Reorganization Of Actin Filaments Research Articles

Cytokinin-induced root growth involves actin filament reorganization

Root architecture is developmentally plastic and affected by many intrinsic factors (e.g. plant hormones) and extrinsic factors (e.g. touch, gravity) in order to maximize nutrient and water acquisition. We have recently shown that asymmetrical exposure of cytokinin (CK) at the root tip causes root growth directional changes that is dependent on ethylene signaling and is potentiated by glucose signaling. Auxin homeostasis as maintained by auxin signaling and transport is also involved in CK-induced root cell elongation and differential growth. The signaling pathways eventually converge at actin filament organization since actin filament organization inhibitor latrunculin B (Lat B) can also induce similar growth. We, show that CK can actually alter actin filament organization as seen in actin binding protein 35S::GFP-ABD2-GFP transgenic lines as is also altered by auxin polar transport inhibitor 1-N-naphthylphthalamic acid (NPA) and Lat B in different manners.

Dynamic actin remodeling during epithelial–mesenchymal transition depends on increased moesin expression

Remodeling of actin filaments is necessary for epithelial-mesenchymal transition (EMT); however, understanding of how this is regulated in real time is limited. We used an actin filament reporter and high-resolution live-cell imaging to analyze the regulated dynamics of actin filaments during transforming growth factor-β-induced EMT of mammary epithelial cells. Progressive changes in cell morphology were accompanied by reorganization of actin filaments from thin cortical bundles in epithelial cells to thick, parallel, contractile bundles that disassembled more slowly but remained dynamic in transdifferentiated cells. We show that efficient actin filament remodeling during EMT depends on increased expression of the ezrin/radixin/moesin (ERM) protein moesin. Cells suppressed for moesin expression by short hairpin RNA had fewer, thinner, and less stable actin bundles, incomplete morphological transition, and decreased invasive capacity. These cells also had less α-smooth muscle actin and phosphorylated myosin light chain in cortical patches, decreased abundance of the adhesion receptor CD44 at membrane protrusions, and attenuated autophosphorylation of focal adhesion kinase. Our findings suggest that increased moesin expression promotes EMT by regulating adhesion and contractile elements for changes in actin filament organization. We propose that the transciptional program driving EMT controls progressive remodeling of actin filament architectures.

High expression of Lifeact in Arabidopsis thaliana reduces dynamic reorganization of actin filaments but does not affect plant development

Lifeact is a novel probe that labels actin filaments in a wide range of organisms. We compared the localization and reorganization of Lifeact:Venus-labeled actin filaments in Arabidopsis root hairs and root epidermal cells of lines that express different levels of Lifeact: Venus with that of actin filaments labeled with GFP:FABD2, a commonly used probe in plants. Unlike GFP:FABD2, Lifeact:Venus labeled the highly dynamic fine F-actin in the subapical region of tip-growing root hairs. Lifeact:Venus expression at varying levels was not observed to affect plant development. However, at expression levels comparable to those of GFP:FABD2 in a well-characterized marker line, Lifeact:Venus reduced reorganization rates of bundles of actin filaments in root epidermal cells. Reorganization rates of cytoplasmic strands, which reflect the reorganization of the actin cytoskeleton, were also reduced in these lines. Moreover, in the same line, Lifeact:Venus-decorated actin filaments were more resistant to depolymerization by latrunculin B than those in an equivalent GFP:FABD2-expressing line. In lines where Lifeact: Venus is expressed at lower levels, these effects are less prominent or even absent. We conclude that Lifeact: Venus reduces remodeling of the actin cytoskeleton in Arabidopsis in a concentration-dependent manner. Since this reduction occurs at expression levels that do not cause defects in plant development, selection of normally growing plants is not sufficient to determine optimal Lifeact expression levels. When correct expression levels of Lifeact have been determined, it is a valuable probe that labels dynamic populations of actin filaments such as fine F-actin, better than FABD2 does.

The Plant-Specific Actin Binding Protein SCAB1 Stabilizes Actin Filaments and Regulates Stomatal Movement inArabidopsis

Microfilament dynamics play a critical role in regulating stomatal movement; however, the molecular mechanism underlying this process is not well understood. We report here the identification and characterization of STOMATAL CLOSURE-RELATED ACTIN BINDING PROTEIN1 (SCAB1), an Arabidopsis thaliana actin binding protein. Plants lacking SCAB1 were hypersensitive to drought stress and exhibited reduced abscisic acid-, H(2)O(2)-, and CaCl(2)-regulated stomatal movement. In vitro and in vivo analyses revealed that SCAB1 binds, stabilizes, and bundles actin filaments. SCAB1 shares sequence similarity only with plant proteins and contains a previously undiscovered actin binding domain. During stomatal closure, actin filaments switched from a radial orientation in open stomata to a longitudinal orientation in closed stomata. This switch took longer in scab1 plants than in wild-type plants and was correlated with the delay in stomatal closure seen in scab1 mutants in response to drought stress. Our results suggest that SCAB1 is required for the precise regulation of actin filament reorganization during stomatal closure.

Responses of endothelial cells to extremely slow flows

The process of blood vessel formation is accompanied by very minimal flow in the beginning, followed by increased flow rates once the vessel develops sufficiently. Many studies have been performed for endothelial cells at shear stress levels of 0.1-60 dyn∕cm(2); however, little is known about the effect of extremely slow flows (shear stress levels of 10(-4)-10(-2) dyn∕cm(2)) that endothelial cells may experience during early blood vessel formation where flow-sensing by indirect mass transport sensing rather than through mechanoreceptor sensing mechanisms would become more important. Here, we show that extremely low flows enhance proliferation, adherens junction protein localization, and nitric oxide secretion of endothelial cells, but do not induce actin filament reorganization. The responses of endothelial cells in different flow microenvironments need more attention because increasing evidence shows that endothelial cell behaviors at the extremely slow flow regimes cannot be linearly extrapolated from observations at faster flow rates. The devices and methods described here provide a useful platform for such studies.

In vitro and in vivo evaluation of the anti-angiogenic actions of 4-hydroxybenzyl alcohol.

4-Hydroxybenzyl alcohol (HBA) is a phenolic plant compound, which has been shown to influence many cellular mechanisms. In the present study, we analysed in vitro and in vivo the anti-angiogenic actions of this pleiotropic agent. Migration and protein expression of HBA- and vehicle-treated endothelial-like eEND2 cells was assessed by cell migration assay and Western blot analyses. HBA action on vascular sprouting was analysed in an aortic ring assay. In vivo anti-angiogenic actions of HBA were studied in the dorsal skinfold chamber model of endometriosis in mice. Western blot analyses demonstrated that HBA inhibited proliferation of eEND2 cells, as indicated by down-regulation of proliferating cell nuclear antigen expression, and reduced expression of vascular endothelial growth factor and matrix metalloproteinase 9. HBA suppressed the migration of eEND2 cells, accompanied by inhibition of actin filament reorganization, revealed by fluorescence staining of the cytoskeleton. In addition, HBA reduced vascular sprouting in the aortic ring assay. Finally, we found, in the dorsal skinfold chamber model in vivo using intravital fluorescence microscopy, that HBA inhibited the vascularization of developing endometriotic lesions, as indicated by a decreased functional capillary density of lesions in HBA-treated mice and a reduced lesion size, compared with control animals. HBA targets several angiogenic mechanisms and therefore represents a promising anti-angiogenic agent for the treatment of angiogenic diseases, such as endometriosis.

Modulation of the Actin Filament Reorganization Alters Cellular Calcium and Surface Adhesion in Wharton's Jelly Cells

Wharton's jelly cells (WJCs), the mesenchymal stem cells derived from umbilical cord, require extensive remodeling of actin filaments by the action of a multitude of actin-binding proteins for their biological functions of self-renewal and differentiation. In this study, molecular approaches by specific siRNAs for caldesmon (CaD), and profilin (Pro) and by gelsolin (GSN) overexpression were used to alter those gene expressions and their subsequent controls of actin polymerization in WJCs and to investigate the effects of modulation of the actin filament reorganization on cellular Ca2+ levels and surface adhesion force in the cell. Ca2+ levels were measured as the fura-2 F340/F380 fluorescence ratio in the cells. Silencing of CaD and Pro by siRNAs raised intracellular Ca2+ and rendered the resistance of decreasing Ca2+ levels by a protein kinase inhibitor, genistein. The similar change in cellular Ca2+ was also found in GSN-overexpressed cells. To determine whether changes in cellular Ca2+ associated with modulation of the actin filament reorganization will affect surface adhesion of the cell, atomic force microscopy (AFM) force-distance measurements were applied to measure the stiffness and the adhesion force in si-control, si-CaD, si-Pro, and GSN overexpressed cells. Results obtained indicate that disturbing actin filament polymerization significantly increase in cell adhesion force but do not alter the stiffness in the cells. Apparently, modulation of the actin filament polymerization might alter cellular Ca2+ and cell adhesion that is important for determining the capacity of cell differentiation in WJCs.

Secretase-Independent and RhoGTPase/PAK/ERK-Dependent Regulation of Cytoskeleton Dynamics in Astrocytes by NSAIDs and Derivatives

Profens like ibuprofen, R-flurbiprofen, or CHF5074 are being considered for the treatment of Alzheimer's disease because epidemiological data indicates that non-steroidal anti-inflammatory drugs are protective against neurodegeneration. Rho-GTPases are small G proteins, including RhoA, Cdc42, and Rac1, which control cytoskeleton dynamics. Because ibuprofen promotes axon growth via RhoA in neurons, we examined whether profens modulate astrocyte plasticity via Rho-GTPases. We report that ibuprofen (100-500 μM), R-flurbiprofen (100-500 μM), and CHF5074 (10-30 μM) caused a concentration-dependent stellation of astrocytes in primary cultures, associated with the reorganization of GFAP and actin filaments. The stellation was independent of COX2, α-, β- or γ-secretase as judged by the lack of effect of inhibitors of these enzymes. RhoA, PAK, and Cdc42, but not Rac1, accounted for the profen-mediated stellation, as concluded from the joint analyses of activities and reversal experiments with adenoviral or pharmacological manipulations. Ibuprofen accelerated migration in a scratch-wound assay, while R-flurbiprofen had no effect and CHF5074 caused deceleration. Cell polarity regulation by Cdc42 and ERK1/2 may underlie the paradoxical effects of profens on migration. We conclude that profens regulate cytoskeleton dynamics in astrocytes via Rho-GTPases, PAK, and ERK1/2. Since migration is a hallmark of astrocyte response during inflammation we propose that, in addition to (or instead of) lowering amyloid-β42 via secretases, ibuprofen and its derivatives may prevent Alzheimer's disease instead of AD by modulating astrocyte reactivity through Rho-GTPase/PAK/ERK-dependent signaling.

Targeted Proteomics of Isolated Glomeruli from the Kidneys of Diabetic Rats: Sorbin and SH3 Domain Containing 2 Is a Novel Protein Associated with Diabetic Nephropathy

To evaluate proteins associated with the development of diabetic nephropathy, a major cause of the end-stage renal disease, we analyzed protein expression in isolated glomeruli from spontaneous type 2 diabetic (OLETF) rats and their age-matched control littermates (LETO) in the early and proteinuric stages of diabetic nephropathy using QSTAR Elite LC-MS/MS. Among the 191 and 218 proteins that were altered significantly in the OLETF rats, twenty-four were actin cytoskeleton-associated proteins implicated in the formation of stress fibers, and the impairment of actin polymerization, intermediate filaments and microtubules. Importantly, sorbin and SH3 domain containing 2 (SORBS2), which is involved in the formation of stress fibers, was significantly upregulated in both stages of diabetic nephropathy (1.49- and 1.97-fold, resp.). Immunohistochemical and quantitative-PCR analyses revealed upregulation of SORBS2 in podocytes of glomeruli of OLETF rats. Our findings suggested that SORBS2 may be associated with the development of diabetic nephropathy possibility by reorganization of actin filaments.

Rho-Rho Kinase Pathway Is Involved in the Protective Effect of Early Ischemic Preconditioning in the Rat Heart

It has been shown that p38 mitogen-activated protein (MAP) kinase is absolutely necessary for the cardioprotection of early ischemic preconditioning in the heart. Reorganization of actin cytoskeleton after translocation of HSP27, which is mediated by p38 MAP kinase, was reported to be necessary for the cardioprotective effect of early ischemic preconditioning. Although Rho and Rho kinase are reported to regulate reorganization of actin filaments, it is unknown whether Rho-Rho kinase pathway is involved in the cardioprotective effect of early ischemic preconditioning. The aim of the present study is to determine the involvement of Rho-Rho kinase pathway in the protective effect of early ischemic preconditioning in the rat hearts. Dominant-negative Rho significantly reduced the hypoxia-reoxygenation-induced activation of p38 MAP kinase, and constitutive active Rho activated p38 MAP kinase in rat myoblast H9c2 cells. Y-27632, a specific Rho kinase inhibitor, concentration-dependently attenuated the post-ischemic recovery of left ventricular developed pressure by early ischemic preconditioning. Thus, Rho-Rho kinase pathway is, at least in part, involved in the mechanism of early ischemic preconditioning.

Invasion of Eukaryotic Cells byBorrelia burgdorferiRequires β1Integrins and Src Kinase Activity

Lyme disease, caused by the bacterium Borrelia burgdorferi, is the most widespread tick-borne infection in the northern hemisphere that results in a multistage disorder with concomitant pathology, including arthritis. During late-stage experimental infection in mice, B. burgdorferi evades the adaptive immune response despite the presence of borrelia-specific bactericidal antibodies. In this study we asked whether B. burgdorferi could invade fibroblasts or endothelial cells as a mechanism to model the avoidance from humorally based clearance. A variation of the gentamicin protection assay, coupled with the detection of borrelial transcripts following gentamicin treatment, indicated that a portion of B. burgdorferi cells were protected in the short term from antibiotic killing due to their ability to invade cultured mammalian cells. Long-term coculture of B. burgdorferi with primary human fibroblasts provided additional support for intracellular protection. Furthermore, decreased invasion of B. burgdorferi in murine fibroblasts that do not synthesize the β(1) integrin subunit was observed, indicating that β(1)-containing integrins are required for optimal borrelial invasion. However, β(1)-dependent invasion did not require either the α(5)β(1) integrin or the borrelial fibronectin-binding protein BBK32. The internalization of B. burgdorferi was inhibited by cytochalasin D and PP2, suggesting that B. burgdorferi invasion required the reorganization of actin filaments and Src family kinases (SFK), respectively. Taken together, these results suggest that B. burgdorferi can invade and retain viability in nonphagocytic cells in a process that may, in part, help to explain the phenotype observed in untreated experimental infection.

The Suppression of CRMP2 Expression by Bone Morphogenetic Protein (BMP)-SMAD Gradient Signaling Controls Multiple Stages of Neuronal Development

The formation of the functional mammalian cerebral cortex requires a concerted control of neurogenesis, neuronal migration, and neuronal morphogenesis. However, molecular mechanisms that control these processes are not well understood. We have found that the BMP signaling downstream transcription factor SMAD1 and CRMP2 (collapsin response mediator protein-2) are inversely and complementarily expressed in the developing neocortex. BMPs can suppress CRMP2 expression in cortical cells. Our ChIP assay demonstrates that both SMAD1 and -4 bind to CRMP2 promoter in the neocortex, and overexpression of SMAD1 and 4 in vivo suppresses CRMP2 expression. RNA interference of CRMP2 and overexpression of dominant negative forms of CRMP2 in utero cause accumulation of multipolar cells in the ventricular, subventricular, and intermediate zones and suppresses neurite outgrowth, suggesting that CRMP2 is required for multipolar to bipolar transition for directional neuronal migration and neurite outgrowth. Thus, our study reveals a novel mechanism that the BMP-SMAD signaling pathway controls neuronal migration and neurite outgrowth by suppressing the transcription of CRMP2.

Cigarette Smoke-Related Hydroquinone Induces Filamentous Actin Reorganization and Heat Shock Protein 27 Phosphorylation through p38 and Extracellular Signal-Regulated Kinase 1/2 in Retinal Pigment Epithelium: Implications for Age-Related Macular Degeneration

Retinal pigment epithelium (RPE)-derived membranous debris named blebs, may accumulate and contribute to sub-RPE deposit formation, which is the earliest sign of age-related macular degeneration (AMD). Oxidative injury to the RPE might play a significant role in AMD. However, the underlying mechanisms are unknown. We previously reported that hydroquinone (HQ), a major pro-oxidant in cigarette smoke, foodstuff, and atmospheric pollutants, induces actin rearrangement and membrane blebbing in RPE cells as well as sub-RPE deposits in mice. Here, we show for the first time that phosphorylated Heat shock protein 27 (Hsp27), a key regulator of actin filaments dynamics, is up-regulated in RPE from patients with AMD. Also, HQ-induced nonlethal oxidative injury led to Hsp27mRNA up-regulation, dimer formation, and Hsp27 phosphorylation in ARPE-19 cells. Furthermore, we found that a cross talk between p38 and extracellular signal-regulated kinase (ERK) mediates HQ-induced Hsp27 phosphorylation and actin aggregate formation, revealing ERK as a novel upstream mediator of Hsp27 phosphorylation. Finally, we demonstrated that Hsp25, p38, and ERK phosphorylation are increased in aging C57BL/6 mice chronically exposed to HQ, whereas Hsp25 expression is decreased. Our data suggest that phosphorylated Hsp27 might be a key mediator in AMD and HQ-induced oxidative injury to the RPE, which may provide helpful insights into the early cellular events associated with actin reorganization and bleb formation involved in sub-RPE deposits formation relevant to the pathogenesis of AMD.

Effect of a calcium-channel blocker (verapamil) on the morphology, cytoskeleton and collagenase activity of human skin fibroblasts

The effects of verapamil modulating collagen biosynthesis have prompted us to study the role of this drug in cultured fibroblasts. In this article, we describe the effects of verapamil on fibroblast behaviour, with special emphasis to phenotypic modifications, reorganisation of actin filaments and secretion of MMP1. Human dermal fibroblasts treated with 50-μM verapamil changed their normal spindle-shaped morphology to stellate. Treated cells showed discrete reorganisation of actin filaments, as revealed by fluorescein isothiocyanate (FITC)-phalloidin staining and confocal microscopy. We hypothesised that these effects would be associated to lower levels of cytosolic Ca 2+. Indeed, short time loading with calcium green confirmed that verapamil-treated fibroblasts exhibited lower intracellular calcium levels compared to controls. We also observed that verapamil increases the secretion of MMP1 in cultured fibroblasts, as demonstrated by zymography, specific substrate assays and immunoblot. The morphological alterations induced by verapamil are neither cytotoxic nor associated with other dramatic cytoskeleton alterations. Thus we may conclude that this drug enhances collagenase secretion and does not disrupt the major tracks necessary to deliver these enzymes in the extracellular space. The present results suggested that verapamil could be used at physiological levels to enhance collagen I breakdown, and may be considered a potential candidate for intralesional therapy of wound healing and fibrocontractive diseases.

Evolutionary selection of new breast cancer cell-targeting peptides and phages with the cell-targeting peptides fully displayed on the major coat and their effects on actin dynamics during cell internalization.

Filamentous phage as a bacteria-specific virus can be conjugated with an anticancer drug and has been proposed to serve as a carrier to deliver drugs to cancer cells for targeted therapy. However, how cell-targeting filamentous phage alone affects cancer cell biology is unclear. Phage libraries provide an inexhaustible reservoir of new ligands against tumor cells and tissues that have potential therapeutic and diagnostic applications in cancer treatment. Some of these identified ligands might stimulate various cell responses. Here we identified new cell internalizing peptides (and the phages with such peptides fused to each of ~3900 copies of their major coat protein) using landscape phage libraries and for the first time investigated the actin dynamics when selected phages are internalized into the SKBR-3 breast cancer cells. Our results show that phages harboring VSSTQDFP and DGSIPWST peptides could selectively internalize into the SKBR-3 breast cancer cells with high affinity, and also show rapid involvement of membrane ruffling and rearrangements of actin cytoskeleton during the phage entry. The actin dynamics was studied by using live cell and fluorescence imaging. The cell-targeting phages were found to enter breast cancer cells through energy dependent mechanism and phage entry interferes with actin dynamics, resulting in reorganization of actin filaments and increased membrane rufflings in SKBR-3 cells. These results suggest that, when phage enters epithelial cells, it triggers transient changes in the host cell actin cytoskeleton. This study also shows that using multivalent phage libraries considerably increases the repertoire of available cell-internalizing ligands with potential applications in targeted drug delivery, imaging, molecular monitoring and profiling of breast cancer cells.

Arp2/3- and Cofilin-coordinated Actin Dynamics Is Required for Insulin-mediated GLUT4 Translocation to the Surface of Muscle Cells

GLUT4 vesicles are actively recruited to the muscle cell surface upon insulin stimulation. Key to this process is Rac-dependent reorganization of filamentous actin beneath the plasma membrane, but the underlying molecular mechanisms have yet to be elucidated. Using L6 rat skeletal myoblasts stably expressing myc-tagged GLUT4, we found that Arp2/3, acting downstream of Rac GTPase, is responsible for the cortical actin polymerization evoked by insulin. siRNA-mediated silencing of either Arp3 or p34 subunits of the Arp2/3 complex abrogated actin remodeling and impaired GLUT4 translocation. Insulin also led to dephosphorylation of the actin-severing protein cofilin on Ser-3, mediated by the phosphatase slingshot. Cofilin dephosphorylation was prevented by strategies depolymerizing remodeled actin (latrunculin B or p34 silencing), suggesting that accumulation of polymerized actin drives severing to enact a dynamic actin cycling. Cofilin knockdown via siRNA caused overwhelming actin polymerization that subsequently inhibited GLUT4 translocation. This inhibition was relieved by reexpressing Xenopus wild-type cofilin-GFP but not the S3E-cofilin-GFP mutant that emulates permanent phosphorylation. Transferrin recycling was not affected by depleting Arp2/3 or cofilin. These results suggest that cofilin dephosphorylation is required for GLUT4 translocation. We propose that Arp2/3 and cofilin coordinate a dynamic cycle of actin branching and severing at the cell cortex, essential for insulin-mediated GLUT4 translocation in muscle cells.

Myosin IIb Regulates Actin Dynamics during Synaptic Plasticity and Memory Formation

Reorganization of the actin cytoskeleton is essential for synaptic plasticity and memory formation. Presently, the mechanisms that trigger actin dynamics during these brain processes are poorly understood. In this study, we show that myosin II motor activity is downstream of LTP induction and is necessary for the emergence of specialized actin structures that stabilize an early phase of LTP. We also demonstrate that myosin II activity contributes importantly to an actin-dependent process that underlies memory consolidation. Pharmacological treatments that promote actin polymerization reversed the effects of a myosin II inhibitor on LTP and memory. We conclude that myosin II motors regulate plasticity by imparting mechanical forces onto the spine actin cytoskeleton in response to synaptic stimulation. These cytoskeletal forces trigger the emergence of actin structures that stabilize synaptic plasticity. Our studies provide a mechanical framework for understanding cytoskeletal dynamics associated with synaptic plasticity and memory formation.

Actin reorganization contributes to loss of cell adhesion in pemphigus vulgaris

In the human autoimmune blistering skin disease pemphigus vulgaris autoantibodies (PV-IgG), which are mainly directed against keratinocyte cell adhesion molecules desmoglein (Dsg) 3 and Dsg1, cause keratinocyte cell dissociation (acantholysis). Recent studies reported that loss of keratinocyte cell adhesion was accompanied by profound alterations of the actin cytoskeleton. Nevertheless, the relevance of actin reorganization in this process is unclear at present. In this study, we provide evidence for an important role of actin reorganization in pemphigus pathogenesis. In parallel to loss of cell adhesion and fragmentation of Dsg3 staining along cell borders, PV-IgG treatment resulted in striking changes in actin cytoskeleton organization. Moreover, in experiments using fluorescence recovery after photobleaching (FRAP), PV-IgG were detected to interfere with actin dynamics. Therefore, we investigated whether pharmacological manipulation of actin polymerization modulates pathogenic effects of PV-IgG. Pharmacological stabilization of actin filaments via jasplakinolide significantly blocked cell dissociation and Dsg3 fragmentation, whereas cytochalasin D-induced actin depolymerization strongly enhanced pathogenic effects of PV-IgG. To substantiate these findings, we studied whether the protective effects of Rho GTPases, which are potent regulators of the actin cytoskeleton and were shown to be involved in pemphigus pathogenesis, were dependent on modulation of actin dynamics. Cytotoxic necrotizing factor-1 (CNF-1)-mediated activation of Rho-GTPases enhanced the cortical junction-associated actin belt and blunted PV-IgG-induced cell dissociation. However, when actin polymerization was blocked under these conditions via addition of latrunculin B, the protective effects of CNF-1 were abrogated. Taken together, these experiments indicate that reorganization of cortical actin filaments is a critical step in PV-IgG-induced keratinocyte dissociation.

Cytoskeletal Changes in Oocytes and Early Embryos During in vitro Fertilization Process in Mice

The cytoskeleton plays crucial roles in the development and fertilization of germ cells and in the early embryo development. The growth, maturation and fertilization of oocytes require an active movement and a correct localization of cellular organelles. This is performed by the re-organization of microtubules and actin filaments. Therefore, the aim of the present study was to determine the changes in cytoskeleton during in vitro fertilization process using appropriate immunofluorescence techniques. While the chromatin content was found to be scattered throughout the nucleus during the oocyte maturation period, it was seen only around nucleolus following the completion of the maturation. Microtubules, during oocyte maturation, were regularly distributed throughout the ooplasm which was then localized in the subcortical region of oocytes. Similarly microfilaments were scattered throughout the ooplasm during the oocyte maturation period whereas they were seen in the subcortical region around the polar body and above the meiotic spindle throughout the late developmental stages. In conclusion, those changes occurred in microtubules and microfilaments might be closely related to the re-organization of the genetic material during the oocyte maturation and early embryo development.

Ca2+/Calmodulin-dependent Protein Kinase IV-mediated LIM Kinase Activation Is Critical for Calcium Signal-induced Neurite Outgrowth

Actin cytoskeletal remodeling is essential for neurite outgrowth. LIM kinase 1 (LIMK1) regulates actin cytoskeletal remodeling by phosphorylating and inactivating cofilin, an actin filament-disassembling factor. In this study, we investigated the role of LIMK1 in calcium signal-induced neurite outgrowth. The calcium ionophore ionomycin induced LIMK1 activation and cofilin phosphorylation in Neuro-2a neuroblastoma cells. Knockdown of LIMK1 or expression of a kinase-dead mutant of LIMK1 suppressed ionomycin-induced cofilin phosphorylation and neurite outgrowth in Neuro-2a cells. Ionomycin-induced cofilin phosphorylation and neurite outgrowth were also blocked by KN-93, an inhibitor of Ca(2+)/calmodulin-dependent protein kinases (CaMKs), and STO-609, an inhibitor of CaMK kinase. An active form of CaMKIV but not CaMKI enhanced Thr-508 phosphorylation of LIMK1 and increased the kinase activity of LIMK1. Moreover, the active form of CaMKIV induced cofilin phosphorylation and neurite outgrowth, and a dominant negative form of CaMKIV suppressed ionomycin-induced neurite outgrowth. Taken together, our results suggest that LIMK1-mediated cofilin phosphorylation is critical for ionomycin-induced neurite outgrowth and that CaMKIV mediates ionomycin-induced LIMK1 activation.