The significance of alpha 5 beta 1 integrin dependent and independent actin cytoskelton organization in cell transformation and survival

Cell-matrix and cell-cell interactions are important physiological determinants of cell growth, survival and transformation. Cell adhesion to the extra cellular matrix (ECM) via integrins also crucially influences the organization of the cytoskeleton. It triggers a cascade of intracellular biochemical events, which regulate cell viability and growth. We have studied the relationship between cell attachment to the substratum and cytoskeletal organization and cell survival and transformation. Our results demonstrate that in the absence of attachment to the substratum, adhesion-dependent fibroblasts exhibit rapid loss of viability. However, a small percentage of cells survive even after remaining non-adherent for 16h. The adherent and non-adherent cells differ from one another both morphologically and physiologically. The latter show a loss of alpha5beta1 integrin expression on their surface and bind non-specifically to the substratum and ECM, thereby activating certain pathways more efficiently than adherent cells. We have also shown that non-adherent cells grow faster and have worse cytoskeletal organization after attachment to the substratum, and do not form focal adhesions or actin stress fibres. Hence, our data suggests that rat fibroblasts in prolonged suspension exhibit some properties that are comparable to cells undergoing transformation, by adapting integrin-dependent or independent signalling pathways for their survival.     

The snake venom disintegrin salmosin induces apoptosis by disassembly of focal adhesions in bovine capillary endothelial cells

Salmosin, a disintegrin purified from a Korean snake (Agkistrodon halys brevicaudus) venom, interacts with integrin alpha(v)beta(3) and inhibits the proliferation of bovine capillary endothelial (BCE) cells induced by basic fibroblast growth factor (bFGF). We investigated salmosin's mechanism of inhibition of BCE cell proliferation by examining changes in the cytoskeleton and activation of integrin-mediated signaling molecules. Salmosin disassembled cortical actins at focal adhesions and induced cells to be rounded and detached, but it did not alter microtubule structures in the early stage of cells being rounded. Immunolocalization of paxillin also demonstrated that focal adhesions were disassembled by salmosin. In salmosin-treated BCE cells, focal adhesion kinase (FAK) was dephosphorylated and expression of paxillin and p130(CAS) was decreased, but PI3 kinase, ILK, and beta-catenin were not expressed in decreased amounts or modified, suggesting that salmosin inactivated FAK-dependent integrin signaling pathways. While BCE cells proliferated normally on plates coated with salmosin, cells treated with salmosin eventually underwent apoptosis. These observations strongly suggest that salmosin disorganizes focal contacts to detach cells by competing with the extracellular matrix (ECM) for direct binding to integrin alpha(v)beta(3) on the cell surface, eventually leading to apoptosis.     

Myosin-5, kinesin-1 and myosin-17 cooperate in secretion of fungal chitin synthase

Plant infection by pathogenic fungi requires polarized secretion of enzymes, but little is known about the delivery pathways. Here, we investigate the secretion of cell wall-forming chitin synthases (CHSs) in the corn pathogen Ustilago maydis. We show that peripheral filamentous actin (F-actin) and central microtubules (MTs) form independent tracks for CHSs delivery and both cooperate in cell morphogenesis. The enzyme Mcs1, a CHS that contains a myosin-17 motor domain, is travelling along both MTs and F-actin. This transport is independent of kinesin-3, but mediated by kinesin-1 and myosin-5. Arriving vesicles pause beneath the plasma membrane, but only ~15% of them get exocytosed and the majority is returned to the cell centre by the motor dynein. Successful exocytosis at the cell tip and, to a lesser extent at the lateral parts of the cell requires the motor domain of Mcs1, which captures and tethers the vesicles prior to secretion. Consistently, Mcs1-bound vesicles transiently bind F-actin but show no motility in vitro. Thus, kinesin-1, myosin-5 and dynein mediate bi-directional motility, whereas myosin-17 introduces a symmetry break that allows polarized secretion.     

Regulatory roles of phosphoinositides in membrane trafficking and their potential impact on cell-wall synthesis and re-modelling

Background

Plant cell walls are complex matrices of carbohydrates and proteins that control cell morphology and provide protection and rigidity for the plant body. The construction and maintenance of this intricate system involves the delivery and recycling of its components through a precise balance of endomembrane trafficking, which is controlled by a plethora of cell signalling factors. Phosphoinositides (PIs) are one class of signalling molecules with diverse roles in vesicle trafficking and cytoskeleton structure across different kingdoms. Therefore, PIs may also play an important role in the assembly of plant cell walls.

Scope

The eukaryotic PI pathway is an intricate network of different lipids, which appear to be divided in different pools that can partake in vesicle trafficking or signalling. Most of our current understanding of how PIs function in cell metabolism comes from yeast and mammalian systems; however, in recent years significant progress has been made towards a better understanding of the plant PI system. This review examines the current state of knowledge of how PIs regulate vesicle trafficking and their potential influence on plant cell-wall architecture. It considers first how PIs are formed in plants and then examines their role in the control of vesicle trafficking. Interactions between PIs and the actin cytoskeleton and small GTPases are also discussed. Future challenges for research are suggested.     

alpha1,3 fucosyltransferase-VII up-regulates the mRNA of alpha5 integrin and its biological function

After transfection of alpha1,3fucosyltransferase (FucT)-VII cDNA into H7721 human hepatocarcinoma cells, the expression of alpha5, but not beta1 integrin was significantly up-regulated. This was evidenced by the increase of alpha5 integrin on cell surface as well as the increase of alpha5 mRNA and protein in the cells. However, the expressions of sialyl Lewis X (SLe(x), the product of alpha1,3FucT-VII) on both alpha5 and beta1 integrin subunits were unchanged. Concomitantly, the tyrosine autophosphorylated FAK and dephosphorylated Src (FAK and Src involve in the signal transduction of integrin alpha5beta1) were up-regulated, while the Tyr-527 phosphorylated Src was down-regulated. The above-mentioned alterations were correlated to the expressions of alpha1,3FucT-VII in different alpha1,3FucT-VII transfected H7721 cell lines. In addition, after alpha1,3FucT-VII transfection, cell adhesion to fibronectin (Fn) and chemotaxic cell migration were obviously promoted. The cell adhesion could be blocked by alpha5 integrin antibody, and cell migration was obviously attenuated by the antibodies to both alpha5 integrin and SLe(x). These findings suggest that the increased surface alpha5 integrin caused by the up-regulation of alpha5 mRNA promotes the cell adhesion to Fn, cell migratiom, and Fn-induced signaling of alpha5beta1 integrin. The up-regulation of surface SLe(x) originated from the over expression of alpha1,3FucT-VII also led to the stimulation of cell migration. This is the first time to report that alpha1,3FucT-VII can regulate the mRNA expression of integrin.     

细丝蛋白A在细胞黏附和迁移中的作用

细胞迁移在发育、伤口愈合、炎症反应和肿瘤转移等多种病理生理过程中发挥重要作用。细丝蛋白A（filamin A,FlnA）是一种在各组织细胞中广泛表达的微丝结合蛋白,其表达异常导致细胞迁移功能障碍。该文回顾了相关的文献,首先介绍生理情况下细丝蛋白A的功能,接着介绍细丝蛋白A基因突变和表达异常导致的多种遗传性疾病及其与肿瘤转移的关系,突出细丝蛋白A对迁移的影响在这些疾病发病中的作用,最后深入探讨了细丝蛋白A影响细胞迁移和黏附的可能机制。     

Actin-dependent deposition of putative endosomes and endoplasmic reticulum during early stages of wound healing in characean internodal cells

We investigated the behaviour of organelles stained with FM1-43 (putative endosomes) and/or LysoTracker Red (LTred; acidic compartments) and of the endoplasmic reticulum (ER) during healing of puncture and UV-induced wounds in internodal cells of Nitella flexilis and Chara corallina. Immediately after puncture, wounds were passively sealed with a plug of solid vacuolar inclusions, onto which a bipartite wound wall was actively deposited. The outer, callose-containing amorphous layer consisted of remnants of FM1-43- and LTred-labelled organelles, ER cisternae and polysaccharide-containing secretory vesicles, which became deposited in the absence of membrane retrieval (compound exocytosis). During formation of the inner cellulosic layer, exocytosis of secretory vesicles with the newly formed plasma membrane is coupled to endocytosis via coated vesicles. Migration of FM1-43- and LTred-stained organelles, ER and secretory vesicles towards the cell cortex and deposition of a bipartite wound wall could also be induced by spot-like irradiation with ultraviolet light. Cytochalasin D reversibly inhibited the accumulation and deposition of organelles. Our study indicates that active actin-dependent deposition of putative recycling endosomes is required for wound healing (plasma membrane repair) and supports the hypothesis that deposition of ER cisternae helps to restore wounding-disturbed Ca(2+) metabolism.     

Membrane protein hMYADM preferentially expressed in myeloid cells is up-regulated during differentiation of stem cells and myeloid leukemia cells

We report here the molecular cloning and characterization of a novel human gene (hMYADM) derived from a human bone marrow stromal cell (BMSC) cDNA library, which shares high homology with mouse myeloid-associated differentiation marker (MYADM). hMYADM is also closely related to many other eukaryotic proteins, which together form a novel and highly conserved MYADM-like family. hMYADM with 322-residue protein contains eight putative transmembrane segments and confocal microscopic analysis confirmed its membrane localization by using anti-hMYADM monoclonal antibody. hMYADM mRNA was selectively expressed in human monocytes, dendritic cells, promyeloid or monocytic leukemia cell lines, but not in CD4+, CD8+, CD19+ cells, nor in T cell leukemia or lymphocytic leukemia cell lines. hMYADM expression was also found in normal human bone marrow enriched for CD34+ stem cells, and the expression was up-regulated when these cells were induced to differentiate toward myeloid cells. The mRNA expression level of hMYADM significantly increased in acute promyelocytic leukemia HL-60 and chronic myelogenous leukemia K562 cell line after phorbol myristate acetate (PMA)-induced differentiation. Our study suggests that hMYADM is selectively expressed in myeloid cells, and involved in the myeloid differentiation process, indicating that hMYADM may be one useful membrane marker to monitor stem cell differentiation or myeloid leukemia differentiation.     

Binding of APC and dishevelled mediates Wnt5a‐regulated focal adhesion dynamics in migrating cells

Wnt5a is a representative ligand that activates the Wnt/β‐catenin‐independent pathway, resulting in the regulation of cell adhesion, migration, and polarity, but its molecular mechanism is poorly understood. This report shows that Dishevelled (Dvl) binds to adenomatous polyposis coli (APC) gene product, and this binding is enhanced by Wnt5a. Dvl was involved in the stabilization of the plus end dynamics of microtubules as well as APC. Frizzled2 (Fz2) was present with Wnt5a at the leading edge of migrating cells and formed a complex with APC through Dvl. Fz2 also interacted with integrins at the leading edge, and Dvl and APC associated with and activated focal adhesion kinase and paxillin. The binding of APC to Dvl enhanced the localization of paxillin to the leading edge and was involved in Wnt5a‐dependent focal adhesion turnover. Furthermore, this new Wnt5a signalling pathway was important for the epithelial morphogenesis in the three‐dimensional culture. These results suggest that the functional and physical interaction of Dvl and APC is involved in Wnt5a/Fz2‐dependent focal adhesion dynamics during cell migration and epithelial morphogenesis.     

Protein kinase C-dependent mobilization of the alpha6beta4 integrin from hemidesmosomes and its association with actin-rich cell protrusions drive the chemotactic migration of carcinoma cells.

We explored the hypothesis that the chemotactic migration of carcinoma cells that assemble hemidesmosomes involves the activation of a signaling pathway that releases the alpha6beta4 integrin from these stable adhesion complexes and promotes its association with F-actin in cell protrusions enabling it to function in migration. Squamous carcinoma-derived A431 cells were used because they express alpha6beta4 and migrate in response to EGF stimulation. Using function-blocking antibodies, we show that the alpha6beta4 integrin participates in EGF-stimulated chemotaxis and is required for lamellae formation on laminin-1. At concentrations of EGF that stimulate A431 chemotaxis ( approximately 1 ng/ml), the alpha6beta4 integrin is mobilized from hemidesmosomes as evidenced by indirect immunofluorescence microscopy using mAbs specific for this integrin and hemidesmosomal components and its loss from a cytokeratin fraction obtained by detergent extraction. EGF stimulation also increased the formation of lamellipodia and membrane ruffles that contained alpha6beta4 in association with F-actin. Importantly, we demonstrate that this mobilization of alpha6beta4 from hemidesmosomes and its redistribution to cell protrusions occurs by a mechanism that involves activation of protein kinase C-alpha and that it is associated with the phosphorylation of the beta4 integrin subunit on serine residues. Thus, the chemotactic migration of A431 cells on laminin-1 requires not only the formation of F-actin-rich cell protrusions that mediate alpha6beta4-dependent cell movement but also the disruption of alpha6beta4-containing hemidesmosomes by protein kinase C.     

The fibronectin-binding integrins alpha5beta1 and alphavbeta3 differentially modulate RhoA-GTP loading,organization of cell matrix adhesions,and fibronectin fibrillogenesis

We have studied the formation of different types of cell matrix adhesions in cells that bind to fibronectin via either alpha5beta1 or alphavbeta3. In both cases, cell adhesion to fibronectin leads to a rapid decrease in RhoA activity. However, alpha5beta1 but not alphavbeta3 supports high levels of RhoA activity at later stages of cell spreading, which are associated with a translocation of focal contacts to peripheral cell protrusions, recruitment of tensin into fibrillar adhesions, and fibronectin fibrillogenesis. Expression of an activated mutant of RhoA stimulates alphavbeta3-mediated fibrillogenesis. Despite the fact that alpha5beta1-mediated adhesion to the central cell-binding domain of fibronectin supports activation of RhoA, other regions of fibronectin are required for the development of alpha5beta1-mediated but not alphavbeta3-mediated focal contacts. Using chimeras of beta1 and beta3 subunits, we find that the extracellular domain of beta1 controls RhoA activity. By expressing both beta1 and beta3 at high levels, we show that beta1-mediated control of the levels of beta3 is important for the distribution of focal contacts. Our findings demonstrate that the pattern of fibronectin receptors expressed on a cell dictates the ability of fibronectin to stimulate RhoA-mediated organization of cell matrix adhesions.     

Direct association between caspase 3 and alpha5beta1 integrin and its role during anoikis of rat fibroblasts

We have demonstrated a direct association between alpha5beta1 integrin and caspase 3, both pro- and mature enzyme, in various sub-cellular compartments of rat fibroblasts undergoing anoikis. Integrin associated caspase 3 showed high activity in the plasma membranes, whereas in the cytosol and microsomal fraction it showed little or no activity. Our results suggest a possible role for recycled un-ligated alpha5beta1 integrin molecules between cytosol and plasma membrane, in regulation of caspase-3 activity and induction of cell death in adhesion-deprived cells.     

Integrating the actin and vimentin cytoskeletons. adhesion-dependent formation of fimbrin-vimentin complexes in macrophages.

Cells adhere to the substratum through specialized structures that are linked to the actin cytoskeleton. Recent studies report that adhesion also involves the intermediate filament (IF) and microtubule cytoskeletons, although their mechanisms of interaction are unknown. Here we report evidence for a novel adhesion-dependent interaction between components of the actin and IF cytoskeletons. In biochemical fractionation experiments, fimbrin and vimentin coprecipitate from detergent extracts of macrophages using vimentin- or fimbrin-specific antisera. Fluorescence microscopy confirms the biochemical association. Both proteins colocalized to podosomes in the earliest stages of cell adhesion and spreading. The complex is also found in filopodia and retraction fibers. After detergent extraction, fimbrin and vimentin staining of podosomes, filopodia, and retraction fibers are lost, confirming that the complex is localized to these structures. A 1:4 stoichiometry of fimbrin binding to vimentin and a low percentage (1%) of the extracted vimentin suggest that fimbrin interacts with a vimentin subunit. A fimbrin-binding site was identified in the NH(2)-terminal domain of vimentin and the vimentin binding site at residues 143-188 in the CH1 domain of fimbrin. Based on these observations, we propose that a fimbrin-vimentin complex may be involved in directing the assembly of the vimentin cytoskeleton at cell adhesion sites.     

Spatio‐temporal analysis of cellulose synthesis during cell plate formation in Arabidopsis

During cytokinesis a new crosswall is rapidly laid down. This process involves the formation at the cell equator of a tubulo‐vesicular membrane network (TVN). This TVN evolves into a tubular network (TN) and a planar fenestrated sheet, which extends at its periphery before fusing to the mother cell wall. The role of cell wall polymers in cell plate assembly is poorly understood. We used specific stains and GFP‐labelled cellulose synthases (CESAs) to show that cellulose, as well as three distinct CESAs, accumulated in the cell plate already at the TVN stage. This early presence suggests that cellulose is extruded into the tubular membrane structures of the TVN. Co‐localisation studies using GFP–CESAs suggest the delivery of cellulose synthase complexes (CSCs) to the cell plate via phragmoplast‐associated vesicles. In the more mature TN part of the cell plate, we observed delivery of GFP–CESA from doughnut‐shaped organelles, presumably Golgi bodies. During the conversion of the TN into a planar fenestrated sheet, the GFP–CESA density diminished, whereas GFP–CESA levels remained high in the TVN zone at the periphery of the expanding cell plate. We observed retrieval of GFP–CESA in clathrin‐containing structures from the central zone of the cell plate and from the plasma membrane of the mother cell, which may contribute to the recycling of CESAs to the peripheral growth zone of the cell plate. These observations, together with mutant phenotypes of cellulose‐deficient mutants and pharmacological experiments, suggest a key role for cellulose synthesis already at early stages of cell plate assembly.     

Drosophila beta spectrin functions independently of alpha spectrin to polarize the Na,K ATPase in epithelial cells

Spectrin has been proposed to function as a sorting machine that concentrates interacting proteins such as the Na,K ATPase within specialized plasma membrane domains of polarized cells. However, little direct evidence to support this model has been obtained. Here we used a genetic approach to directly test the requirement for the beta subunit of the alphabeta spectrin molecule in morphogenesis and function of epithelial cells in Drosophila. beta Spectrin mutations were lethal during late embryonic/early larval development and they produced subtle defects in midgut morphology and stomach acid secretion. The polarized distributions of alphabeta(H) spectrin and ankyrin were not significantly altered in beta spectrin mutants, indicating that the two isoforms of Drosophila spectrin assemble independently of one another, and that ankyrin is upstream of alphabeta spectrin in the spectrin assembly pathway. In contrast, beta spectrin mutations had a striking effect on the basolateral accumulation of the Na,K ATPase. The results establish a role for beta spectrin in determining the subcellular distribution of the Na, K ATPase and, unexpectedly, this role is independent of alpha spectrin.     

Cdk5 regulates multiple cellular events in neural development,function and disease

Cyclin‐dependent kinases (CDKs) generally regulate cell proliferation in dividing cells, including neural progenitors. In contrast, an unconventional CDK, Cdk5, is predominantly activated in post‐mitotic cells, and involved in various cellular events, such as microtubule and actin cytoskeletal organization, cell–cell and cell–extracellular matrix adhesions, and membrane trafficking. Interestingly, recent studies have indicated that Cdk5 is associated with several cell cycle‐related proteins, Cyclin‐E and p27^kip1. Taking advantage of multiple functionality, Cdk5 plays important roles in neuronal migration, layer formation, axon elongation and dendrite arborization in many regions of the developing brain, including cerebral cortex and cerebellum. Cdk5 is also required for neurogenesis at least in the cerebral cortex. Furthermore, Cdk5 is reported to control neurotransmitter release at presynaptic sites, endocytosis of the NMDA receptor at postsynaptic sites and dendritic spine remodeling, and thereby regulate synaptic plasticity and memory formation and extinction. In addition to these physiological roles in brain development and function, Cdk5 is associated with many neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. In this review, I will introduce the physiological and pathological roles of Cdk5 in mammalian brains from the viewpoint of not only in vivo phenotypes but also its molecular and cellular functions.     

Differential transport and processing of variant mouse mammary tumor virus glycoproteins.

The transport and proteolytic processing of two individual gene isolates of the mouse mammary tumor virus (MMTV) glycoprotein were compared in transfected rat HTC hepatoma cells. Plasmids were constructed such that the MMTV glycoprotein genes were constitutively expressed from the promoter within the Rous Sarcoma Virus 5' Long Terminal Repeat in the absence of other MMTV proteins. An isolate of the GR strain MMTV glycoprotein was efficiently transported and processed resulting in the localization of MMTV glycoproteins at the cell surface and in the extracellular environment. Moreover, the kinetics of acquisition of endoglycosidase H resistant oligosaccharide side chains and the rate of endoproteolytic cleavage of the glycosylated polyprotein expressed in transfected cells were virtually identical to that observed in viral-infected rat hepatoma cells. In contrast, a natural variant of the C3H strain MMTV glycoprotein expressed in transfected cells was retained in an intracellular compartment by a heavy chain binding protein (BiP)-independent pathway in an endoglycosidase H sensitive and uncleaved form. This MMTV glycoprotein isolate was retained early in the exocytic pathway and displayed a half-life of approximately 45 min in transfected cells. Only a minor fraction of the expressed C3H variant glycoprotein was detected at the cell surface but was not externalized. Our results suggest that the variant C3H MMTV glycoprotein contains one or more mutations that preclude its efficient transport through the exocytic pathway.