Anillin binds nonmuscle myosin II and regulates the contractile ring

We demonstrate that the contractile ring protein anillin interacts directly with nonmuscle myosin II and that this interaction is regulated by myosin light chain phosphorylation. We show that despite their interaction, anillin and myosin II are independently targeted to the contractile ring. Depletion of anillin in Drosophila or human cultured cells results in cytokinesis failure. Human cells depleted for anillin fail to properly regulate contraction by myosin II late in cytokinesis and fail in abscission. We propose a role for anillin in spatially regulating the contractile activity of myosin II during cytokinesis.     

Anillin is a scaffold protein that links RhoA, actin, and myosin during cytokinesis

Cell division after mitosis is mediated by ingression of an actomyosin-based contractile ring. The active, GTP-bound form of the small GTPase RhoA is a key regulator of contractile-ring formation. RhoA concentrates at the equatorial cell cortex at the site of the nascent cleavage furrow. During cytokinesis, RhoA is activated by its RhoGEF, ECT2. Once activated, RhoA promotes nucleation, elongation, and sliding of actin filaments through the coordinated activation of both formin proteins and myosin II motors (reviewed in [1, 2]). Anillin is a 124 kDa protein that is highly concentrated in the cleavage furrow in numerous animal cells in a pattern that resembles that of RhoA [3-7]. Although anillin contains conserved N-terminal actin and myosin binding domains and a PH domain at the C terminus, its mechanism of action during cytokinesis remains unclear. Here, we show that human anillin contains a conserved C-terminal domain that is essential for its function and localization. This domain shares homology with the RhoA binding protein Rhotekin and directly interacts with RhoA. Further, anillin is required to maintain active myosin in the equatorial plane during cytokinesis, suggesting it functions as a scaffold protein to link RhoA with the ring components actin and myosin. Although furrows can form and initiate ingression in the absence of anillin, furrows cannot form in anillin-depleted cells in which the central spindle is also disrupted, revealing that anillin can also act at an early stage of cytokinesis.     

Drosophila nonmuscle myosin II promotes the asymmetric segregation of cell fate determinants by cortical exclusion rather than active transport

Cell fate diversity can be achieved through the asymmetric segregation of cell fate determinants. In the Drosophila embryo, neuroblasts divide asymmetrically and in a stem cell fashion. The determinants Prospero and Numb localize in a basal crescent and are partitioned from neuroblasts to their daughters (GMCs). Here we show that nonmuscle myosin II regulates asymmetric cell division by an unexpected mechanism, excluding determinants from the apical cortex. Myosin II is activated by Rho kinase and restricted to the apical cortex by the tumor suppressor Lethal (2) giant larvae. During prophase and metaphase, myosin II prevents determinants from localizing apically. At anaphase and telophase, myosin II moves to the cleavage furrow and appears to "push" rather than carry determinants into the GMC. Therefore, the movement of myosin II to the contractile ring not only initiates cytokinesis but also completes the partitioning of cell fate determinants from the neuroblast to its daughter.     

Optogenetic EB1 inactivation shortens metaphase spindles by disrupting cortical force-producing interactions with astral microtubules

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Loss of Necdin impairs myosin activation and delays cell polarization

NDN is one of several genes inactivated in Prader–Willi syndrome (PWS), a developmental disorder characterized by obesity, hypotonia, and developmental delay. We demonstrate that loss of Necdin in murine and human fibroblasts impairs polarity initiation through a Cdc42‐myosin‐dependent pathway, thereby reducing cell migration. We identified defective polarization in both primary neuron cultures and in the developing limb in Ndn‐null mice. Ndn‐null neurons fail to activate myosin light chain and display defective polarization with respect to a brain‐derived neurotrophic factor gradient. Pax3+ muscle progenitors in Ndn‐null developing forelimbs display defective polarization, do not adequately migrate into the dorsal limb bud, and extensor muscles are consequently smaller. These results provide strong evidence that Necdin is a key protein regulating polarization of the cytoskeleton during development. Furthermore, this is the first demonstration of a cellular defect in PWS and suggests a novel molecular mechanism to explain neurological and muscular pathophysiologies in PWS. genesis 48:540–553, 2010. © 2010 Wiley‐Liss, Inc.     

Effect of local polarization of after-discharges of cortical neurones

After strong tetanization epileptiform after-discharges occur in the neurones of the sensorimotor cortex of unanaesthetized rabbits, they take the form of bursts of impulses occurring at intervals of 150–600 msec. The bursts are caused by paroxysmal depolarization shifts (PDS) of the membrane potential (MP). When the MP is reduced to 10–20 mV, on account of the considerable damage to the neurone the PDS give way to hyperpolarization oscillations. Unlike the prolonged action potentials (AP), which are quite frequently recorded in damaged cells, the intracellular PDS and the extracellular bursts of after-discharges show no change in frequency when a current is passed through the recording microelectrode. It was found impossible to suppress the generation of PDS by means of a hyperpolarizing current (1–3·10^–9 A), or to evoke PDS by a depolarizing current. Therefore we were unable to confirm the hypothesis that PDS occur as a result of reorganization of the generation of the electrical impulse. Support is given to the hypothesis that the PDS are altered and enormously potentiated excitatory postsynaptic potentials (EPSP)Brain Institute, Academy of Medical Sciences of the USSR, Moscow, Translated from Neirofiziologiya, Vol. 2, No. 5, pp. 460–468, September–October, 1970.     

Anillin is a substrate of anaphase-promoting complex/cyclosome (APC/C) that controls spatial contractility of myosin during late cytokinesis

Anillin, an actin-binding protein localized at the cleavage furrow, is required for cytokinesis. Through an in vitro expression screen, we identified anillin as a substrate of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that controls mitotic progression. We found that the levels of anillin fluctuate in the cell cycle, peaking in mitosis and dropping drastically during mitotic exit. Ubiquitination of anillin required a destruction-box and was mediated by Cdh1, an activator of APC/C. Overexpression of Cdh1 reduced the levels of anillin, whereas inactivation of APC/C(Cdh1) increased the half-life of anillin. Functionally, anillin was required for the completion of cytokinesis. In anillin knockdown cells, the cleavage furrow ingressed but failed to complete the ingression. At late cytokinesis, the cytosol and DNA in knockdown cells underwent rapid myosin-based oscillatory movement across the furrow. During this movement, RhoA and active myosin were absent from the cleavage furrow, and myosin was redistributed to cortical patches, which powers the random oscillatory movement. We concluded that anillin functions to maintain the localization of active myosin, thereby ensuring the spatial control of concerted contraction during cytokinesis.     

Retinoic acid differentiation of HL-60 cells promotes cytoskeletal polarization

Retinoic acid (RA) treatment of HL-60 cells in vitro induces granulocytic differentiation, involving reorganization of the nucleus and cytoplasm, development of chemoattractant-directed migration, and eventual apoptosis. The present studies with HL-60/S4 cells document that major elements of the cytoskeleton are changed: actin increases by 50%; vimentin decreases by more than 95%. The cellular content of alpha-tubulin does not significantly change; but the centrosomal-microtubule (MT) array moves away from the lobulating nucleus. Cytoskeletal-modifying chemicals modulate this polarized reorganization: Taxol and cytochalasin D enhance centrosome movement; nocodazole reverses it. Cytoskeletal-modifying chemicals do not appear to affect nuclear lobulation or the integrity of envelope-limited chromatin sheets (ELCS). Employing bcl-2-overexpressing HL-60 cells permitted demonstration of nuclear lobulation, ELCS formation, and centrosome-MT movement concomitantly during RA-induced differentiation, implying independence between the cellular reorganization and apoptotic programs. RA appears to promote an inherent potential in HL-60 cells for cytoskeletal polarization, likely to be important for chemoattractant-directed cell migration, an established characteristic of mature granulocytes.     

LKB1/STRAD promotes axon initiation during neuronal polarization

Axon/dendrite differentiation is a critical step in neuronal development. In cultured hippocampal neurons, the accumulation of LKB1 and STRAD, two interacting proteins critical for establishing epithelial polarity, in an undifferentiated neurite correlates with its subsequent axon differentiation. Downregulation of either LKB1 or STRAD by siRNAs prevented axon differentiation, and overexpression of these proteins led to multiple axon formation. Furthermore, interaction of STRAD with LKB1 promoted LKB1 phosphorylation at a PKA site S431 and elevated the LKB1 level, and overexpressing LKB1 with a serine-to-alanine mutation at S431 (LKB1(S431A)) prevented axon differentiation. In developing cortical neurons in vivo, downregulation of LKB1 or overexpression of LKB1(S431A) also abolished axon formation. Finally, local exposure of the undifferentiated neurite to brain-derived neurotrophic factor or dibutyryl-cAMP promoted axon differentiation in a manner that depended on PKA-dependent LKB1 phosphorylation. Thus local LKB1/STRAD accumulation and PKA-dependent LKB1 phosphorylation represents an early signal for axon initiation.     

Gamma-interferon promotes differentiation of cultured cortical and hippocampal neurons

Clinical and experimental evidence suggests that the development of the brain may be modulated by soluble growth factors traditionally associated with cells of the immune system. As part of an investigation into agents modulating early neural differentiation, we examined the effects of the lymphokine gamma-interferon (IFN-gamma) on the development of cultured cortical and hippocampal neurons from embryonic rats and mice. We report here that recombinant IFN-gamma, at concentrations of 0.2-10 U/ml (50-2500 pg/ml, 3-150 pM), affects the differentiation of embryonic central neurons. IFN-gamma increased the number of cells expressing neurofilament (NF) protein, the growth of primary and secondary neurites on NF-expressing somas, and the extent of cell aggregation observed in culture. IFN-gamma-induced increases in the numbers of NF-positive cells were seen in the virtual absence of differentiated astrocytes, and in mixed neuron-glia cultures. Our results thus indicate that at physiologically relevant concentrations IFN-gamma acts, either directly on neurons and their precursor cells and/or indirectly via nonneuronal cell stimulation, to promote the differentiation of immature neurons.     

Leukocyte-endothelium interaction promotes SDF-1-dependent polarization of CXCR4

Chemokine-driven migration is accompanied by polarization of the cell body and of the intracellular signaling machinery. The extent to which chemokine receptors polarize during chemotaxis is currently unclear. To analyze the distribution of the chemokine receptor CXCR4 during SDF-1 (CXCL12)-induced chemotaxis, we retrovirally expressed a CXCR4-GFP fusion protein in the CXCR4-deficient human hematopoietic progenitor cell line KG1a. This KG1a CXCR4-GFP cell line showed full restoration of SDF-1 responsiveness in assays detecting activation of ERK1/2 phosphorylation, actin polymerization, adhesion to endothelium under conditions of physiological flow, and (transendothelial) chemotaxis. When adhered to cytokine-activated endothelium in the absence of SDF-1, CXCR4 did not localize to the leading edge of the cell but was uniformly distributed over the plasma membrane. In contrast, when SDF-1 was immobilized on cytokine-activated endothelium, the CXCR4-GFP receptors that were present on the cell surface markedly redistributed to the leading edge of migrating cells. In addition, CXCR4-GFP co-localized with lipid rafts in the leading edge of SDF-1-stimulated cells, at the sites of contact with the endothelial surface. Inhibition of lipid raft formation prevents SDF-1-dependent migration, internalization of CXCR4, and polarization to the leading edge of CXCR4, indicating that CXCR4 surface expression and signaling requires lipid rafts. These data show that SDF-1, immobilized on activated human endothelium, induces polarization of CXCR4 to the leading edge of migrating cells, revealing co-operativity between chemokine and substrate in the control of cell migration.     

The astral relaxation theory of cytokinesis revisited

Cytokinesis in animal cells is accomplished by the active constriction of the equatorial regions of a cell by an actomyosin-containing contractile ring. The mitotic apparatus specifies the position and orientation of the furrow such that the mitotic spindle is always bisected. Global cortical contractions occur in the cortex of a cell prior to cytokinesis that are independent of the presence of the mitotic apparatus. It was proposed some years ago that the asters of the mitotic apparatus could act to relax the preformed cortical tension in their vicinity. This would produce a differential in tension between the equatorial regions and the adjacent regions of the cortex so that the equatorial regions would contract, forming a cleavage furrow. It can be shown that, as it stands, this theory cannot explain cleavage. However, if cortical contractile elements are assumed to be laterally mobile in the plane of the cortex, then the astral relaxation theory can account for many of the aspects of cleavage, including the formation of the contractile ring. Similar schemes may account for the behaviour of the lamellapodia of motile cells.     

Convergence and extension at gastrulation require a myosin IIB-dependent cortical actin network

Force-producing convergence (narrowing) and extension (lengthening) of tissues by active intercalation of cells along the axis of convergence play a major role in axial morphogenesis during embryo development in both vertebrates and invertebrates, and failure of these processes in human embryos leads to defects including spina bifida and anencephaly. Here we use Xenopus laevis, a system in which the polarized cell motility that drives this active cell intercalation has been related to the development of forces that close the blastopore and elongate the body axis, to examine the role of myosin IIB in convergence and extension. We find that myosin IIB is localized in the cortex of intercalating cells, and show by morpholino knockdown that this myosin isoform is essential for the maintenance of a stereotypical, cortical actin cytoskeleton as visualized with time-lapse fluorescent confocal microscopy. We show that this actin network consists of foci or nodes connected by cables and is polarized relative to the embryonic axis, preferentially cyclically shortening and lengthening parallel to the axis of cell polarization, elongation and intercalation, and also parallel to the axis of convergence forces during gastrulation. Depletion of MHC-B results in disruption of this polarized cytoskeleton, loss of the polarized protrusive activity characteristic of intercalating cells, eventual loss of cell-cell and cell-matrix adhesion, and dose-dependent failure of blastopore closure, arguably because of failure to develop convergence forces parallel to the myosin IIB-dependent dynamics of the actin cytoskeleton. These findings bridge the gap between a molecular-scale motor protein and tissue-scale embryonic morphogenesis.     

p116Rip promotes myosin phosphatase activity in airway smooth muscle cells

Myosin phosphatase-Rho interacting protein (p116^Rip) was originally found as a RhoA-binding protein. Subsequent studies by us and others revealed that p116^Rip facilitates myosin light chain phosphatase (MLCP) activity through direct and indirect manners. However, it is unclear how p116^Rip regulates myosin phosphatase activity in cells. To elucidate the role of p116^Rip in cellular contractile processes, we suppressed the expression of p116^Rip by RNA interference in human airway smooth muscle cells (HASMCs). We found that knockdown of p116^Rip in HASMCs led to increased di-phosphorylated MLC (pMLC), that is phosphorylation at both Ser19 and Thr18. This was because of a change in the interaction between MLCP and myosin, but not an alteration of RhoA/ROCK signaling. Attenuation of Zipper-interacting protein kinase (ZIPK) abolished the increase in di-pMLC, suggesting that ZIPK is involved in this process. Moreover, suppression of p116^Rip expression in HASMCs substantially increased the histamine-induced collagen gel contraction. We also found that expression of the p116^Rip was decreased in the airway smooth muscle tissue from asthmatic patients compared with that from non-asthmatic patients, suggesting a potential role of p116^Rip expression in asthma pathogenesis.     

A lipid-signaled myosin phosphatase surge disperses cortical contractile force early in cell spreading

When cells cease migrating through the vasculature, adhere to extracellular matrix, and begin to spread, they exhibit rapid changes in contraction and relaxation at peripheral regions newly contacting the underlying substrata. We describe here a requirement in this process for myosin II disassembly at the cell cortex via the action of myosin phosphatase (MP), which in turn is regulated by a plasma membrane signaling lipid. Cells in suspension exhibit high levels of activity of the signaling enzyme phospholipase D2 (PLD2), elevating production of the lipid second messenger phosphatidic acid (PA) at the plasma membrane, which in turn recruits MP and stores it there in a presumed inactive state. On cell attachment, down-regulation of PLD2 activity decreases PA production, leading to MP release, myosin dephosphorylation, and actomyosin disassembly. This novel model for recruitment and restraint of MP provides a means to effect a rapid cytoskeletal reorganization at the cell cortex upon demand.