Wound closure in the lamellipodia of single cells: mediation by actin polymerization in the absence of an actomyosin purse string

The actomyosin purse string is an evolutionarily conserved contractile structure that is involved in cytokinesis, morphogenesis, and wound healing. Recent studies suggested that an actomyosin purse string is crucial for the closure of wounds in single cells. In the present study, morphological and pharmacological methods were used to investigate the role of this structure in the closure of wounds in the peripheral cytoplasm of sea urchin coelomocytes. These discoidal shaped cells underwent a dramatic form of actin-based centripetal/retrograde flow and occasionally opened and closed spontaneous wounds in their lamellipodia. Fluorescent phalloidin staining indicated that a well defined fringe of actin filaments assembles from the margin of these holes, and drug studies with cytochalasin D and latrunculin A indicated that actin polymerization is required for wound closure. Additional evidence that actin polymerization is involved in wound closure was provided by the localization of components of the Arp2/3 complex to the wound margin. Significantly, myosin II immunolocalization demonstrated that it is not associated with wound margins despite being present in the perinuclear region. Pharmacological evidence for the lack of myosin II involvement in wound closure comes from experiments in which a microneedle was used to produce wounds in cells in which actomyosin contraction was inhibited by treatment with kinase inhibitors. Wounds produced in kinase inhibitor-treated cells closed in a manner similar to that seen with control cells. Taken together, our results suggest that an actomyosin purse string mechanism is not responsible for the closure of lamellar wounds in coelomocytes. We hypothesize that the wounds heal by means of a combination of the force produced by actin polymerization alone and centripetal flow. Interestingly, these cells did assemble an actomyosin structure around the margin of phagosome-like membrane invaginations, indicating that myosin is not simply excluded from the periphery by some general mechanism. The results indicate that the actomyosin purse string is not the only mechanism that can mediate wound closure in single cells.     

Essential roles of myosin phosphatase in the maintenance of epithelial cell integrity of Drosophila imaginal disc cells

Reorganization of the actin cytoskeleton and contraction of actomyosin play pivotal roles in controlling cell shape changes and motility in epithelial morphogenesis. Dephosphorylation of the myosin regulatory light chain (MRLC) by myosin phosphatase is one of the key events involved. Allelic combinations producing intermediate strength mutants of the Drosophila myosin-binding subunit (DMBS) of myosin phosphatase showed imaginal discs with multilayered disrupted morphologies, and extremely mislocated cells, suggesting that DMBS is required to maintain proper epithelial organization. Clonal analyses revealed that DMBS null mutant cells appear to retract basally and localization of apical junction markers such as DE-cadherin is indetectable in most cells, whereas phosphorylated MRLC and F-actin become heavily concentrated apically, indicating misconfiguration of the apical cytoskeleton. In agreement with these findings, DMBS was found to concentrate at the apical domain suggesting its function is localized. Phenotypes similar to DMBS mutants including increased migration of cells were obtained by overexpressing the constitutive active form of MRLC or Rho-associated kinase signifying that the phenotypes are indeed caused through activation of Myosin II. The requirement of DMBS for the integrity of static epithelial cells in imaginal discs suggests that the regulation of Myosin II by DMBS has a role more general than its previously demonstrated functions in morphogenetic events.     

Actomyosin contraction during cellularization is regulated in part by Src64 control of Actin 5C protein levels

Src64 is required for actomyosin contraction during cellularization of the Drosophila embryonic blastoderm. The mechanism of actomyosin ring constriction is poorly understood even though a number of cytoskeletal regulators have been implicated in the assembly, organization, and contraction of these microfilament rings. How these cytoskeletal processes are regulated during development is even less well understood. To investigate the role of Src64 as an upstream regulator of actomyosin contraction, we conducted a proteomics screen to identify proteins whose expression levels are controlled by src64. Global levels of actin are reduced in src64 mutant embryos. Furthermore, we show that reduction of the actin isoform Actin 5C causes defects in actomyosin contraction during cellularization similar to those caused by src64 mutation, indicating that a relatively high level of Actin 5C is required for normal actomyosin contraction and furrow canal structure. However, reduction of Actin 5C levels only slows down actomyosin ring constriction rather than preventing it, suggesting that src64 acts not only to modulate actin levels, but also to regulate the actomyosin cytoskeleton by other means.     

Schizosaccharomyces pombe Pxl1 is a paxillin homologue that modulates Rho1 activity and participates in cytokinesis

Schizosaccharomyces pombe Rho GTPases regulate actin cytoskeleton organization and cell integrity. We studied the fission yeast gene SPBC4F6.12 based on its ability to suppress the thermosensitivity of cdc42-1625 mutant strain. This gene, named pxl1(+), encodes a protein with three LIM domains that is similar to paxillin. Pxl1 does not interact with Cdc42 but it interacts with Rho1, and it negatively regulates this GTPase. Fission yeast Pxl1 forms a contractile ring in the cell division region and deletion of pxl1(+) causes a delay in cell-cell separation, suggesting that it has a function in cytokinesis. Pxl1 N-terminal region is required and sufficient for its localization to the medial ring, whereas the LIM domains are necessary for its function. Pxl1 localization requires actin polymerization and the actomyosin ring, but it is independent of the septation initiation network (SIN) function. Moreover, Pxl1 colocalizes and interacts with Myo2, and Cdc15, suggesting that it is part of the actomyosin ring. Here, we show that in cells lacking Pxl1, the myosin ring is not correctly assembled and that actomyosin ring contraction is delayed. Together, these data suggest that Pxl1 modulates Rho1 GTPase signaling and plays a role in the formation and contraction of the actomyosin ring during cytokinesis.     

Two components of actin-based retrograde flow in sea urchin coelomocytes

Sea urchin coelomocytes represent an excellent experimental model system for studying retrograde flow. Their extreme flatness allows for excellent microscopic visualization. Their discoid shape provides a radially symmetric geometry, which simplifies analysis of the flow pattern. Finally, the nonmotile nature of the cells allows for the retrograde flow to be analyzed in the absence of cell translocation. In this study we have begun an analysis of the retrograde flow mechanism by characterizing its kinetic and structural properties. The supramolecular organization of actin and myosin II was investigated using light and electron microscopic methods. Light microscopic immunolocalization was performed with anti-actin and anti-sea urchin egg myosin II antibodies, whereas transmission electron microscopy was performed on platinum replicas of critical point-dried and rotary-shadowed cytoskeletons. Coelomocytes contain a dense cortical actin network, which feeds into an extensive array of radial bundles in the interior. These actin bundles terminate in a perinuclear region, which contains a ring of myosin II bipolar minifilaments. Retrograde flow was arrested either by interfering with actin polymerization or by inhibiting myosin II function, but the pathway by which the flow was blocked was different for the two kinds of inhibitory treatments. Inhibition of actin polymerization with cytochalasin D caused the actin cytoskeleton to separate from the cell margin and undergo a finite retrograde retraction. In contrast, inhibition of myosin II function either with the wide-spectrum protein kinase inhibitor staurosporine or the myosin light chain kinase-specific inhibitor KT5926 stopped flow in the cell center, whereas normal retrograde flow continued at the cell periphery. These differential results suggest that the mechanism of retrograde flow has two, spatially segregated components. We propose a "push-pull" mechanism in which actin polymerization drives flow at the cell periphery, whereas myosin II provides the tension on the actin cytoskeleton necessary for flow in the cell interior.     

Zonula occludens-1 and -2 regulate apical cell structure and the zonula adherens cytoskeleton in polarized epithelia

The structure and function of both adherens (AJ) and tight (TJ) junctions are dependent on the cortical actin cytoskeleton. The zonula occludens (ZO)-1 and -2 proteins have context-dependent interactions with both junction types and bind directly to F-actin and other cytoskeletal proteins, suggesting ZO-1 and -2 might regulate cytoskeletal activity at cell junctions. To address this hypothesis, we generated stable Madin-Darby canine kidney cell lines depleted of both ZO-1 and -2. Both paracellular permeability and the localization of TJ proteins are disrupted in ZO-1/-2-depleted cells. In addition, immunocytochemistry and electron microscopy revealed a significant expansion of the perijunctional actomyosin ring associated with the AJ. These structural changes are accompanied by a recruitment of 1-phosphomyosin light chain and Rho kinase 1, contraction of the actomyosin ring, and expansion of the apical domain. Despite these changes in the apical cytoskeleton, there are no detectable changes in cell polarity, localization of AJ proteins, or the organization of the basal and lateral actin cytoskeleton. We conclude that ZO proteins are required not only for TJ assembly but also for regulating the organization and functional activity of the apical cytoskeleton, particularly the perijunctional actomyosin ring, and we speculate that these activities are relevant both to cellular organization and epithelial morphogenesis.     

The role of Rho family proteins in controlling the migration of crawling cells

Migration of crawling cells (amoebae and some kinds of the tissue cells) is a process related to the dynamic reorganization of actomyosin cytoskeleton. That reorganization engages actin polymerization and de-polymerization, branching of actin network and interaction of myosin II with actin filaments. All those cytoskeleton changes lead to the cell progression, contraction and shifting of the uropod and the cell adhesion. Numerous external stimuli, which activate various surface receptors and signal transduction pathways, can promote migration. Rho family proteins play an important role in the regulation of actin cytoskeleton organization. The most known members of this family are Rho, Rac and Cdc42 proteins, present in all mammalian tissue cells. These proteins control three different stages of cell migration: progression of the frontal edge, adhesion which stabilizes the frontal area, and de-adhesion and shifting of the uropod. Cdc42 and Rac control cell polarization, lamellipodium formation and expansion, organization of focal complexes. Rho protein regulates contractile activity of actomyosin cytoskeleton outside the frontal area, and thus contraction and de-adhesion of the uropod.     

Distinct roles for ROCK1 and ROCK2 in the regulation of cell detachment

This study, using mouse embryonic fibroblast (MEF) cells derived from ROCK1^−/− and ROCK2^−/− mice, is designed to dissect roles for ROCK1 and ROCK2 in regulating actin cytoskeleton reorganization induced by doxorubicin, a chemotherapeutic drug. ROCK1^−/− MEFs exhibited improved actin cytoskeleton stability characterized by attenuated periphery actomyosin ring formation and preserved central stress fibers, associated with decreased myosin light chain 2 (MLC2) phosphorylation but preserved cofilin phosphorylation. These effects resulted in a significant reduction in cell shrinkage, detachment, and predetachment apoptosis. In contrast, ROCK2^−/− MEFs showed increased periphery membrane folding and impaired cell adhesion, associated with reduced phosphorylation of both MLC2 and cofilin. Treatment with inhibitor of myosin (blebbistatin), inhibitor of actin polymerization (cytochalasin D), and ROCK pan-inhibitor (Y27632) confirmed the contributions of actomyosin contraction and stress fiber instability to stress-induced actin cytoskeleton reorganization. These results support a novel concept that ROCK1 is involved in destabilizing actin cytoskeleton through regulating MLC2 phosphorylation and peripheral actomyosin contraction, whereas ROCK2 is required for stabilizing actin cytoskeleton through regulating cofilin phosphorylation. Consequently, ROCK1 and ROCK2 can be functional different in regulating stress-induced stress fiber disassembly and cell detachment.     

Phosphorylation of myosin II regulatory light chain controls its accumulation, not that of actin, at the contractile ring in HeLa cells

During cytokinesis in eukaryotic cells, an actomyosin-based contractile ring (CR) is assembled along the equator of the cell. Myosin II ATPase activity is stimulated by the phosphorylation of the myosin II regulatory light chain (MRLC) in vitro, and phosphorylated MRLC localizes at the CR in various types of cells. Previous studies have determined that phosphorylated MRLC plays an important role in CR furrowing. However, the role of phosphorylated MRLC in CR assembly remains unknown. Here, we have used confocal microscopy to observe dividing HeLa cells expressing fluorescent protein-tagged MRLC mutants and actin during CR assembly near the cortex. Di-phosphomimic MRLC accumulated at the cell equator earlier than non-phosphorylatable MRLC and actin. Interestingly, perturbation of myosin II activity by non-phosphorylatable MRLC expression or treatment with blebbistatin, a myosin II inhibitor, did not alter the time of actin accumulation at the cell equator. Furthermore, inhibition of actin polymerization by treatment with latrunculin A had no effect on MRLC accumulation at the cell equator. Taken together, these data suggest that phosphorylated MRLC temporally controls its own accumulation, but not that of actin, in cultured mammalian cells.     

Thrombin-induced contraction in alveolar epithelial cells probed by traction microscopy.

Contractile tension of alveolar epithelial cells plays a major role in the force balance that regulates the structural integrity of the alveolar barrier. The aim of this work was to study thrombin-induced contractile forces of alveolar epithelial cells. A549 alveolar epithelial cells were challenged with thrombin, and time course of contractile forces was measured by traction microscopy. The cells exhibited basal contraction with total force magnitude 55.0 +/- 12.0 nN (mean +/- SE, n = 12). Traction forces were exerted predominantly at the cell periphery and pointed to the cell center. Thrombin (1 U/ml) induced a fast and sustained 2.5-fold increase in traction forces, which maintained peripheral and centripetal distribution. Actin fluorescent staining revealed F-actin polymerization and enhancement of peripheral actin rim. Disruption of actin cytoskeleton with cytochalasin D (5 microM, 30 min) and inhibition of myosin light chain kinase with ML-7 (10 microM, 30 min) and Rho kinase with Y-27632 (10 microM, 30 min) markedly depressed basal contractile tone and abolished thrombin-induced cell contraction. Therefore, the contractile response of alveolar epithelial cells to the inflammatory agonist thrombin was mediated by actin cytoskeleton remodeling and actomyosin activation through myosin light chain kinase and Rho kinase signaling pathways. Thrombin-induced contractile tension might further impair alveolar epithelial barrier integrity in the injured lung.     

Rho signaling pathway and apical constriction in the early lens placode

Epithelial invagination in many model systems is driven by apical cell constriction, mediated by actin and myosin II contraction regulated by GTPase activity. Here we investigate apical constriction during chick lens placode invagination. Inhibition of actin polymerization and myosin II activity by cytochalasin D or blebbistatin prevents lens invagination. To further verify if lens placode invaginate through apical constriction, we analyzed the role of Rho-ROCK pathway. Rho GTPases expression at the apical portion of the lens placode occurs with the same dynamics as that of the cytoskeleton. Overexpression of the pan-Rho inhibitor C3 exotoxin abolished invagination and had a strong effect on apical myosin II enrichment and a mild effect on apical actin localization. In contrast, pharmacological inhibition of ROCK activity interfered significantly with apical enrichment of both actin and myosin. These results suggest that apical constriction in lens invagination involves ROCK but apical concentration of actin and myosin are regulated through different pathways upstream of ROCK. genesis 49:368-379, 2011.     

Coordinated waves of actomyosin flow and apical cell constriction immediately after wounding

Epithelial wound healing relies on tissue movements and cell shape changes. Our work shows that, immediately after wounding, there was a dramatic cytoskeleton remodeling consisting of a pulse of actomyosin filaments that assembled in cells around the wound edge and flowed from cell to cell toward the margin of the wound. We show that this actomyosin flow was regulated by Diaphanous and ROCK and that it elicited a wave of apical cell constriction that culminated in the formation of the leading edge actomyosin cable, a structure that is essential for wound closure. Calcium signaling played an important role in this process, as its intracellular concentration increased dramatically immediately after wounding, and down-regulation of transient receptor potential channel M, a stress-activated calcium channel, also impaired the actomyosin flow. Lowering the activity of Gelsolin, a known calcium-activated actin filament–severing protein, also impaired the wound response, indicating that cleaving the existing actin filament network is an important part of the cytoskeleton remodeling process.     

Micron-scale supramolecular myosin arrays help mediate cytoskeletal assembly at mature adherens junctions

Epithelial cells assemble specialized actomyosin structures at E-Cadherin–based cell–cell junctions, and the force exerted drives cell shape change during morphogenesis. The mechanisms that build this supramolecular actomyosin structure remain unclear. We used ZO-knockdown MDCK cells, which assemble a robust, polarized, and highly organized actomyosin cytoskeleton at the zonula adherens, combining genetic and pharmacologic approaches with superresolution microscopy to define molecular machines required. To our surprise, inhibiting individual actin assembly pathways (Arp2/3, formins, or Ena/VASP) did not prevent or delay assembly of this polarized actomyosin structure. Instead, as junctions matured, micron-scale supramolecular myosin arrays assembled, with aligned stacks of myosin filaments adjacent to the apical membrane, overlying disorganized actin filaments. This suggested that myosin arrays might bundle actin at mature junctions. Consistent with this idea, inhibiting ROCK or myosin ATPase disrupted myosin localization/organization and prevented actin bundling and polarization. We obtained similar results in Caco-2 cells. These results suggest a novel role for myosin self-assembly, helping drive actin organization to facilitate cell shape change.     

The role of actin, actomyosin and microtubules in defining cell shape during the differentiation of Naegleria amebae into flagellates

Differentiation of Naegleria amebae into flagellates was used to examine the interaction between actin, actomyosin and microtubules in defining cell shape. Amebae, which lack microtubules except during mitosis, differentiate into flagellates with a fixed shape and a complex microtubule cytoskeleton in 120 min. Based on earlier models of ameboid motility it has been suggested that actomyosin is quiescent in flagellates. This hypothesis was tested by following changes in the cytoskeleton using three-dimensional reconstructions prepared by confocal microscopy of individual cells stained with antibodies against actin and tubulin as well as with phalloidin and DNase I. F-actin as defined by phalloidin staining was concentrated in expanding pseudopods. Most phalloidin staining was lost as cells rounded up before the onset of flagellum formation. Actin staining with a Naegleria-specific antibody that recognizes both F- and G-actin was confined to the cell cortex of both amebae and flagellates. DNase I demonstrated G-actin throughout all stages. Most of the actin in the cortex was not bound by phalloidin yet was resistant to detergent extraction suggesting that it was polymerized. The microtubule cytoskeleton of flagellates was intimately associated with this actin cortex. Treatment of flagellates with cytochalasin D produced a rapid loss of flagellate shape and the appearance of phalloidin staining while latrunculin A stabilized the flagellate shape. These results suggest that tension produced by an actomyosin network is required to maintain the flagellate shape. The rapid loss of the flagellate shape induced by drugs, which specifically block myosin light chain kinase, supports this hypothesis.     

Distinct cytoskeleton populations and extensive crosstalk control Ciona notochord tubulogenesis

Cell elongation is a fundamental process that allows cells and tissues to adopt new shapes and functions. During notochord tubulogenesis in the ascidian Ciona intestinalis, a dramatic elongation of individual cells takes place that lengthens the notochord and, consequently, the entire embryo. We find a novel dynamic actin- and non-muscle myosin II-containing constriction midway along the anteroposterior aspect of each notochord cell during this process. Both actin polymerization and myosin II activity are required for the constriction and cell elongation. Discontinuous localization of myosin II in the constriction indicates that the actomyosin network produces local contractions along the circumference. This reveals basal constriction by the actomyosin network as a novel mechanism for cell elongation. Following elongation, the notochord cells undergo a mesenchymal-epithelial transition and form two apical domains at opposite ends. Extracellular lumens then form at the apical surfaces. We show that cortical actin and Ciona ezrin/radixin/moesin (ERM) are essential for lumen formation and that a polarized network of microtubules, which contributes to lumen development, forms in an actin-dependent manner at the apical cortex. Later in notochord tubulogenesis, when notochord cells initiate a bi-directional crawling movement on the notochordal sheath, the microtubule network rotates 90° and becomes organized as parallel bundles extending towards the leading edges of tractive lamellipodia. This process is required for the correct organization of actin-based protrusions and subsequent lumen coalescence. In summary, we establish the contribution of the actomyosin and microtubule networks to notochord tubulogenesis and reveal extensive crosstalk and regulation between these two cytoskeleton components.     

Arp2/3 complex inhibition radically alters lamellipodial actin architecture,suspended cell shape,and the cell spreading process

Recent studies have investigated the dendritic actin cytoskeleton of the cell edge''s lamellipodial (LP) region by experimentally decreasing the activity of the actin filament nucleator and branch former, the Arp2/3 complex. Here we extend these studies via pharmacological inhibition of the Arp2/3 complex in sea urchin coelomocytes, cells that possess an unusually broad LP region and display correspondingly exaggerated centripetal flow. Using light and electron microscopy, we demonstrate that Arp2/3 complex inhibition via the drug CK666 dramatically altered LP actin architecture, slowed centripetal flow, drove a lamellipodial-to-filopodial shape change in suspended cells, and induced a novel actin structural organization during cell spreading. A general feature of the CK666 phenotype in coelomocytes was transverse actin arcs, and arc generation was arrested by a formin inhibitor. We also demonstrate that CK666 treatment produces actin arcs in other cells with broad LP regions, namely fish keratocytes and Drosophila S2 cells. We hypothesize that the actin arcs made visible by Arp2/3 complex inhibition in coelomocytes may represent an exaggerated manifestation of the elongate mother filaments that could possibly serve as the scaffold for the production of the dendritic actin network.     

Microtubules and mitotic cycle phase modulate spatiotemporal distributions of F-actin and myosin II in Drosophila syncytial blastoderm embryos

We studied cyclic reorganizations of filamentous actin, myosin II and microtubules in syncytial Drosophila blastoderms using drug treatments, time-lapse movies and laser scanning confocal microscopy of fixed stained embryos (including multiprobe three-dimensional reconstructions). Our observations imply interactions between microtubules and the actomyosin cytoskeleton. They provide evidence that filamentous actin and cytoplasmic myosin II are transported along microtubules towards microtubule plus ends, with actin and myosin exhibiting different affinities for the cell's cortex. Our studies further reveal that cell cycle phase modulates the amounts of both polymerized actin and myosin II associated with the cortex. We analogize pseudocleavage furrow formation in the Drosophila blastoderm with how the mitotic apparatus positions the cleavage furrow for standard cytokinesis, and relate our findings to polar relaxation/global contraction mechanisms for furrow formation.     

Actin depolymerization drives actomyosin ring contraction during budding yeast cytokinesis

Actin filaments and myosin II are evolutionarily conserved force-generating components of the contractile ring during cytokinesis. Here we show that in budding yeast, actin filament depolymerization plays a major role in actomyosin ring constriction. Cofilin mutation or chemically stabilizing actin filaments attenuate actomyosin ring constriction. Deletion of myosin II motor domain or the myosin regulatory light chain reduced the contraction rate and also the rate of actin depolymerization in the ring. We constructed a quantitative microscopic model of actomyosin ring constriction via filament sliding driven by both actin depolymerization and myosin II motor activity. Model simulations based on experimental measurements support the notion that actin depolymerization is the predominant mechanism for ring constriction. The model predicts invariability of total contraction time regardless of the initial ring size, as originally reported for C. elegans embryonic cells. This prediction was validated in yeast cells of different sizes due to different ploidies.     

Antiepileptic teratogen valproic acid (VPA) modulates organisation and dynamics of the actin cytoskeleton

The antiepileptic drug valproic acid (VPA) and teratogenic VPA analogues have been demonstrated to inhibit cell motility and affect cell morphology. We here show that disruption of microtubules or of microfilaments by exposure to nocodazole or cytochalasin D had different effects on morphology of control cells and cells treated with VPA, indicating that VPA affected the cytoskeletal determinants of cell morphology. Furthermore, VPA treatment induced an increase of F-actin, and of FAK, paxillin, vinculin, and phosphotyrosine in focal adhesion complexes. These changes were accompanied by increased adhesion of VPA-treated cells to the extracellular matrix. Treatment with an RGD-containing peptide reducing integrin binding to components of the extracellular matrix partially reverted the motility inhibition induced by VPA, indicating that altered adhesion contributed to, but was not the sole reason for the VPA mediated inhibition of motility. In addition it is shown that the actomyosin cytoskeleton of VPA-treated cells was capable of contraction upon exposure to ATP, indicating that the reduced motility of VPA-treated cells was not caused by an inhibition of actomyosin contraction. On the other hand, VPA caused a redistribution of the actin severing protein gelsolin, and left the cells unable to respond to treatment with a gelsolin-peptide known to reduce the amount of gelsolin bound to phosphatidylinositol bisphosphate (PIP2), leaving a larger amount of the protein in a potential actin binding state. These findings indicate that VPA affects cell morphology and motility through interference with the dynamics of the actin cytoskeleton.     

Pxl1p, a paxillin-related protein, stabilizes the actomyosin ring during cytokinesis in fission yeast

Paxillins are a family of conserved LIM domain-containing proteins that play important roles in the function and integrity of the actin cytoskeleton. Although paxillins have been extensively characterized by cell biological and biochemical approaches, genetic studies are relatively scarce. Here, we identify and characterize a paxillin-related protein Pxl1p in the fission yeast Schizosaccharomyces pombe. Pxl1p is a component of the fission yeast actomyosin ring, a structure that is essential for cytokinesis. Cells deleted for pxl1 display a novel phenotype characterized by a splitting of the actomyosin ring in late anaphase, leading to the formation of two rings of which only one undergoes constriction. In addition, the rate of actomyosin ring constriction is slower in the absence of Pxl1p. pxl1Delta mutants display strong genetic interactions with mutants defective in IQGAP-related protein Rng2p and mutants defective in components of the fission yeast type II myosin machinery. Collectively, these results suggest that Pxl1p might cooperate with type II myosin and Rng2p-IQGAP to regulate actomyosin ring constriction as well as to maintain its integrity during constriction.