It was observed that pulp cell number is a determining factor in pulp dry weight variability within a bunch. On the other hand, the cell filling rate was identical for all fruits in the bunch and was influenced by the source/sink ratio. A Michaelis-Menten relationship was invoked to relate the cell filling rate in a bunch to the source/sink ratio during bunch filling.