Delay In Cytokinesis Research Articles

Human Endonuclease ANKLE1 Localizes at the Midbody and Processes Chromatin Bridges to Prevent DNA Damage and cGAS-STING Activation.

Chromatin bridges connecting the two segregating daughter nuclei arise from chromosome fusion or unresolved interchromosomal linkage. Persistent chromatin bridges are trapped in the cleavage plane, triggering cytokinesis delay. The trapped bridges occasionally break during cytokinesis, inducing DNA damage and chromosomal rearrangements. Recently, Caenorhabditis elegans LEM-3 and human TREX1 nucleases have been shown to process chromatin bridges. Here, it is shown that ANKLE1 endonuclease, the human ortholog of LEM-3, accumulates at the bulge-like structure of the midbody via its N-terminal ankyrin repeats. Importantly, ANKLE1-/- knockout cells display an elevated level of G1-specific 53BP1 nuclear bodies, prolonged activation of the DNA damage response, and replication stress. Increased DNA damage observed in ANKLE1-/- cells is rescued by inhibiting actin polymerization or reducing actomyosin contractility. ANKLE1 does not act in conjunction with structure-selective endonucleases, GEN1 and MUS81 in resolving recombination intermediates. Instead, ANKLE1 acts on chromatin bridges by priming TREX1 nucleolytic activity and cleaving bridge DNA to prevent the formation of micronuclei and cytosolic dsDNA that activate the cGAS-STING pathway. It is therefore proposed that ANKLE1 prevents DNA damage and autoimmunity by cleaving chromatin bridges to avoid catastrophic breakage mediated by actomyosin contractile forces.

A novel checkpoint pathway controls actomyosin ring constriction trigger in fission yeast.

In fission yeast, the septation initiation network (SIN) ensures temporal coordination between actomyosin ring (CAR) constriction with membrane ingression and septum synthesis. However, questions remain about CAR regulation under stress conditions. We show that Rgf1p (Rho1p GEF), participates in a delay of cytokinesis under cell wall stress (blankophor, BP). BP did not interfere with CAR assembly or the rate of CAR constriction, but did delay the onset of constriction in the wild type cells but not in the rgf1Δ cells. This delay was also abolished in the absence of Pmk1p, the MAPK of the cell integrity pathway (CIP), leading to premature abscission and a multi-septated phenotype. Moreover, cytokinesis delay correlates with maintained SIN signaling and depends on the SIN to be achieved. Thus, we propose that the CIP participates in a checkpoint, capable of triggering a CAR constriction delay through the SIN pathway to ensure that cytokinesis terminates successfully.

Delayed cytokinesis generates multinuclearity and potential advantages in the amoeba Acanthamoebacastellanii Neff strain

Multinuclearity is a widespread phenomenon across the living world, yet how it is achieved, and the potential related advantages, are not systematically understood. In this study, we investigate multinuclearity in amoebae. We observe that non-adherent amoebae are giant multinucleate cells compared to adherent ones. The cells solve their multinuclearity by a stretchy cytokinesis process with cytosolic bridge formation when adherence resumes. After initial adhesion to a new substrate, the progeny of the multinucleate cells is more numerous than the sibling cells generated from uninucleate amoebae. Hence, multinucleate amoebae show an advantage for population growth when the number of cells is quantified over time. Multiple nuclei per cell are observed in different amoeba species, and the lack of adhesion induces multinuclearity in diverse protists such as Acanthamoebacastellanii, Vermamoebavermiformis, Naegleriagruberi and Hartmannellarhysodes. In this study, we observe that agitation induces a cytokinesis delay, which promotes multinuclearity. Hence, we propose the hypothesis that multinuclearity represents a physiological adaptation under non-adherent conditions that can lead to biologically relevant advantages.

Cleavage-furrow formation without F-actin in Chlamydomonas

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 alga Chlamydomonas 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 large Chlamydomonas chloroplast 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.

REI-1, a Novel Rab11 GEF with a SH3BP5 domain

ABSTRACTThe small GTPase Rab proteins are key regulators of membrane trafficking. Rab11 is one of the best-characterized molecules among the Rab family proteins and it plays multiple roles in endocytic recycling, exocytosis, and cytokinesis. However, it remains unclear how Rab11 is activated at a precise timing and location and regulates its diverse functions. Specifically, our knowledge of the upstream regulatory factors that activate Rab11 is limited. In this regard, we have identified the RAB-11-interacting protein-1 (REI-1) as a novel guanine nucleotide exchange factor (GEF) for RAB-11 in Caenorhabditis elegans (C. elegans). REI-1 family proteins are conserved among metazoans, and its human homolog, SH3BP5, also exhibits strong GEF activity toward human Rab11. In C. elegans, REI-1 is expressed in the germline and co-localizes with RAB-11 on late-Golgi membranes. The loss of REI-1 impaired the targeting of RAB-11 to the late-Golgi compartment, as well as the recycling endosomes in embryos and further reduced the recruitment of RAB-11 to the cleavage furrow, resulting in the delay of cytokinesis. We suggest that REI-1 is the GEF responsible for regulating RAB-11 localization and function in early embryos.

REI-1 Is a Guanine Nucleotide Exchange Factor Regulating RAB-11 Localization and Function in C. elegans Embryos

The small GTPase Rab11 dynamically changes its location to regulate various cellular processes such as endocytic recycling, secretion, and cytokinesis. However, our knowledge of its upstream regulators is still limited. Here, we identify the RAB-11-interacting protein-1 (REI-1) as a unique family of guanine nucleotide exchange factors (GEFs) for RAB-11 in Caenorhabditis elegans. Although REI-1 and its human homolog SH3-binding protein 5 do not contain any known Rab-GEF domains, they exhibited strong GEF activity toward Rab11 in vitro. In C. elegans, REI-1 is expressed in the germline and co-localizes with RAB-11 on the late-Golgi membranes. The loss of REI-1 specifically impaired the targeting of RAB-11 to the late-Golgi compartment and the recycling endosomes in embryos and further reduced the RAB-11 distribution to the cleavage furrow, which resulted in cytokinesis delay. These results suggest that REI-1 is a GEF specifically regulating the RAB-11 localization and functions in early embryos.

Phosphorylation of myosin II regulatory light chain by ZIP kinase is responsible for cleavage furrow ingression during cell division in mammalian cultured cells

Zipper-interacting protein kinase (ZIPK) is known to regulate several functions such as apoptosis, smooth muscle contraction, and cell migration. While exogenously expressed GFP-ZIPK localizes to the cleavage furrow, role of ZIPK in cytokinesis is obscure. Here, we show that ZIPK is a major MRLC kinase during mitosis. Moreover, ZIPK siRNA-mediated knockdown causes delay of cytokinesis. The delay in cytokinesis of ZIPK-knockdown cells was rescued by the exogenous diphosphorylation-mimicking MRLC mutant. Taken together, these findings suggest that ZIPK plays a role in the progression and completion of cytokinesis through MRLC phosphorylation.

Cnb1 involved in cytokinesis in Schizosaccharomyces pombe

Serine/Threonine-specific calcineurin (CN) is highly conserved in eukaryotes, which plays an important role in transcriptional regulation. In Schizosaccharomyces pombe, CN exists as a heterodimer composed by catalytic subunit Ppb1 and regulatory subunit Cnb1. Deletion of cnb1+ reduced the growth rate of cells, and caused a chained phenotype, and had delay in cytokinesis. In cytokinesis, Cnb1 could form CN complex with Ppb1 and could colocalize and constrict with the contractile ring at division plane. Tubulin could cross the septum in cnb1Δ strain, suggesting that the septum is not fully matured. These results suggest Cnb1 might be involved in maturation of septum. The signals of septins in cnb1Δ strain were also analyzed. Septins include Spn1, Spn2, Spn3, and Spn4. Septins help to guide hydrolytic enzymes for septum degrada-tion. Eighty percent of cnb1Δ cells lacked the signals of Spn2 or Spn3 at septum, and twenty percent of cnb1Δ cells lacked the signals of Spn1 or Spn4 at septum. The reduction of the septin signals was not due to impaired transcription of septins, since the protein levels of septins in the cnb1Δ cells were not decreased. These results imply that Cnb1 might regulate the stability of septin ring in a transcription-independent manner. In general, our study showed that Cnb1 contributes to the maturation of septum and the stability of septin ring and is important in the cytokinesis.

Targeting telomere-containing chromosome ends with a near-infrared femtosecond laser to study the activation of the DNA damage response and DNA damage repair pathways

Telomeres are at the ends of chromosomes. Previous evidence suggests that laser-induced deoxyribose nucleic acid (DNA) breaks at chromosome ends during anaphase results in delayed cytokinesis. A possible explanation for this delay is that the DNA damage response (DDR) mechanism has been activated. We describe a live imaging method to study the effects of DDR activation following focal point near-infrared femtosecond laser microirradiation either at a single chromosome end or at a chromosome arm in mitotic anaphase cells. Laser microirradiation is used in combination with dual fluorescent labeling to monitor the co-localization of double-strand break marker γH2AX along with the DDR factors in PtK2 (Potorous tridactylus) cells. Laser-induced DNA breaks in chromosome ends as well as in chromosome arms results in recruitment of the following: poly(ADP-ribose) polymerase 1, checkpoint sensors (p-Chk1, p-Chk2), DNA repair protein Ku70/Ku80, and proliferating cell nuclear antigen. However, phosphorylated p53 at serine 15 is detected only at chromosome ends and not at chromosome arms. Full activation of DDR on damaged chromosome ends may explain previously published results that showed the delay of cytokinesis.

Condensin and the Spindle Midzone Prevent Cytokinesis Failure Induced by Chromatin Bridges in C. elegans Embryos

During cell division, chromosomes must clear the path of the cleavage furrow before the onset of cytokinesis. The abscission checkpoint in mammalian cells stabilizes the cleavage furrow in the presence of a chromatin obstruction. This provides time to resolve the obstruction before the cleavage furrow regresses or breaks the chromosomes, preventing aneuploidy or DNA damage. Two unanswered questions in the proposed mechanistic pathway of the abscission checkpoint concern factors involved in (1) resolving the obstructions and (2) coordinating obstruction resolution with the delay in cytokinesis. We found that the one-cell and two-cell C. elegans embryos suppress furrow regression following depletion of essential chromosome-segregation factors: topoisomerase II(TOP-2), CENP-A(HCP-3), cohesin, and to a lesser degree, condensin. Chromatin obstructions activated Aurora B(AIR-2) at the spindle midzone, which is needed for the abscission checkpoint in other systems. Condensin I, but not condensin II, localizes to the spindle midzone in anaphase and to the midbody during normal cytokinesis. Interestingly, condensin I is enriched on chromatin bridges and near the midzone/midbody in an AIR-2-dependent manner. Disruption of AIR-2, the spindle midzone, or condensin leads to cytokinesis failure in a chromatin-obstruction-dependent manner. Examination of the condensin-deficient embryos uncovered defects in both the resolution of the chromatin obstructions and the maintenance of the stable cleavage furrow. We postulate that condensin I is recruited by Aurora B(AIR-2) to aid in the resolution of chromatin obstructions and also helps generate a signal to maintain the delay in cytokinesis.

Tissue inducible Lifeact expression allows visualization of actin dynamics in vivo and ex vivo

We describe here the development and characterization of a conditionally inducible mouse model expressing Lifeact-GFP, a peptide that reports the dynamics of filamentous actin. We have used this model to study platelets, megakaryocytes and melanoblasts and we provide evidence that Lifeact-GFP is a useful reporter in these cell types ex vivo. In the case of platelets and megakaryocytes, these cells are not transfectable by traditional methods, so conditional activation of Lifeact allows the study of actin dynamics in these cells live. We studied melanoblasts in native skin explants from embryos, allowing the visualization of live actin dynamics during cytokinesis and migration. Our study revealed that melanoblasts lacking the small GTPase Rac1 show a delay in the formation of new pseudopodia following cytokinesis that accounts for the previously reported cytokinesis delay in these cells. Thus, through use of this mouse model, we were able to gain insights into the actin dynamics of cells that could only previously be studied using fixed specimens or following isolation from their native tissue environment.

The involvement of MCT-1 oncoprotein in inducing mitotic catastrophe and nuclear abnormalities

Centrosome amplification and chromosome abnormality are frequently identified in neoplasia and tumorigenesis. However, the mechanisms underlying these defects remain unclear. We here identify that MCT-1 is a centrosomal oncoprotein involved in mitosis. Knockdown of MCT-1 protein results in intercellular bridging, chromosome mis-congregation, cytokinesis delay, and mitotic death. Introduction of MCT-1 oncogene into the p53 deficient cells (MCT-1-p53), the mitotic checkpoint kinases and proteins are deregulated synergistically. These biochemical alterations are accompanied with increased frequencies of cytokinesis failure, multi-nucleation, and centrosome amplification in subsequent cell cycle. As a result, the incidences of polyploidy and aneuploidy are progressively induced by prolonged cell cultivation or further promoted by sustained spindle damage on MCT-1-p53 background. These data show that the oncoprotein perturbs centrosome structure and mitotic progression, which provide the molecular aspect of chromsomal abnormality in vitro and the information for understanding the stepwise progression of tumors under oncogenic stress.

Abstract A68: MCT-1 oncoprotein induces chromosomal aberrations through impairing centrosomal and mitotic-spindle checkpoints.

Abstract Centrosome amplification and chromosome abnormalities are frequently detected in neoplasia and during tumorigenesis; however, the mechanism underlying these defects is still unclear. We here identify that the translational regulator, MCT-1, is a novel proto-oncoprotein located at the spindle-organizing apparatus. Decrease of cellular MCT-1 level results in intercellular bridging, chromosome mis-congregation, cytokinesis delay, and post-mitotic apoptosis. MCT-1 induction alongside p53 deficiency (MCT-1-p53) synergistically deregulates mitotic checkpoint kinases and induces Cyclin E protein degradation. These are implicated with increased frequency of cytokinesis failure, multi-nucleation, and centrosome amplification in the subsequent cell cycle. Consistent with the incidences of polyploidy and aneuploidy are progressively induced by the continued cell cultivation and further promoted by the disruption of microtubule structure, array CGH analysis confirms that the pronounced chromosome amplifications and deletions occur on MCT-1-p53 cellular background. This is the first demonstration that MCT-1 plays an important role in the regulation of centrosome integrity and mitotic progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A68.

The Peptidyl-Prolyl Isomerase Pin1 Regulates Cytokinesis through Cep55

Failure of cytokinesis results in tetraploidy and can increase the genomic instability frequently observed in cancer. The peptidyl-prolyl isomerase Pin1, which is deregulated in many tumors, regulates several processes, including cell cycle progression. Here, we show a novel role for Pin1 in cytokinesis. Pin1 knockout mouse embryonic fibroblasts show a cytokinesis delay, and depletion of Pin1 from HeLa cells also causes a cytokinesis defect. Furthermore, we provide evidence that Pin1 localizes to the midbody ring and regulates the final stages of cytokinesis by binding to centrosome protein 55 kDa (Cep55), an essential component of this ring. This interaction induces Polo-like kinase 1-mediated phosphorylation of Cep55, which is critical for the function of Cep55 during cytokinesis. Importantly, Pin1 knockdown does not enhance the cytokinesis defect in Cep55-depleted cells, indicating that Pin1 and Cep55 act in the same pathway. These data are the first evidence that Pin1 regulates cytokinesis and may provide a mechanistic explanation as to how pathologic levels of Pin1 can stimulate tumorigenesis.

Disruption of actin cytoskeleton reorganization after fertilization affects cell cycle time in sea urchin eggs

We have investigated the effects of cytochalasin D on first cleavage of fertilized eggs from the sea urchin Strongylocentrotus purpuratus. Cytochalasin D inhibits the cortical actin filament reorganization that occurs immediately after fertilization in sea urchin eggs, and arrests cell division by preventing normal contractile ring formation which inhibits cytokinesis. In this study, eggs were incubated in 2 or 4 μg/mL cytochalasin D within the first 5 minutes post-fertilization, for 10, 20, or 30 minutes, followed by washout with artificial sea water. At 2 μg/mL, two groups of results were obtained; a 20–25 minute delay in cytokinesis irrespective of exposure time, and a time-dependent response where longer incubation times produced lengthier delays in cytokinesis. The time-dependent delays in cell division also occurred at 4 μg/mL cytochalasin D, along with cytotoxic effects that correlated to the length of drug exposure. The results show that disruption of the normal actin cytoskeleton reorganization which occurs within the first 30 minutes after fertilization can affect the timing of first cleavage, suggesting that the state of the actin cytoskeleton is somehow monitored as cells enter cytokinesis.

Regulation of mitotic exit by the RNF8 ubiquitin ligase

RNF8 is a ubiquitin ligase with a FHA domain near its N terminus, and a RING-finger domain at its C terminus, through which it recruits several ubiquitin-conjugating enzymes. In metazoans, only the mitotic checkpoint regulator CHFR shares this domain architecture. Here we show that RNF8 is a nuclear protein that follows a cell-cycle-dependent turnover, reaching its highest levels in mitosis, followed by a strong decline in late mitotic stages. Overexpression of RNF8 caused a delay in cytokinesis and the frequent appearance of aberrant mitotic figures. These effects were dependent on the ubiquitin ligase activity of RNF8, since they were significantly attenuated when a RING-finger mutant, inactive as an E3, was overexpressed. Depletion of RNF8 also caused a delay in the exit from the mitotic arrest induced by nocodazole, associated with a reduced turnover of the APC/C substrate cyclin B1. These observations suggest that RNF8 regulates the rate of exit from mitosis and cytokinesis.

A Specific Isoform of Nonmuscle Myosin II-C Is Required for Cytokinesis in a Tumor Cell Line

Nonmuscle myosin IIs play an essential role during cytokinesis. Here, we explore the function of an alternatively spliced isoform of nonmuscle myosin heavy chain (NMHC) II-C, called NMHC II-C1, in the A549 human lung tumor cell line during cytokinesis. NMHC II-C1 contains an insert of 8 amino acids in the head region of NMHC II-C. First, we show that there is a marked increase in both the mRNA encoding NMHC II-C1 and protein in tumor cell lines compared with nontumor cell lines derived from the same tissue. Quantification of the amount of myosin II isoforms in the A549 cells shows that the amounts of NMHC II-A and II-C1 protein are about equal and substantially greater than NMHC II-B. Using specific siRNAs to decrease NMHC II-C1 in cultured A549 cells resulted in a 5.5-fold decrease in the number of cells at 120 h, whereas decreasing NMHC II-A with siRNA does not affect cell proliferation. This decreased proliferation can be rescued by reintroducing NMHC II-C1 but not NMHC II-A or II-B into A549 cells, although noninserted NMHC II-C does rescue to a limited extent. Time lapse video microscopy revealed that loss of NMHC II-C1 leads to a delay in cytokinesis and prolongs it from 2 to 8-10 h. These findings are consistent with the localization of NMHC II-C1 to the intercellular bridge that attaches the two dividing cells during the late phases of cytokinesis. The results suggest a specific function for NMHC II-C1 in cytokinesis in the A549 tumor cell line.

Histone H2B mutations in inner region affect ubiquitination, centromere function, silencing and chromosome segregation

The reiterated nature of histone genes has hampered genetic approach to dissect the role of histones in chromatin dynamics. We here report isolation of three temperature-sensitive (ts) Schizosaccharomyces pombe strains, containing amino-acid substitutions in the sole histone H2B gene (htb1+). The mutation sites reside in the highly conserved, non-helical residues of H2B, which are implicated in DNA-protein or protein-protein interactions in the nucleosome. In the allele of htb1-72, the substitution (G52D) occurs at the DNA binding loop L1, causing disruption of the gene silencing in heterochromatic regions and lagging chromosomes in anaphase. In another allele htb1-223 (P102L) locating in the junction between alpha3 and alphaC, the mutant residue is in contact with H2A and other histones, leading to structural aberrations in the central centromere chromatin and unequal chromosome segregation in anaphase. The third allele htb1-442 (E34K) near alpha1 displayed little defect. Evidence is provided that monoubiquitinated H2B is greatly unstable in P102L mutant, possibly owing to proteasome-independent destruction or enhanced deubiquitination. Histone H2B thus plays an important role in centromere/kinetochore formation.

Interaction between EB1 and p150 glued Is Required for Anaphase Astral Microtubule Elongation and Stimulation of Cytokinesis

In animal cells, microtubules (MTs) of the mitotic apparatus (MA) communicate with the cell cortex to stimulate cytokinesis; however, the molecular nature of this stimulus remains elusive . A signal for cytokinesis likely involves the MT plus end binding family of proteins, which includes EB1, p150glued, APC, LIS1, and CLIP-170. These proteins modulate MT dynamics and facilitate interactions between growing MTs and their intracellular targets, including kinetochores, organelles, and the cell cortex . The dynein-dynactin complex mediates many of these microtubule capture events . We report that EB1 and p150glued interactions are required for stimulation of cytokinesis in dividing sea urchin eggs. Injected antibodies against EB1 or p150glued suppressed furrow ingression but did not prevent elongation of anaphase astral MTs toward the cortex, suggesting that EB1 and dynactin are both required for communication between the MA and the cortex. Targeted disruption of the interaction between EB1 and p150glued suppressed anaphase astral MT elongation and resulted in a delay of cytokinesis that could not be overcome by manipulation of the asters toward the cortex. We conclude that EB1 and dynactin participate in stimulation of the cleavage furrow, and their interaction promotes elongation of astral MTs at anaphase onset.

Chromosomal Instability Induced by Pim-1 Is Passage-dependent and Associated with Dysregulation of Cyclin B1

Overexpression of the oncogenic serine/threonine kinase Pim-1 has been shown to induce chromosomal missegregation and polyploidy in prostate epithelial cell lines (1). Here we demonstrated that Pim-1-induced polyploidy develops in a passage-dependent manner in culture consistent with a stochastic mode of progression. Induction of chromosomal instability by Pim-1 was not restricted to prostate cells as it was also observed in telomerase-immortalized normal human mammary epithelial cells. Elevated levels of cyclin B1 protein, but not its messenger RNA, were evident in early passage Pim-1 overexpressing cells, suggesting that increased cyclin B1 levels contribute to the development of polyploidy. Furthermore, regulation of cyclin B1 protein and cyclin B1/CDK1 activity after treatment with anti-microtubule agents was impaired. Small interfering RNA targeting cyclin B1 reversed the cytokinesis delay but not the mitotic checkpoint defect in Pim-1 overexpressing cells. These results indicated that chronic Pim-1 overexpression dysregulates cyclin B1 protein expression, which contributes to the development of polyploidy by delaying cytokinesis.