Dismantling Cell–Cell Contacts during Apoptosis Is Coupled to a Caspase-dependent Proteolytic Cleavage of β-Catenin

Cell death by apoptosis is a tightly regulated process that requires coordinated modification in cellular architecture. The caspase protease family has been shown to play a key role in apoptosis. Here we report that specific and ordered changes in the actin cytoskeleton take place during apoptosis.

In this context, we have dissected one of the first hallmarks in cell death, represented by the severing of contacts among neighboring cells. More specifically, we provide demonstration for the mechanism that could contribute to the disassembly of cytoskeletal organization at cell–cell adhesion. In fact, β-catenin, a known regulator of cell–cell adhesion, is proteolytically processed in different cell types after induction of apoptosis. Caspase-3 (cpp32/apopain/yama) cleaves in vitro translated β-catenin into a form which is similar in size to that observed in cells undergoing apoptosis. β-Catenin cleavage, during apoptosis in vivo and after caspase-3 treatment in vitro, removes the amino- and carboxy-terminal regions of the protein. The resulting β-catenin product is unable to bind α-catenin that is responsible for actin filament binding and organization. This evidence indicates that connection with actin filaments organized at cell–cell contacts could be dismantled during apoptosis. Our observations suggest that caspases orchestrate the specific and sequential changes in the actin cytoskeleton occurring during cell death via cleavage of different regulators of the microfilament system.

     

Cytoskeleton as a target in menadione-induced oxidative stress in cultured mammalian cells: alterations underlying surface bleb formation

Several in vitro and in vivo studies have suggested that surface bleb formation during oxidative cell injury is related to alteration in cytoskeleton organization. Various cell lines different in origin and growth characteristics were exposed to 2-methyl-1,4-naphthoquinone (menadione) which is known to induce bleb formation and cytotoxicity by generating considerable amounts of oxygen-reactive species. Treated cells were analyzed by means of immunocytochemistry and electron microscopy in order to investigate the morphological and molecular features underlying bleb generation. The results obtained indicate that menadione-induced bleb formation is a widely observed phenomenon present mainly in round or mitotic cells. Surface blebs appear free of organelles and contain only few ribosomes and amorphous material. Occasionally, they undergo detachment from the cell surface as large cytoplasmic vesicles. Bleb surfaces with protein clusters as well as bald blisters with an almost exclusive localization of intramembrane particles on their narrow base were detected using freeze-fracture techniques. Immunocytochemical investigations performed on menadione-exposed cells revealed that some surface proteins (collagen IV, sialo-proteins, beta 2 microglobulin and fibronectin) and adhesion molecules (vinculin) underwent changes in their expression over the bleb surface. Moreover, different behavioural characteristics of actin microfilaments, vimentin and keratin intermediate filaments and microtubules was observed. Alpha-actinin, vimentin and microtubular proteins (tubulin, MAPs and tau) were detected within the blebs. On the other hand, actin and keratin filaments appeared to be absent. The results presented here demonstrate that cytoskeletal structures and the microfilament system in particular, represent important targets in menadione-induced morphological changes in cultured cells. These changes appear to lead to the redistribution of several cytoskeletal and membrane proteins as well as dissociation of the cytoskeleton network from its anchoring domains in the plasma membrane thus generating sites of structural weakness where blebs would arise and progressively grow. Experimental evidence supporting a crucial role of thiol oxidation and elevation of cytoplasmic calcium concentration in bleb formation is also provided.     

Function of Rho family proteins in actin dynamics during phagocytosis and engulfment

Phagocytosis is the uptake of large particles by cells by a mechanism that is based on local rearrangement of the actin microfilament cytoskeleton. In higher organisms, phagocytic cells are essential for host defence against invading pathogens, and phagocytosis contributes to inflammation and the immune response. In addition, engulfment, defined as the phagocytic clearance of cell corpses generated by programmed cell death or apoptosis, has an essential role in tissue homeostasis. Although morphologically distinct phagocytic events can be observed depending on the type of surface receptor engaged, work over the past two years has revealed the essential underlying role of Rho family proteins and their downstream effectors in controlling actin dynamics during phagocytosis.     

The molecular basis of induction and formation of tunneling nanotubes

Tunneling nanotubes (TNTs) and associated structures are recently recognized structures for intercellular communication. They are F-actin-containing thin protrusions of the plasma membrane of a cell and allow a direct physical connection to the plasma membranes of remote cells. TNTs and associated structures serve as mediators for intercellular transfer of organelles as well as membrane components and cytoplasmic molecules. Moreover, several pathogens have been shown to exploit these structures to spread among cells. Because of their contribution to normal cellular functions and importance in pathological conditions, studies on TNTs and related structures have accelerated over the past few years. These studies have revealed key molecules for their induction and/or formation; HIV Nef and M-Sec can induce the formation of TNTs in coordination with the remodeling of the actin cytoskeleton and vesicle trafficking.     

Microfilament reorganization during apoptosis: the role of Gas2, a possible substrate for ICE-like proteases.

Gas2, a component of the microfilament system, belongs to the class of gas genes whose expression is induced at growth arrest. After serum or growth factor addition to quiescent NIH 3T3 cells, Gas2 is hyperphosphorylated and relocalized at the membrane ruffles. By overexpressing gas2wt and a series of deletion mutants of the C-terminal region, we have analysed its role in the organization of the actin cytoskeleton in different cell lines. Overexpression of Gas2 deleted at its C-terminal region (delta 276-314 and delta 236-314), but not its wild-type form, induces dramatic changes in the actin cytoskeleton and cell morphology. These effects are not due to interference of the deleted forms with the endogenous Gas2wt function but could be ascribed to a gain of function. We demonstrate that during apoptosis the C-terminal domain of Gas2 is removed by proteolytic cleavage, resulting in a protein that is similar in size to the described delta 276-314. Moreover, by using in vitro mutagenesis, we also demonstrate that the proteolytic processing of Gas2 during apoptosis is dependent on an aspartic acid residue at position 279. The evidence accumulated here could thus represent a first example of a mechanism linking apoptosis with the co-ordinated microfilament-dependent cell shape changes, as possibly mediated by an interleukin-1 beta-converting enzyme (ICE)-like dependent proteolytic cleavage of the Gas2 protein.     

Cytoskeletal changes in mouse embryonic fibroblasts exposed to colcemid. Electron microscopic research on platinum replicas

Cytoskeletons of colcemid-treated mouse embryo fibroblasts were studied using platinum replica technique. In the control cells, cytoskeletal components were oriented along direction of cell polarization. Structure of the control cytoskeleton changed regularly from the cell active edge to its centre forming several zones. Distribution of microtubules by colcemid led to significant changes in the organization of actin cytoskeleton. Both orientation and zonal differentiation of cytoskeleton disappeared in colcemid-treated fibroblasts. Changes in the fine structure of microfilament sheath were most prominent. Control sheath was composed of stretched tightly packed microfilaments. Colcemid treatment transformed it into fine microfilament meshwork, normally characteristic only for ruffle zone. Alterations of the fine structure of focal contacts and ruffles were also observed in treated cells. The role of microtubules in the organization of intracellular tensions and in the distribution of sites of actin polymerization is discussed.     

Human MCF10A mammary epithelial cells undergo apoptosis following actin depolymerization that is independent of attachment and rescued by Bcl-2

Many tumor cells are impaired in adhesion-regulated apoptosis, which contributes to their metastatic potential. However, suppression of this apoptotic pathway in untransformed cells is not mediated only by adhesion to the extracellular matrix but also through the resulting ability to spread and adopt a distinct morphology. Since cell spreading is dependent on the integrity of the actin microfilament cytoskeleton, we sought to determine if actin depolymerization was sufficient to induce apoptosis, even in the presence of continuous attachment. For this study, we used a human mammary epithelial cell line (MCF10A), which is immortalized but remains adhesion dependent for survival. Treatment of MCF10A cells with latrunculin-A (LA), an inhibitor of actin polymerization, rapidly led to disruption of the actin cytoskeleton and caused cell rounding but preserved attachment. Initiation of apoptosis in LA-treated MCF10A cells was detected by mitochondrial localization of the Bax apoptotic protein, which was prevented by overexpression of Bcl-2. DNA fragmentation and poly(ADP-ribose) polymerase (PARP) cleavage in LA-treated MCF10A cells indicated progression to the execution phase of apoptosis. The MDA-MB-453 cell line, which was derived from a metastatic human mammary tumor, was resistant to PARP cleavage and loss of viability in response to actin depolymerization. Stable overexpression of Bcl-2 in the untransformed MCF10A cells was able to recapitulate the resistance to apoptosis found in the tumor cell line. We demonstrate that inhibition of actin polymerization is sufficient to stimulate apoptosis in attached MCF10A cells, and we present a novel role for Bcl-2 in cell death induced by direct disruption of the actin cytoskeleton.     

Staurosporine induces dissolution of microfilament bundles by a protein kinase C-independent pathway

The protein kinase C (PKC) inhibitor staurosporine was found to dramatically alter the actin microfilament cytoskeleton of a variety of cultured cells, including PTK2 epithelial cells, Swiss 3T3 fibroblasts, and human foreskin fibroblasts. For example, PTK2 cells exposed to 20 nM staurosporine exhibited a progressive thinning and loss of cytoplasmic actin microfilament bundles over a 60-min period. During this time microtubule and intermediate filament systems remained intact (as shown by immunofluorescence and at higher resolution by photoelectron microscopy), and the cells remained spread even though microfilament bundles were absent. Higher doses of staurosporine or longer exposure times at lower doses resulted in morphological alterations, but even severely arborized cells recovered normal morphology and actin patterns after a wash and an incubation for several hours in fresh medium. The actin filament disruption induced by staurosporine was distinguishable from the actin reorganization induced by exposure to the tumor promoter (and activator of PKC) phorbol myristate acetate (PMA). Swiss 3T3 cells made deficient in PKC by prolonged exposure to PMA (PKC down-regulation) exhibited actin alterations in response to staurosporine which were comparable to those in cells which had not been exposed to the phorbol ester. In a parallel control experiment, the actin cytoskeleton of PKC-deficient 3T3 cells was unaffected in response to PMA, consistent with down-regulation of this kinase. While the exact mechanism of staurosporine-induced actin reorganization remains to be determined, the observed effects of staurosporine on PKC-deficient cells make a role for PKC unlikely. These results indicate the need for care when staurosporine is employed as an inhibitor of protein kinase C in studies involving intact cells.     

MOESIN crosslinks actin and cell membrane in Drosophila oocytes and is required for OSKAR anchoring

In Drosophila, development of the embryonic germ cells depends on posterior transport and site-specific translation of oskar (osk) mRNA and on interdependent anchoring of the osk mRNA and protein within the posterior subcortical region of the oocyte. Transport of the osk mRNA is mediated by microtubules, while anchoring of the osk gene products at the posterior pole of the oocyte is suggested to be microfilament dependent. To date, only a single actin binding protein (TropomyosinII) has been identified with a putative role in osk mRNA and protein anchoring. This communication demonstrates that mutations in the Drosophila moesin (Dmoe) gene that encodes another actin binding protein result in delocalization of osk mRNA and protein from the posterior subcortical region and, as a consequence, in failure of embryonic germ cell development. In Dmoe mutant oocytes, the subcortical actin network is detached from the cell membrane, while the polarized microtubule cytoskeleton is unaffected. In line with the earlier observations, colocalization of ectopic actin and OSK protein in Dmoe mutants suggests that the actin cytoskeleton anchors OSK protein to the subcortical cytoplasmic area of the Drosophila oocyte.     

Actin-binding Protein Drebrin Regulates HIV-1-triggered Actin Polymerization and Viral Infection

HIV-1 contact with target cells triggers F-actin rearrangements that are essential for several steps of the viral cycle. Successful HIV entry into CD4⁺ T cells requires actin reorganization induced by the interaction of the cellular receptor/co-receptor complex CD4/CXCR4 with the viral envelope complex gp120/gp41 (Env). In this report, we analyze the role of the actin modulator drebrin in HIV-1 viral infection and cell to cell fusion. We show that drebrin associates with CXCR4 before and during HIV infection. Drebrin is actively recruited toward cell-virus and Env-driven cell to cell contacts. After viral internalization, drebrin clustering is retained in a fraction of the internalized particles. Through a combination of RNAi-based inhibition of endogenous drebrin and GFP-tagged expression of wild-type and mutant forms, we establish drebrin as a negative regulator of HIV entry and HIV-mediated cell fusion. Down-regulation of drebrin expression promotes HIV-1 entry, decreases F-actin polymerization, and enhances profilin local accumulation in response to HIV-1. These data underscore the negative role of drebrin in HIV infection by modulating viral entry, mainly through the control of actin cytoskeleton polymerization in response to HIV-1.     

Sensitivity of Fibroblasts and Their Cytoskeletons to Substratum Topographies: Topographic Guidance and Topographic Compensation by Micromachined Grooves of Different Dimensions

Fibroblasts alter their shape, orientation, and direction of movement to align with the direction of micromachined grooves, exhibiting a phenomenon termed topographic guidance. In this study we examined the ability of the microtubule and actin microfilament bundle systems, either in combination with or independently from each other, to affect alignment of human gingival fibroblasts on sets of micromachined grooves of different dimensions. To assess specifically the role of microtubules and actin microfilament bundles, we examined cell alignment, over time, in the presence or absence of specific inhibitors of microtubules (colcemid) and actin microfilament bundles (cytochalasin B). Using time-lapse videomicroscopy, computer-assisted morphometry and confocal microscopy of the cytoskeleton we found that the dimensions of the grooves influenced the kinetics of cell alignment irrespective of whether cytoskeletons were intact or disturbed. Either an intact microtubule or an intact actin microfilament-bundle system could produce cell alignment with an appropriate substratum. Cells with intact microtubules aligned to smaller topographic features than cells deficient in microtubules. Moreover, cells deficient in microtubules required significantly more time to become aligned. An unexpected finding was that very narrow 0.5-μm-wide and 0.5-μm-deep grooves aligned cells deficient in actin microfilament bundles (cytochalasin B-treated) better than untreated control cells but failed to align cells deficient in microtubules yet containing microfilament bundles (colcemid treated). Thus, the microtubule system appeared to be the principal but not sole cytoskeletal substratum-response mechanism affecting topographic guidance of human gingival fibroblasts. This study also demonstrated that micromachined substrata can be useful in dissecting the role of microtubules and actin microfilament bundles in cell behaviors such as contact guidance and cell migration without the use of drugs such as cytochalasin and colcemid.     

Identification of the cytolinker plectin as a major early in vivo substrate for caspase 8 during CD95- and tumor necrosis factor receptor-mediated apoptosis

Caspase 8 plays an essential role in the execution of death receptor-mediated apoptosis. To determine the localization of endogenous caspase 8, we used a panel of subunit-specific anti-caspase 8 monoclonal antibodies in confocal immunofluorescence microscopy. In the human breast carcinoma cell line MCF7, caspase 8 predominantly colocalized with and bound to mitochondria. After induction of apoptosis through CD95 or tumor necrosis factor receptor I, active caspase 8 translocated to plectin, a major cross-linking protein of the three main cytoplasmic filament systems, whereas the caspase 8 prodomain remained bound to mitochondria. Plectin was quantitatively cleaved by caspase 8 at Asp 2395 in the center of the molecule in all cells tested. Cleavage of plectin clearly preceded that of other caspase substrates such as poly(ADP-ribose) polymerase, gelsolin, cytokeratins, or lamin B. In primary fibroblasts from plectin-deficient mice, apoptosis-induced reorganization of the actin cytoskeleton, as seen in wild-type cells, was severely impaired, suggesting that during apoptosis, plectin is required for the reorganization of the microfilament system.     

植物微丝骨架动态变化的调节

由球形肌动蛋白聚合而成的微丝骨架,又称肌动蛋白纤维,它在细胞运动、细胞形态建成以及物质运输等诸多生命活动中发挥重要作用。细胞内微丝的解聚和聚合动态特性是微丝骨架行使功能的重要基础,并受到如微丝结合蛋白、金属离子、小G蛋白等各种因素的严格控制。植物细胞微丝骨架的研究虽然晚于动物细胞,但也取得了飞速发展。本文对植物细胞内微丝骨架动态变化的作用机制及一些主要调节因子的最新研究进展做一介绍。     

Patterning of the membrane cytoskeleton by the extracellular matrix

The extracellular matrices of different tissues contain components which affect the migration, morphology and differentiation of many types of cells. These forms of cell behavior often involve dramatic changes in cytoskeletal organization. Extracellular matrix components are recognized by specific cell surface receptors which span the membrane and interact with the actin cytoskeleton. In cultured cells, the matrix receptors are concentrated in sites of cell attachment called focal adhesions. Information that is conveyed from the extracellular matrix to the cytoskeleton may involve matrix components, cell surface receptors, as well as the proteins at the cytoplasmic face of the focal adhesion which link the receptors to the actin cytoskeleton.     

Function and interactions of integrins

Integrins are heterodimeric cell adhesion molecules that link the extracellular matrix to the cytoskeleton. The integrin family in man comprises 24 members, which are the result of different combinations of 1 of 18 alpha- and 1 of 8 beta-subunits. Alternative splicing of mRNA of some alpha- and beta-subunits and postranslational modifications of integrin subunits further increase the diversity of the integrin family. In their capacity as adhesion receptors that organize the cytoskeleton, integrins play an important role in controlling various steps in the signaling pathways that regulate processes as diverse as proliferation, differentiation, apoptosis, and cell migration. The intracellular signals that lead to these effects may be transduced via cytoplasmic components, which have been identified as integrin-binding proteins in yeast two-hybrid screens and which could mediate the coupling of integrins to intracellular signaling pathways. In this review an overview is given of the function and ligand-binding properties of integrins as well as of proteins that associate with integrins and may play a role in their signaling function.     

LIM kinase 1 modulates cortical actin and CXCR4 cycling and is activated by HIV-1 to initiate viral infection

Almost all viral pathogens utilize a cytoskeleton for their entry and intracellular transport. In HIV-1 infection, binding of the virus to blood resting CD4 T cells initiates a temporal course of cortical actin polymerization and depolymerization, a process mimicking the chemotactic response initiated from chemokine receptors. The actin depolymerization has been suggested to promote viral intracellular migration through cofilin-mediated actin treadmilling. However, the role of the virus-mediated actin polymerization in HIV infection is unknown, and the signaling molecules involved remain unidentified. Here we describe a pathogenic mechanism for triggering early actin polymerization through HIV-1 envelope-mediated transient activation of the LIM domain kinase (LIMK), a protein that phosphorylates cofilin. We demonstrate that HIV-mediated LIMK activation is through gp120-triggered transient activation of the Rack-PAK-LIMK pathway, and that knockdown of LIMK through siRNA decreases filamentous actin, increases CXCR4 trafficking, and diminishes viral DNA synthesis. These results suggest that HIV-mediated early actin polymerization may directly regulate the CXCR4 receptor during viral entry and is involved in viral DNA synthesis. Furthermore, we also demonstrate that in resting CD4 T cells, actin polymerization can be triggered through transient treatment with a pharmacological agent, okadaic acid, that activates LIMK and promotes HIV latent infection of resting CD4 T cells. Taken together, our results suggest that HIV hijacks LIMK to control the cortical actin dynamics for the initiation of viral infection of CD4 T cells.     

Embryogenesis in <Emphasis Type="Italic">Sedum acre</Emphasis> L.: structural and immunocytochemical aspects of suspensor development

The changes in the formation of both the actin and the microtubular cytoskeleton during the differentiation of the embryo-suspensor in Sedum acre were studied in comparison with the development of the embryo-proper. The presence and distribution of the cytoskeletal elements were examined ultrastructurally and with the light microscope using immunolabelling and rhodamine-phalloidin staining. At the globular stage of embryo development extensive array of actin filaments is present in the cytoplasm of basal cell, the microfilament bundles generally run parallel to the long axis of basal cell and pass in close to the nucleus. Microtubules form irregular bundles in the cytoplasm of the basal cell. A strongly fluorescent densely packed microtubules are present in the cytoplasmic layer adjacent to the wall separating the basal cell from the first layer of the chalazal suspensor cells. At the heart-stage of embryo development, in the basal cell, extremely dense arrays of actin materials are located near the micropylar and chalazal end of the cell. At this stage of basal cell formation, numerous actin filaments congregate around the nucleus. In the fully differentiated basal cell and micropylar haustorium, the tubulin cytoskeleton forms a dense prominent network composed of numerous cross-linked filaments. In the distal region of the basal cell, a distinct microtubular cytoskeleton with numerous microtubules is observed in the cytoplasmic layer adjacent to the wall, separating the basal cell from the first layer of the chalazal suspensor cells. The role of cytoskeleton during the development of the suspensor in S. acre is discussed.     

Microfilament disruption in a noncycling organized tissue, the corneal endothelium, initiates mitosis

The adult corneal endothelium represents a noncycling cell population that resides as a monolayer on its basement membrane, Descemet's membrane. Evidence is presented for the first time, showing that mitotic regulation in this organized tissue, residing on its natural basement membrane, is coupled to microfilament integrity. When mitotically quiescent rat corneal endothelia are organ cultured in medium containing serum and cytochalasin B, low levels of mitosis are initiated. Supplementing the culture medium with either insulin or IGF-2 augments this response and results in increased cell density within the tissue monolayer. Fluorescence microscopy of actin using TRITC-conjugated phalloidin revealed that cellular circumferential microfilament bundles appear unaffected by cytochalasin B treatment, whereas the cytoplasmic microfilaments appear to be completely disrupted. These results suggest the possibility that the actin cytoskeleton is involved with the regulation of cell growth in the corneal endothelium.     

Proteomics analysis of differential expression of cellular proteins in response to avian H9N2 virus infection in human cells

We present the first proteomic analysis on the cellular responses to avian influenza virus (H9N2) infection in a human cell line in different time courses in order to search for target proteins for viral pathogenesis/adaptation studies. By using 2-DE coupled with MALDI-TOF MS and nano-ESI-MS/MS, we identified a set of differentially expressed cellular proteins, including cytoplasmic actin, cytokeratin, prohibitin, enoyl-CoA hydratase, peptide-prolyl cis-trans isomerase A (PPIase A), chloride intracellular channel protein 1, pyruvate dehydrogenase E1 component subunit beta, adenine phosphoribosyltransferase, guanine nucleotide-binding protein subunit beta, nucleoside diphosphate kinase A, elongation factor 1-beta and splicing factor, arginine/serine rich 1. The most significant changes in different time courses were found in cytoplasmic actin and cytokeratin, both of which constituted the major components of cytoskeleton network in the cells. The obtained data suggested a possible role of the cytoskeleton during avian influenza virus infection of mammalian cells, which might help for better understanding of the dynamics of avian influenza virus and host interaction in mammalian cell setting.