Formation Of Contractile Ring Research Articles

Non-Muscle Myosin IIC as a Prognostic and Therapeutic Target in Cancer

Cancer metastasis is associated with epithelial-mesenchymal transition (EMT), and NMII is linked to EMT. Calcium sensitization through ROCK activates myosin II, contributing to metastasis. NMII undergoes phosphorylation at Thr18/Ser19, affecting its function and involvement in processes like exocytosis, apoptosis, and transcellular intravasations. Inhibition of myosin II ATPase activity blocks cytokinesis, highlighting its role in cell cycle regulation. NMII has distinct binding motifs and sites for regulation, including Thr18/Ser19 via kinases/phosphatases, Ser1/Ser2/Thr9 via phosphokinase C, and involvement in cytokinesis via contractile ring formation. These characteristics make NMII a potential therapeutic target. NMII-C is found to be less expressive in glioma (40% low expression, 60% high expression), not showing prognostic value. However, in stomach cancer (35–65% expression) and renal cancer (40–60% expression), NMII-C appears to have prognostic significance. NMII-C’s prognostic value has not been reported in various other cancers, including thyroid, lung, liver, colorectal, urothelial, endometrial, head and neck, melanoma, prostate, testicular, pancreatic, breast, cervical, and ovarian cancers, highlighting a need for further research in these areas. Some pharmacological agents such as Blebbistatin, DT-13, Statins, and Y27632 have shown promising potential against NMII-C in cancer therapy, with positive results in pre-clinical studies and ongoing clinical trials. Understanding the specific functions and regulation of NMII isoforms, particularly NMII-C, may provide valuable insights into the development of targeted cancer therapies. Additionally, the study suggests that NMII-C may have prognostic importance in specific cancer stages, offering a foundation for further exploration of NMII-C’s mechanisms of action for future medical interventions.

A role for the carbon source of the cell and protein kinase A in regulating the S. pombe septation initiation network.

The septation initiation network (SIN) is a conserved signal transduction network, which is important for cytokinesis in Schizosaccharomyces pombe. The SIN component Etd1p is required for association of some SIN proteins with the spindle pole body (SPB) during anaphase and for contractile ring formation. We show that tethering of Cdc7p or Sid1p to the SIN scaffold Cdc11p at the SPB, rescues etd1-Δ. Analysis of a suppressor of the mutant etd1-M9 revealed that SIN signalling is influenced by the carbon source of the cell. Growth on a non-fermentable carbon source glycerol reduces the requirement for SIN signalling but does not bypass it. The decreased need for SIN signalling is mediated largely by reduction of protein kinase A activity, and it is phenocopied by deletion of pka1 on glucose medium. We conclude that protein kinase A is an important regulator of the SIN, and that SIN signalling is regulated by the carbon source of the cell.

Modelling contractile ring formation and division to daughter cells for simulating proliferative multicellular dynamics.

Cell proliferation is a fundamental process underlying embryogenesis, homeostasis, wound healing, and cancer. The process involves multiple events during each cell cycle, such as cell growth, contractile ring formation, and division to daughter cells, which affect the surrounding cell population geometrically and mechanically. However, existing methods do not comprehensively describe the dynamics of multicellular structures involving cell proliferation at a subcellular resolution. In this study, we present a novel model for proliferative multicellular dynamics at the subcellular level by building upon the nonconservative fluid membrane (NCF) model that we developed in earlier research. The NCF model utilizes a dynamically-rearranging closed triangular mesh to depict the shape of each cell, enabling us to analyze cell dynamics over extended periods beyond each cell cycle, during which cell surface components undergo dynamic turnover. The proposed model represents the process of cell proliferation by incorporating cell volume growth and contractile ring formation through an energy function and topologically dividing each cell at the cleavage furrow formed by the ring. Numerical simulations demonstrated that the model recapitulated the process of cell proliferation at subcellular resolution, including cell volume growth, cleavage furrow formation, and division to daughter cells. Further analyses suggested that the orientation of actomyosin stress in the contractile ring plays a crucial role in the cleavage furrow formation, i.e., circumferential orientation can form a cleavage furrow but isotropic orientation cannot. Furthermore, the model replicated tissue-scale multicellular dynamics, where the successive proliferation of adhesive cells led to the formation of a cell sheet and stratification on the substrate. Overall, the proposed model provides a basis for analyzing proliferative multicellular dynamics at subcellular resolution.

Two RhoGEF isoforms with distinct localisation control furrow position during asymmetric cell division

Cytokinesis partitions cellular content between daughter cells. It relies on the formation of an acto-myosin contractile ring, whose constriction induces the ingression of the cleavage furrow between the segregated chromatids. Rho1 GTPase and its RhoGEF (Pbl) are essential for this process. However, how Rho1 is regulated to sustain furrow ingression while maintaining correct furrow position remains poorly defined. Here, we show that during asymmetric division of Drosophila neuroblasts, Rho1 is controlled by two Pbl isoforms with distinct localisation. Spindle midzone- and furrow-enriched Pbl-A focuses Rho1 at the furrow to sustain efficient ingression, while Pbl-B pan-plasma membrane localization promotes the broadening of Rho1 activity and the subsequent enrichment of myosin on the entire cortex. This enlarged zone of Rho1 activity is critical to adjust furrow position, thereby preserving correct daughter cell size asymmetry. Our work highlights how the use of isoforms with distinct localisation makes an essential process more robust.

Measurement of Microtubule Stability in Mammalian Cells.

The microtubule cytoskeleton is essential for various biological processes such as the intracellular distribution of molecules and organelles, cell morphogenesis, chromosome segregation, and specification of the location of contractile ring formation. Distinct cell types contain microtubules with different extents of stability. For example, microtubules in neurons are highly stabilized to support organelle (or vesicular) transport over large distances, and microtubules in motile cells are more dynamic. In some cases, such as the mitotic spindle, both dynamic and stable microtubules coexist. Alteration of microtubule stability is connected to disease states, making understanding microtubule stability an important area of research. Methods to measure microtubule stability in mammalian cells are described here. Together, these approaches allow microtubule stability to be measured qualitatively or semiquantitatively following staining for post-translational modifications of tubulin or treating cells with microtubule destabilizing agents such as nocodazole. Microtubule stability can also be measured quantitatively by performing fluorescence recovery after photobleaching or fluorescence photoactivation of tubulin in live cells. These methods should be helpful for those seeking to understand microtubule dynamics and stabilization. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Fixing and staining cells for tubulin post-translational modifications Basic Protocol 2: Evaluating microtubule stability following treatment with nocodazole in live or fixed cells Basic Protocol 3: Measurement of microtubule dynamic turnover by quantification of fluorescence recovery after photobleaching Basic Protocol 4: Measurement of microtubule dynamic turnover by quantification of dissipation of fluorescence after photoactivation.

Targeting USP10 induces degradation of oncogenic ANLN in esophageal squamous cell carcinoma

Anillin (ANLN) is a mitosis-related protein that promotes contractile ring formation and cytokinesis, but its cell cycle-dependent degradation mechanisms in cancer cells remain unclear. Here, we show that high expression of ANLN promotes cytokinesis and proliferation in esophageal squamous cell carcinoma (ESCC) cells and is associated with poor prognosis in ESCC patients. Furthermore, the findings of the study showed that the deubiquitinating enzyme USP10 interacts with ANLN and positively regulates ANLN protein levels. USP10 removes the K11- and K63-linked ubiquitin chains of ANLN through its deubiquitinase activity and prevents ANLN ubiquitin-mediated degradation. Importantly, USP10 promotes contractile ring assembly at the cytokinetic furrow as well as cytokinesis by stabilizing ANLN. Interestingly, USP10 and the E3 ubiquitin ligase APC/C co-activator Cdh1 formed a functional complex with ANLN in a non-competitive manner to balance ANLN protein levels. In addition, the macrolide compound FW-04-806 (F806), a natural compound with potential for treating ESCC, inhibited the mitosis of ESCC cells by targeting USP10 and promoting ANLN degradation. F806 selectively targeted USP10 and inhibited its catalytic activity but did not affect the binding of Cdh1 to ANLN and alters the balance of the USP10-Cdh1-ANLN complex. Additionally, USP10 expression was positively correlated with ANLN level and poor prognosis of ESCC patients. Overall, targeting the USP10-ANLN axis can effectively inhibit ESCC cell-cycle progression.

Delivery of ceramide phosphoethanolamine lipids to the cleavage furrow through the endocytic pathway is essential for male meiotic cytokinesis.

Cell division, wherein 1 cell divides into 2 daughter cells, is fundamental to all living organisms. Cytokinesis, the final step in 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 specified spatiotemporal manner generates a cleavage furrow that physically separates the cytoplasm. Unique lipids with specific biophysical properties have been shown to localize to intercellular bridges (also called midbody) connecting the 2 dividing cells; however, their biological roles and delivery mechanisms remain largely unknown. In this study, we show that ceramide phosphoethanolamine (CPE), the structural analog of sphingomyelin, has unique acyl chain anchors in Drosophila spermatocytes and is essential for meiotic cytokinesis. The head group of CPE is also important for spermatogenesis. We find that aberrant central spindle and contractile ring behavior but not mislocalization of phosphatidylinositol phosphates (PIPs) at the plasma membrane is responsible for the male meiotic cytokinesis defect in CPE-deficient animals. Further, we demonstrate the enrichment of CPE in multivesicular bodies marked by Rab7, which in turn localize to cleavage furrow. Volume electron microscopy analysis using correlative light and focused ion beam scanning electron microscopy shows that CPE-enriched Rab7 positive endosomes are juxtaposed on contractile ring material. Correlative light and transmission electron microscopy reveal Rab7 positive endosomes as a multivesicular body-like organelle that releases its intraluminal vesicles in the vicinity of ingressing furrows. Genetic ablation of Rab7 or Rab35 or expression of dominant negative Rab11 results in significant meiotic cytokinesis defects. Further, we show that Rab11 function is required for localization of CPE positive endosomes to the cleavage furrow. Our results imply that endosomal delivery of CPE to ingressing membranes is crucial for meiotic cytokinesis.

On the role of mechanosensitive binding dynamics in the pattern formation of active surfaces

The actin cortex of an animal cell is a thin polymeric layer attached to the inner side of the plasma membrane. It plays a key role in shape regulation and pattern formation on the cellular and tissue scale and, in particular, generates the contractile ring during cell division. Experimental studies showed that the cortex is fluid-like but highly viscous on long time scales with a mechanics that is sensitively regulated by active and passive cross-linker molecules that tune active stress and shear viscosity. Here, we use an established minimal model of active surface dynamics of the cell cortex supplemented with the experimentally motivated feature of mechanosensitivity in cross-linker binding dynamics. Performing linear stability analysis and computer simulations, we show that cross-linker mechanosensitivity significantly enhances the versatility of pattern formation and enables self-organized formation of contractile rings. Furthermore, we address the scenario of concentration-dependent shear viscosities as a way to stabilize ring-like patterns and constriction in the mid-plane of the active surface.

Measurement of Contractile Ring Tension Using Two-photon Laser Ablation during Drosophila Cellularization.

Cytokinesis occurs at the final step of cell division and leads to the separation of daughter cells. It requires assembly and constriction of the actomyosin contractile ring. The phases of assembly and constriction of the contractile ring show an increase in tension in the actomyosin complex. The measurement of tension in the contractile ring is of interest to probe the mechanics of contractile ring formation. Drosophila cellularization is a powerful genetic model system to study the mechanisms regulating actomyosin contractility during contractile ring constriction. Cellularization occurs in the interphase of syncytial cycle 14, where the plasma membrane extends around individual nuclei and forms complete cells with the help of a contractile ring at the bottom. The contractile ring forms at the furrow tip during the extension around individual nuclei and its assembly requires the coordinated action of cytoskeletal and plasma membrane-associated proteins. Laser ablation of the contractile ring enables the measurement of the contractility of the actomyosin network during cytokinesis. This protocol outlines the method used for estimating the contractility at the actomyosin ring during cellularization by laser ablation, in both control and mutant embryos for a Rho guanosine triphosphatase activating protein (RhoGAP) containing protein called GRAF (GTPase regulator associated with focal adhesion kinase-1). Physical cutting of the contractile ring by a two-photon laser at 800 nm leads to the displacement of the actomyosin ring edges, at a rate dependent upon the tension. This can be carried out at distinct steps of the contractile ring assembly during furrow extension in cellularization. Quantification of the extent of displacement and recoil velocity of movement of the edges at different stages of cellularization provides a quantitative measure of contractility in the system. This protocol describes the experimental procedure containing the preparation of live embryos, optimization of laser power, acquisition settings, and post-acquisition analysis of actomyosin contractility during Drosophila cellularization.

A picket fence function for adherens junctions in epithelial cell polarity.

Adherens junctions are a defining feature of all epithelial cells, providing cell-cell adhesion and contractile ring formation that is essential for cell and tissue morphology. In Drosophila, adherens junctions are concentrated between the apical and basolateral plasma membrane domains, defined by aPKC-Par6-Baz and Lgl/Dlg/Scrib, respectively. Whether adherens junctions contribute to apical-basal polarization itself has been unclear because neuroblasts exhibit apical-basal polarization of aPKC-Par6-Baz and Lgl in the absence of adherens junctions. Here we show that, upon disruption of adherens junctions in epithelial cells, apical polarity determinants such as aPKC can still segregate from basolateral Lgl, but lose their sharp boundaries and also overlap with Dlg and Scrib - similar to neuroblasts. In addition, control of apical versus basolateral domain size is lost, along with control of cell shape, in the absence of adherens junctions. Manipulating the levels of apical Par3/Baz or basolateral Lgl polarity determinants in experiments and in computer simulations confirms that adherens junctions provide a 'picket fence' diffusion barrier that restricts the spread of polarity determinants along the membrane to enable precise domain size control. Movement of adherens junctions in response to mechanical forces during morphogenetic change thus enables spontaneous adjustment of apical versus basolateral domain size as an emergent property of the polarising system.

First person – Alaina Willet

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Alaina Willet is first author on ‘ Phosphorylation in the intrinsically disordered region of F-BAR protein Imp2 regulates its contractile ring recruitment’, published in JCS. Alaina is a Research Instructor in the lab of Kathy Gould at Vanderbilt University, Nashville, TN, USA, investigating the mechanisms regulating contractile ring formation in fission yeast.

Formin Cdc12’s specific actin assembly properties are tailored for cytokinesis in fission yeast

Formins generate unbranched actin filaments by a conserved, processive actin assembly mechanism. Most organisms express multiple formin isoforms that mediate distinct cellular processes and facilitate actin filament polymerization by significantly different rates, but how these actin assembly differences correlate to cellular activity is unclear. We used a computational model of fission yeast cytokinetic ring assembly to test the hypothesis that particular actin assembly properties help tailor formins for specific cellular roles. Simulations run in different actin filament nucleation and elongation conditions revealed that variations in formin’s nucleation efficiency critically impact both the probability and timing of contractile ring formation. To probe the physiological importance of nucleation efficiency, we engineered fission yeast formin chimera strains in which the FH1-FH2 actin assembly domains of full-length cytokinesis formin Cdc12 were replaced with the FH1-FH2 domains from functionally and evolutionarily diverse formins with significantly different actin assembly properties. Although Cdc12 chimeras generally support life in fission yeast, quantitative live-cell imaging revealed a range of cytokinesis defects from mild to severe. In agreement with the computational model, chimeras whose nucleation efficiencies are least similar to Cdc12 exhibit more severe cytokinesis defects, specifically in the rate of contractile ring assembly. Together, our computational and experimental results suggest that fission yeast cytokinesis is ideally mediated by a formin with properly tailored actin assembly parameters.

Adherens junctions are involved in polarized contractile ring formation in dividing epithelial cells of Xenopus laevis embryos

Cells dividing in the plane of epithelial tissues proceed by polarized constriction of the actomyosin contractile ring, leading to asymmetric ingression of the plasma mem brane. Asymmetric cytokinesis results in the apical positioning of the actomyosin contractile ring and ultimately of the midbody. Studies have indicated that the contractile ring is associated with adherens junctions, whose role is to maintain epithelial tissue cohesion. However, it is yet unknown when the contractile ring becomes associated with adherens junctions in epithelial cells. Here, we examined contractile ring formation and activation in the epithelium of Xenopus embryos and explored the implication of adherens junctions in the contractile ring formation. We show that accumulation of proteins involved in contractile ring formation and activation is polarized, starting at apical cell-cell contacts at the presumptive division site and spreading within seconds towards the cell basal side. We also show that adherens junctions are involved in the kinetics of contractile ring formation. Our study reveals that the link between the adherens junctions and the contractile ring is established from the onset of cytokinesis.

CAP1, a target of miR-144/451, negatively regulates erythroid differentiation and enucleation.

The exact molecular mechanism underlying erythroblast enucleation has been a fundamental biological question for decades. In this study, we found that miR‐144/451 critically regulated erythroid differentiation and enucleation. We further identified CAP1, a G‐actin‐binding protein, as a direct target of miR‐144/451 in these processes. During terminal erythropoiesis, CAP1 expression declines along with gradually increased miR‐144/451 levels. Enforced CAP1 up‐regulation inhibits the formation of contractile actin rings in erythroblasts and prevents their terminal differentiation and enucleation. Our findings reveal a negative regulatory role of CAP1 in miR‐144/451‐mediated erythropoiesis and thus shed light on how microRNAs fine‐tune terminal erythroid development through regulating actin dynamics.

C. elegans CLASP/CLS-2 negatively regulates membrane ingression throughout the oocyte cortex and is required for polar body extrusion.

The requirements for oocyte meiotic cytokinesis during polar body extrusion are not well understood. In particular, the relationship between the oocyte meiotic spindle and polar body contractile ring dynamics remains largely unknown. We have used live cell imaging and spindle assembly defective mutants lacking the function of CLASP/CLS-2, kinesin-12/KLP-18, or katanin/MEI-1 to investigate the relationship between meiotic spindle structure and polar body extrusion in C. elegans oocytes. We show that spindle bipolarity and chromosome segregation are not required for polar body contractile ring formation and chromosome extrusion in klp-18 mutants. In contrast, oocytes with similarly severe spindle assembly defects due to loss of CLS-2 or MEI-1 have penetrant and distinct polar body extrusion defects: CLS-2 is required early for contractile ring assembly or stability, while MEI-1 is required later for contractile ring constriction. We also show that CLS-2 both negatively regulates membrane ingression throughout the oocyte cortex during meiosis I, and influences the dynamics of the central spindle-associated proteins Aurora B/AIR-2 and MgcRacGAP/CYK-4. We suggest that proper regulation by CLS-2 of both oocyte cortical stiffness and central spindle protein dynamics may influence contractile ring assembly during polar body extrusion in C. elegans oocytes.

A stochastic model for dynamics of FtsZ filaments and the formation of Z -ring.

Understanding the mechanisms responsible for the formation and growth of FtsZ polymers and their subsequent formation of the Z -ring is important for gaining insight into the cell division in prokaryotic cells. In this work, we present a minimal stochastic model that qualitatively reproduces in vitro observations of polymerization, formation of dynamic contractile ring that is stable for a long time and depolymerization shown by FtsZ polymer filaments. In this stochastic model, we explore different mechanisms for ring breaking and hydrolysis. In addition to hydrolysis, which is known to regulate the dynamics of other tubulin polymers like microtubules, we find that the presence of the ring allows for an additional mechanism for regulating the dynamics of FtsZ polymers. Ring breaking dynamics in the presence of hydrolysis naturally induce rescue and catastrophe events in this model irrespective of the mechanism of hydrolysis.

How cortical waves drive fission of motile cells

Cytokinesis-the division of a cell into two daughter cells-is a key step in cell growth and proliferation. It typically occurs in synchrony with the cell cycle to ensure that a complete copy of the genetic information is passed on to the next generation of daughter cells. In animal cells, cytokinesis commonly relies on an actomyosin contractile ring that drives equatorial furrowing and separation into the two daughter cells. However, also contractile ring-independent forms of cell division are known that depend on substrate-mediated traction forces. Here, we report evidence of an as yet unknown type of contractile ring-independent cytokinesis that we termed wave-mediated cytofission. It is driven by self-organized cortical actin waves that travel across the ventral membrane of oversized, multinucleated Dictyostelium discoideum cells. Upon collision with the cell border, waves may initiate the formation of protrusions that elongate and eventually pinch off to form separate daughter cells. They are composed of a stable elongated wave segment that is enclosed by a cell membrane and moves in a highly persistent fashion. We rationalize our observations based on a noisy excitable reaction-diffusion model in combination with a dynamic phase field to account for the cell shape and demonstrate that daughter cells emerging from wave-mediated cytofission exhibit a well-controlled size.

An interphase contractile ring reshapes primordial germ cells to allow bulk cytoplasmic remodeling.

Some cells discard undesired inherited components in bulk by forming large compartments that are subsequently eliminated. Caenorhabditis elegans primordial germ cells (PGCs) jettison mitochondria and cytoplasm by forming a large lobe that is cannibalized by intestinal cells. Although PGCs are nonmitotic, we find that lobe formation is driven by constriction of a contractile ring and requires the RhoGEF ECT-2, a RhoA activator also essential for cytokinesis. Whereas centralspindlin activates ECT-2 to promote cytokinetic contractile ring formation, we show that the ECT-2 regulator NOP-1, but not centralspindlin, is essential for PGC lobe formation. We propose that lobe contractile ring formation is locally inhibited by the PGC nucleus, which migrates to one side of the cell before the cytokinetic ring assembles on the opposite cortex. Our findings reveal how components of the cytokinetic contractile ring are reemployed during interphase to create compartments used for cellular remodeling, and they reveal differences in the spatial cues that dictate where the contractile ring will form.

Cell cycle-independent furrowing triggered by phosphomimetic mutations of the INCENP STD motif requires Plk1.

ABSTRACTTimely and precise control of Aurora B kinase, the chromosomal passenger complex (CPC) catalytic subunit, is essential for accurate chromosome segregation and cytokinesis. Post-translational modifications of CPC subunits are directly involved in controlling Aurora B activity. Here, we identified a highly conserved acidic STD-rich motif of INCENP that is phosphorylated during mitosis in vivo and by Plk1 in vitro and is involved in controlling Aurora B activity. By using an INCENP conditional-knockout cell line, we show that impairing the phosphorylation status of this region disrupts chromosome congression and induces cytokinesis failure. In contrast, mimicking constitutive phosphorylation not only rescues cytokinesis but also induces ectopic furrows and contractile ring formation in a Plk1- and ROCK1-dependent manner independent of cell cycle and microtubule status. Our experiments identify the phospho-regulation of the INCENP STD motif as a novel mechanism that is key for chromosome alignment and cytokinesis.This article has an associated First Person interview with the first author of the paper.

Mechanical stress induces a scalable switch in cortical flow polarization during cytokinesis.

ABSTRACTDuring animal development, cells need to sense and adapt to mechanical forces from their environment. Ultimately, these forces are transduced through the actomyosin cortex. How the cortex simultaneously responds to and creates forces during cytokinesis is not well understood. Here we show that, under mechanical stress, cortical actomyosin flow can switch polarization during cytokinesis in the C. elegans embryo. In unstressed embryos, longitudinal cortical flow contributes to contractile ring formation, while rotational cortical flow is additionally induced in uniaxially loaded embryos, i.e. embryos compressed between two plates. Rotational flow depends on astral microtubule signals and is required for the redistribution of the actomyosin cortex in loaded embryos. Rupture of longitudinally aligned cortical fibers during cortex rotation releases tension, initiates orthogonal longitudinal flow and, thereby, contributes to furrowing in loaded embryos. Moreover, actomyosin regulators involved in RhoA regulation, cortical polarity and chirality are all required for rotational flow, and become essential for cytokinesis under mechanical stress. In sum, our findings extend the current framework of mechanical stress response during cell division and show scaling of orthogonal cortical flows to the amount of mechanical stress.