Drosophila singed, a fascin homolog, is required for actin bundle formation during oogenesis and bristle extension

Drosophila singed mutants were named for their gnarled bristle phenotype but severe alleles are also female sterile. Recently, singed protein was shown to have 35% peptide identity with echinoderm fascin. Fascin is found in actin filament bundles in microvilli of sea urchin eggs and in filopodial extensions in coelomocytes. We show that Drosophila singed is required for actin filament bundle formation in the cytoplasm of nurse cells during oogenesis; in severe mutants, the absence of cytoplasmic actin filament bundles allows nurse cell nuclei to lodge in ring canals and block nurse cell cytoplasm transport. Singed is also required for organized actin filament bundle formation in the cellular extension that forms a bristle; in severe mutants, the small disorganized actin filament bundles lack structural integrity and allow bristles to bend and branch during extension. Singed protein is also expressed in migratory cells of the developing egg chamber and in the socket cell of the developing bristle, but no defect is observed in these cells in singed mutants. Purified, bacterially expressed singed protein bundles actin filaments in vitro with the same stoichiometry reported for purified sea urchin fascin. Singed-saturated actin bundles have a molar ratio of singed/actin of approximately 1:4.3 and a transverse cross-banding pattern of 12 nm seen using electron microscopy. Our results suggest that singed protein is required for actin filament bundle formation and is a Drosophila homolog of echinoderm fascin.     

Dissection of pathways implicated in integrin-mediated actin cytoskeleton assembly. Involvement of protein kinase C, Rho GTPase, and tyrosine phosphorylation

A panel of antibodies to the alphaIIbbeta3 integrin was used to promote adhesion of Chinese hamster ovary cells transfected with the alphaIIbbeta3 fibrinogen receptor. While some alphaIIbbeta3 antibodies were not able to induce p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation, all the antibodies equally support cell adhesion but not spreading and assembly of actin stress fibers. Absence of stress fibers was also obtained by plating on antibodies directed to the hamster beta1 integrin. In contrast, cells plated on matrix proteins spread organizing actin stress fibers. Treatment with phorbol esters phorbol 12-myristate 13-acetate (PMA) induced cells to spread on antibodies-coated dishes but not to organize actin in stress fibers. The combination of PMA and cytotoxic necrotizing factor 1 (CNF1), a specific Rho activator, induced cell spreading and organization of stress fibers. PMA or the combination of PMA and CNF1 also increases tyrosine phosphorylation of p125FAK in response to antibodies that were otherwise unable to trigger this response. These data show that: 1) matrix proteins and antibodies differ in their ability to induce integrin-dependent actin cytoskeleton organization (while matrix induced stress fibers formation, antibodies did not); 2) p125FAK tyrosine phosphorylation is insufficient per se to trigger actin stress fibers formation since antibodies that activate p125FAK tyrosine phosphorylation did not lead to actin stress fibers assembly; and 3) the inability of anti-integrin antibodies to trigger stress fibers organization is overcome by concomitant activation of the protein kinase C (PKC) and Rho pathways; PKC activation leads to cell spreading and Rho activation is required to organize actin stress fibers.     

The actin-bundling protein fascin is involved in the formation of dendritic processes in maturing epidermal Langerhans cells

Dendritic cells (DC) are characterized by their unique potential to prime naive T cells. Epidermal Langerhans cells (LC), the DC resident in the epidermis, gain this immunostimulatory capacity following Ag contact in vivo or during in vitro culture of epidermal cell suspensions. To analyze differential gene expression in maturing LC, we constructed a highly representative cDNA library of cultivated LC (cLC) in lambda ZAP II containing 18 x 10(6) independent clones. This library was screened with freshly isolated Langerhans cell (fLC)- and cLC-derived probes for cLC-specific cDNAs. The cDNAs identified were sequenced and analyzed by database searches. Two cDNA fragments were identified as fragments of fascin, indicating that fascin is differentially expressed in LC. By competitive RT-PCR, we confirmed that fascin is highly expressed in cLC cultivated for 1, 2, and 3 days, while no signals were obtained with fLC. Western blot and immunofluorescence analysis revealed cLC-specific expression of fascin on the protein level as well. Fascin is known to be involved in the organization of the actin cytoskeleton in cytoplasmatic extensions of nerve growth cones. Its differential expression in maturing LC coincides with the formation of numerous dendritic projections in LC. Their formation was inhibited by incubation of LC with fascin antisense oligonucleotides during cultivation. Therefore, we conclude that fascin is necessary for the formation of the dendritic processes of maturing Langerhans cells and may thus influence T cell-LC interaction.     

Tyrosine phosphorylation of p130Cas is involved in actin organization in osteoclasts

Integrin-mediated interaction with the extracellular matrix plays a critical role in the function of osteoclasts, the bone-resorbing cells. This study examines the role of p130Cas (Crk-associated substrate (Cas)) in actin organization in osteoclasts. Multinucleated osteoclast-like cells (OCLs) were obtained in a co-culture of murine bone marrow cells and primary osteoblasts. After plating on culture dishes, OCLs formed a ringlike structure consisting of F-actin dots at cell periphery (actin ring). The percentage of OCLs with actin rings and its diameter increased with time and cell spreading. Tyrosine phosphorylation of a protein (p130) increased with actin ring formation. Treatment with cytochalasin D disrupted actin rings and reduced tyrosine phosphorylation of p130. Using specific antibodies, p130 was identified as Cas. By immunocytochemistry, Cas was localized to the peripheral regions of OCLs and its distribution overlapped that of F-actin. In OCLs derived from Src(-/-) mice, in which osteoclast activity is severely compromised, tyrosine phosphorylation of Cas was markedly reduced. Moreover, Cas was diffusely distributed in the cytoplasm and actin ring formation is not observed. These findings suggest that Src-dependent tyrosine phosphorylation of Cas is involved in the adhesion-induced actin organization associated with osteoclast activation.     

Activation of Rac and Cdc42 by integrins mediates cell spreading

Adhesion to ECM is required for many cell functions including cytoskeletal organization, migration, and proliferation. We observed that when cells first adhere to extracellular matrix, they spread rapidly by extending filopodia-like projections and lamellipodia. These structures are similar to the Rac- and Cdc42-dependent structures observed in growth factor-stimulated cells. We therefore investigated the involvement of Rac and Cdc42 in adhesion and spreading on the ECM protein fibronectin. We found that integrin-dependent adhesion led to the rapid activation of p21-activated kinase, a downstream effector of Cdc42 and Rac, suggesting that integrins activate at least one of these GTPases. Dominant negative mutants of Rac and Cdc42 inhibit cell spreading in such a way as to suggest that integrins activate Cdc42, which leads to the subsequent activation of Rac; both GTPases then contribute to cell spreading. These results demonstrate that initial integrin-dependent activation of Rac and Cdc42 mediates cell spreading.     

Growth behavior of fibroblasts on microgrooved polystyrene

We investigated the contact guidance phenomenon of rat dermal fibroblasts (RDF) on microgrooved polystyrene substrates. Grooves were 1 microm deep, and between 1 and 10 microm wide. Light microscopy and digital image analysis (DIA) showed that RDF were oriented on all microgrooved substrates. Scanning electron microscopy showed that RDF cultured on 1 or 2 microm wide grooves were positioned on top of the ridges. On the wider 5 and 10 microm grooves, the cells were able to descend into the grooves. In confocal laser scanning microscopy, focal adhesions were lying in the same direction as the actin filament where they attached to. DIA confirmed an orientational behavior of focal adhesions and actin filaments on microgrooves. There were no differences in the measured orientation between the different grooves. Besides, no obvious preference was found for focal adhesions to lie along edges of the surface ridges. Transmission electron microscopy showed that focal adhesions were able to bend along the edges of ridges. On the basis of our observations, we suggest that the breakdown and formation of fibrous cellular components, especially in the filopodium, is influenced by the microgrooves. The microgrooves create a pattern of mechanical stress, which influences cell spreading and cause the cell to be aligned with surface microgrooves.     

Integrin-mediated entry into S phase of human gastric adenocarcinoma cells

The integrins are a family of integral membrane receptors that participate in binding to various extracellular and cell surface proteins during adhesion, migration, and homing of normal and neoplastic cells. In this study, we characterized the involvement of integrins in mediating the growth of an adhesion-dependent gastric adenocarcinoma line, ST2. This line was distinguished and selected for study based on its inability to grow when suspended in soft agar or plated on poly(2-hydroxyethyl methacrylate)-coated dishes. ST2 cells arrested in G0/G1 of the cell cycle when deprived of adhesion to substrate. Using purified matrix components, collagen was found to be highly active in promoting beta 1 integrin-mediated cell attachment and spreading. Subsequent to spreading on collagen, the cells were released from G0/G1 block and progressed into S phase. Monoclonal antibodies to alpha 2 or beta 1 integrin blocked the reinduction of both cell spreading and entry into S phase. These studies suggest that during the metastatic process, integrin receptor interaction with the insoluble matrix may be an important step leading to proliferation of some tumors.     

Single amino acid mutations in Drosophila fascin disrupt actin bundling function in vivo

Fascins bundle actin filaments into large, tightly packed hexagonal arrays that support diverse cellular processes including microvillar projections and filopodial extensions. In Drosophila, fascin is encoded by the singed locus. Severe singed mutants have gnarled bristles and are female sterile due to a defect in rapid cytoplasm transport during oogenesis. In this paper, we report the results of a large EMS mutagenesis screen to generate new singed alleles. A mutation that changes glycine 409 to glutamic acid results in partial inactivation of fascin in vivo; singedG409E mutants have kinked bristles and are fertile with a mild nurse cell cytoplasm transport defect. This mutation is in a small conserved domain near the C-terminus of fascin. A mutation that changes serine 289 to asparagine almost completely inactivates fascin in vivo; singedS289N mutants have gnarled bristles and are sterile due to a severe defect in nurse cell cytoplasm transport caused by the absence of nurse cell cytoplasmic actin bundles. A subsequent EMS mutagenesis screen for dominant suppressors of singedS289N sterility revealed an intragenic suppressor mutation that changes serine 251 to phenylalanine and restores much of fascin's function. These two mutations, S289N and S251F, draw attention to a central domain in fascin.     

Extracellular calcium regulates HeLa cell morphology during adhesion to gelatin: role of translocation and phosphorylation of cytosolic phospholipase A2

Attachment of HeLa cells to gelatin induces the release of arachidonic acid (AA), which is essential for cell spreading. HeLa cells spreading in the presence of extracellular Ca2+ released more AA and formed more distinctive lamellipodia and filopodia than cells spreading in the absence of Ca2+. Addition of exogenous AA to cells spreading in the absence of extracellular Ca2+ restored the formation of lamellipodia and filopodia. To investigate the role of cytosolic phospholipase A2 (cPLA2) in regulating the differential release of AA and subsequent formation of lamellipodia and filopodia during HeLa cell adhesion, cPLA2 phosphorylation and translocation from the cytosol to the membrane were evaluated. During HeLa cell attachment and spreading in the presence of Ca2+, all cPLA2 became phosphorylated within 2 min, which is the earliest time cell attachment could be measured. In the absence of extracellular Ca2+, the time for complete cPLA2 phosphorylation was lengthened to <4 min. Maximal translocation of cPLA2 from cytosol to membrane during adhesion of cells to gelatin was similar in the presence or absence of extracellular Ca2+ and remained membrane associated throughout the duration of cell spreading. The amount of total cellular cPLA2 translocated to the membrane in the presence of extracellular Ca2+ went from <20% for unspread cells to >95% for spread cells. In the absence of Ca2+ only 55-65% of the total cPLA2 was translocated to the membrane during cell spreading. The decrease in the amount translocated could account for the comparable decrease in the amount of AA released by cells during spreading without extracellular Ca2+. Although translocation of cPLA2 from cytosol to membrane was Ca2+ dependent, phosphorylation of cPLA2 was attachment dependent and could occur both on the membrane and in the cytosol. To elucidate potential activators of cPLA2, the extracellular signal-related protein kinase 2 (ERK2) and protein kinase C (PKC) were investigated. ERK2 underwent a rapid phosphorylation upon early attachment followed by a dephosphorylation. Both rates were enhanced during cell spreading in the presence of extracellular Ca2+. Treatment of cells with the ERK kinase inhibitor PD98059 completely inhibited the attachment-dependent ERK2 phosphorylation but did not inhibit cell spreading, cPLA2 phosphorylation, translocation, or AA release. Activation of PKC by phorbol ester (12-O-tetradecanoylphorbol-13-acetate) induced and attachment-dependent phosphorylation of both cPLA2 and ERK2 in suspension cells. However, in cells treated with the PKC inhibitor Calphostin C before attachment, ERK2 phosphorylation was inhibited, whereas cPLA2 translocation and phosphorylation remained unaffected. In conclusion, although cPLA2-mediated release of AA during HeLa cell attachment to a gelatin substrate was essential for cell spreading, neither ERK2 nor PKC appeared to be responsible for the attachment-induced cPLA2 phosphorylation and the release of AA.     

The integrin alpha6beta4 functions in carcinoma cell migration on laminin-1 by mediating the formation and stabilization of actin-containing motility structures

Functional studies on the alpha6beta4 integrin have focused primarily on its role in the organization of hemidesmosomes, stable adhesive structures that associate with the intermediate filament cytoskeleton. In this study, we examined the function of the alpha6beta4 integrin in clone A cells, a colon carcinoma cell line that expresses alpha6beta4 but no alpha6beta1 integrin and exhibits dynamic adhesion and motility on laminin-1. Time-lapse videomicroscopy of clone A cells on laminin-1 revealed that their migration is characterized by filopodial extension and stabilization followed by lamellae that extend in the direction of stabilized filopodia. A function-blocking mAb specific for the alpha6beta4 integrin inhibited clone A migration on laminin-1. This mAb also inhibited filopodial formation and stabilization and lamella formation. Indirect immunofluorescence microscopy revealed that the alpha6beta4 integrin is localized as discrete clusters in filopodia, lamellae, and retraction fibers. Although beta1 integrins were also localized in the same structures, a spatial separation of these two integrin populations was evident. In filopodia and lamellae, a striking colocalization of the alpha6beta4 integrin and F-actin was seen. An association between alpha6beta4 and F-actin is supported by the fact that alpha6beta4 integrin and actin were released from clone A cells by treatment with the F-actin- severing protein gelsolin and that alpha6beta4 immunostaining at the marginal edges of clone A cells on laminin-1 was resistant to solubilization with Triton X-100. Cytokeratins were not observed in filopodia and lamellipodia. Moreover, alpha6beta4 was extracted from these marginal edges with a Tween-40/deoxycholate buffer that solubilizes the actin cytoskeleton but not cytokeratins. Three other carcinoma cell lines (MIP-101, CCL-228, and MDA-MB-231) exhibited alpha6beta4 colocalized with actin in filopodia and lamellae. Formation of lamellae in these cells was inhibited with an alpha6-specific antibody. Together, these results indicate that the alpha6beta4 integrin functions in carcinoma migration on laminin-1 through its ability to promote the formation and stabilization of actin-containing motility structures.     

Identification of a novel integrin signaling pathway involving the kinase Syk and the guanine nucleotide exchange factor Vav1

BACKGROUND:. Integrins induce the formation of large complexes of cytoskeletal and signaling proteins, which regulate many intracellular processes. The activation and assembly of signaling complexes involving focal adhesion kinase (FAK) occurs late in integrin signaling, downstream from actin polymerization. Our previous studies indicated that integrin-mediated activation of the non-receptor tyrosine kinase Syk in hematopoietic cells is independent of FAK and actin polymerization, and suggested the existence of a distinct signaling pathway regulated by Syk. RESULTS:. Multiple proteins were found to be activated by Syk, downstream of engagement of the platelet/megakaryocyte-specific integrin alphaIIbbeta3. The guanine nucleotide exchange factor Vav1 was inducibly phosphorylated in a Syk-dependent manner in cells following their attachment to fibrinogen. Together, Syk and Vav1 triggered lamellipodia formation in fibrinogen-adherent cells and both Syk and Vav1 colocalized with alphaIIbbeta3 in lamellipodia but not in focal adhesions. Additionally, Syk and Vav1 cooperatively induced activation of Jun N-terminal kinase (JNK), extracellular-signal-regulated kinase 2 (ERK2) and the kinase Akt, and phosphorylation of the oncoprotein Cbl in fibrinogen-adherent cells. Activation of all of these proteins by Syk and Vav1 was not dependent on actin polymerization. CONCLUSIONS:. Syk and Vav1 regulate a unique integrin signaling pathway that differs from the FAK pathway in its proximity to the integrin itself, its localization to lamellipodia, and its activation, which is independent of actin polymerization. This pathway may regulate multiple downstream events in hematopoietic cells, including Rac-induced lamellipodia formation, tyrosine phosphorylation of Cbl, and activation of JNK, ERK2 and the phosphatidylinositol 3'-kinase-regulated kinase Akt.     

Expression of integrin and organization of F-actin in epithelial cells depends on the underlying surface

PURPOSE: To evaluate the role of ionic interactions in the cell surface expression of integrins and the organization of F-actin. Understanding these interactions will allow the development of surfaces for prosthetic purposes that will promote the normal expression of adhesion proteins. METHODS: Hema (hydroxyethylmethacrylate) hydrogels were used to mimic the charges present on extracellular matrix proteins. The surfaces were modified by the addition of amines (N,N-dimethylaminoethylmethacrylate; NDAM) or carboxyl moieties (methacrylic acid). The effects of ionic interactions on cellular spreading and on the expression of proteins were examined by modification of the stoichiometrically defined amounts of positive and negative charges on the Hemas. Changes in intracellular pH and the distribution and localization of protein were monitored using fluorescent markers, spectrofluorometry, and confocal laser scanning microscopy, respectively. The immunohistochemical studies were confirmed by flow cytometric analysis. RESULTS: The data indicate that although cells adhered to all the surfaces, the number of cells possessing adhesion receptors is significantly greater on surfaces with amine functionalities. Cell seeding and plating efficiency after 2 hours were identical on all surfaces. The intracellular pH of epithelial cells grown on surfaces containing NDAM, a tertiary amine, was higher than that of cells grown on Hemas containing only methacrylic acid. Lamellipodial extensions and an extensive actin network were present on surfaces containing 5% NDAM. The alpha 6 subunit was localized along the lateral cell membranes. The alpha 2 and 3 subunits were present along cell membranes and at lamellipodial extensions. Cells cultured on surfaces containing only methacrylic acid did not spread. Actin filaments were not detected, and alpha 6 was negligible on these surfaces. CONCLUSIONS: This is a novel approach to understanding cell-substrate interactions, and one that allows quantitative evaluation of the response of cells to defined surfaces. The organization of F-actin is altered by the substrates containing only carboxyl moieties. The distribution of integrin subunits is also altered by the substrate. These results indicate that epithelial cell spreading and protein expression may be regulated by ionic interactions.     

Rac and Cdc42 induce actin polymerization and G1 cell cycle progression independently of p65PAK and the JNK/SAPK MAP kinase cascade

Rac and Cdc42 regulate a variety of responses in mammalian cells including formation of lamellipodia and filopodia, activation of the JNK MAP kinase cascade, and induction of G1 cell cycle progression. Rac is also one of the downstream targets required for Ras-induced malignant transformation. Rac and Cdc42 containing a Y40C effector site substitution no longer intact with the Ser/Thr kinase p65PAK and are unable to activate the JNK MAP kinase pathway. However, they still induce cytoskeletal changes and G1 cell cycle progression. Rac containing an F37A effector site substitution, on the other hand, no longer interacts with the Ser/Thr kinase p160ROCK and is unable to induce lamellipodia or G1 progression. We conclude that Rac and Cdc42 control MAP kinase pathways and actin cytoskeleton organization independently through distinct downstream targets.     

Evidence that cell surface beta 1,4-galactosyltransferase spontaneously galactosylates an underlying laminin substrate during fibroblast migration

beta 1,4-Galactosyltransferase is unusual among the glycosyltransferases in that a subpopulation exists on the cell surface in addition to its traditional biosynthetic location within the Golgi complex. On the cell surface, galactosyltransferase is expressed in spatially restricted, cell type-specific domains, where it functions as a receptor for extracellular oligosaccharide ligands during selected cellular interactions. For example, galactosyltransferase is found on the leading and trailing edges of migrating cells, where it facilitates lamellipodia formation and cell spreading by binding to specific N-linked oligosaccharides within laminin. Although the ability of galactosyltransferase to serve as a laminin receptor is well documented, it is unclear whether it functions solely in a lectin-like capacity to bind laminin glycoside ligands or uses its intrinsic catalytic activity to release itself from and modify its oligosaccharide substrate. In this study, we determined whether cell surface galactosyltransferase spontaneously galactosylates laminin matrices during cell migration using endogenous galactose donors. Cells were prelabeled with [3H]galactose, washed, and transferred in small clusters onto laminin matrices. The prelabeled cells migrated out from the cell cluster, during which time they deposited covalently bound [3H]galactose residues onto the laminin matrix. The degree of galactosylation was both laminin- and time-dependent and required actively migrating, intact cells. The radioactivity released from the 3H-galactosylated laminin by acid hydrolysis comigrated with authentic galactose standards on paper chromatography. In parallel assays, there was no radioactivity deposited on laminin matrices when cells were prelabeled with [3H]fucose or [3H]leucine. Furthermore, [3H]galactosylation was dependent upon galactosyltransferase-mediated cell migration, since prelabeled cells did not deposit [3H]galactose when migrating on fibronectin, upon which migration is integrin-dependent and galactosyltransferase-independent. These results raise the possibility that galactosyltransferase functions catalytically during cell migration, either to dissociate from its oligosaccharide ligand and/or to modify the extracellular matrix.     

Close linkage of three merozoite surface protein genes on chromosome 2 of Plasmodium falciparum

Actin the main component of the cellular microfilament network, is present in human respiratory syncytial virus (HRSV) purified virions, as an internal component. This fact and the results of immunoprecipitation studies indicate that during HRSV infection in HEp-2 cells there are interactions between cellular actin and viral components, that can promote a transitory increase in the polymerization of synthetized actin, mainly of the beta isotype. This increased actin polymerization can be related with the formation of cytoplasmic extensions, that contain beta actin and viral particles observed in the HRSV infected HEp-2 cells. The formation of these structures may indicate that HRSV has developed an actin-based motility system similar to that described for other viral and bacterial systems.     

Topographic compensation: guidance and directed locomotion of fibroblasts on grooved micromachined substrata in the absence of microtubules

Fibroblasts cultured on grooved substrata align themselves and migrate in the direction of the grooves, a phenomenon called contact guidance. Microtubules have been deemed important for cell polarization, directed locomotion, and contact guidance. Because microtubules were the first cytoskeletal element to align with the grooves when fibroblasts spread on grooved substrata, we investigated the consequences of eliminating the influence of microtubules by seeding fibroblasts onto smooth and grooved micromachined substrata in the presence of colcemid. Fibroblasts were examined by time-lapse cinematography and epifluorescence or confocal microscopy to determine cell shape and orientation and the distribution of cytoskeletal or associated elements including actin filaments, vinculin, intermediate filaments, microtubules, and kinesin. As expected, cells spreading on smooth surfaces in the presence of colcemid did not polarize or locomote. Surprisingly however, by 24 hours, cells spread on grooves in the presence of colcemid were morphologically indistinguishable from controls spread on grooves. Both groups were aligned and polarized with the direction of the grooves and demonstrated directional locomotion along the grooves. In the absence of microtubules, kinesin localized to some of the aligned stress fibers and to leading edges of cells spreading on grooves. The grooved substratum compensated for the microtubule deficiency by organizing and maintaining an aligned actin filament framework. Thus, microtubules are not required to establish or maintain stable, polarized cell shapes or directed locomotion, provided an alternate oriented cytoskeletal component is available.     

Muskelin, a novel intracellular mediator of cell adhesive and cytoskeletal responses to thrombospondin-1

We have used an expression cloning strategy based on a cell-attachment assay screen to seek identification of molecules required in cellular responses to thrombospondin-1, a regulated macromolecular component of extracellular matrix. We report the identification and functional characterization of a novel, widely expressed, intracellular protein, named muskelin, which contains dispersed motifs with homology to the tandem repeats first identified in the Drosophila kelch ORF1 protein. In adherent C2C12 cells, muskelin localizes in the cytoplasm and at cell margins. Over-expression of muskelin in C2C12 cells promotes cell attachment to the thrombospondin-1 C-terminal domain, alters the mechanisms of attachment to intact thrombospondin-1 and correlates with decreased formation of fascin microspikes and increased assembly of focal contacts by cells adherent on thrombospondin-1. Reciprocally, cell attachment, spreading and cytoskeletal organization are specifically reduced in TSP-1-adherent cells after antisense depletion of muskelin. These results establish a requirement for muskelin in cell responses to thrombospondin-1 and demonstrate that such responses involve a novel process which is integrated into the regulation of cell-adhesive behaviour and cytoskeletal organization.     

Virus-induced cell motility

Many viruses induce profound changes in cell metabolism and function. Here we show that vaccinia virus induces two distinct forms of cell movement. Virus-induced cell migration was demonstrated by an in vitro wound healing assay in which infected cells migrated independently into the wound area while uninfected cells remained relatively static. Time-lapse microscopy showed that the maximal rate of migration occurred between 9 and 12 h postinfection. Virus-induced cell migration was inhibited by preinactivation of viral particles with trioxsalen and UV light or by the addition of cycloheximide but not by addition of cytosine arabinoside or rifampin. The expression of early viral genes is therefore necessary and sufficient to induce cell migration. Following migration, infected cells developed projections up to 160 microm in length which had growth-cone-like structures and were frequently branched. Time-lapse video microscopy showed that these projections were formed by extension and condensation of lamellipodia from the cell body. Formation of extensions was dependent on late gene expression but not the production of intracellular enveloped (IEV) particles. The requirements for virus-induced cell migration and for the formation of extensions therefore differ from each other and are distinct from the polymerization of actin tails on IEV particles. These data show that poxviruses encode genes which control different aspects of cell motility and thus represent a useful model system to study and dissect cell movement.     

Actin-dependent anterograde movement of growth-cone-like structures along growing hippocampal axons: a novel form of axonal transport?

In time-lapse video recordings of hippocampal neurons in culture, we have identified previously uncharacterized structures, nicknamed "waves," that exhibit lamellipodial activity closely resembling that of growth cones, but which periodically emerge at the base of axons and travel distally at an average rate of 3 microm/min. In electron micrographs of identified waves, the cortical region of the axon appears expanded to either side, forming lamellipodia like those at growth cones. No other gross differences were noted in the ultrastructural features of the axon shaft at the site of a wave. Immunocytochemistry revealed that waves contain a marked concentration of F-actin, GAP-43, cortactin, and ezrin or a related protein, constituents that are also concentrated in growth cones. Treatment with the actin-disrupting agent cytochalasin B caused a reversible collapse of lamellipodia and cessation of the forward movement of individual waves along the axon, indicating that their anterograde transport is dependent on intact actin filaments. Treatment with the microtubule-depolymerizing agent nocodazole led to a rapid disorganization of wave structure and a subsequent suppression of wave activity that may reflect a role of microtubules in actin organization. The results suggest that actin and other cytoskeletal components concentrated in growth cones may be transported together as growth-cone-like structures from the cell body to the axon tip via an actin-dependent mechanism.     

Embryonic mesoderm cells spread in response to platelet-derived growth factor and signaling by phosphatidylinositol 3-kinase

Abnormal mesoderm movement, leading to defects in axial organization, is observed in mouse and Xenopus laevis embryos deprived of platelet-derived growth factor (PDGF) AA signaling. However, neither the cellular response to PDGF nor the signaling pathways involved are understood. Herein we describe an in vitro assay to examine the direct effect of PDGF AA on aggregates of Xenopus embryonic mesoderm cells. We find that PDGF AA stimulates aggregates to spread on fibronectin. This behavior is similar to that of migrating mesoderm cells in vivo that spread and form lamellipodia and filipodia on contact with fibronectin-rich extracellular matrix. We go on to show two lines of evidence that implicate phosphatidylinositol 3-kinase (PI3K) as an important component of PDGF-induced mesoderm cell spreading. (i) The fungal metabolite wortmannin, which inhibits signaling by PI3K, blocks mesoderm spreading in response to PDGF AA. (ii) Activation of a series of receptors with specific tyrosine-to-phenylalanine mutations revealed PDGF-induced spreading of mesoderm cells depends on PI3K but not on other signaling molecules that interact with PDGF receptors including phospholipase C gamma, Ras GTPase-activating protein, and phosphotyrosine phosphatase SHPTP2. These results indicate that a PDGF signal, medicated by PI3K, can facilitate embryonic mesoderm cell spreading on fibronectin. We propose that PDGF, produced by the ectoderm, influences the adhesive properties of the adjacent mesoderm cells during gastrulation.