Role of the Pi3k Regulatory Subunit in the Control of Actin Organization and Cell Migration

Cell migration represents an important cellular response that utilizes cytoskeletal reorganization as its driving force. Here, we describe a new signaling cascade linking PDGF receptor stimulation to actin rearrangements and cell migration. We demonstrate that PDGF activates Cdc42 and its downstream effector N-WASP to mediate filopodia formation, actin stress fiber disassembly, and a reduction in focal adhesion complexes. Induction of the Cdc42 pathway is independent of phosphoinositide 3-kinase (PI3K) enzymatic activity, but it is dependent on the p85α regulatory subunit of PI3K. Finally, data are provided showing that activation of this pathway is required for PDGF-induced cell migration on collagen. These observations show the essential role of the PI3K regulatory subunit p85α in controlling PDGF receptor–induced cytoskeletal changes and cell migration, illustrating a novel signaling pathway that links receptor stimulation at the cell membrane with actin dynamics

Estudios citoquímicos sobre los mecanismos de tinción y fluorescencia de estructuras celulares por pironina Y

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura 18-03-198

The centromere protein CENP-B behaves as a microtubule-associated protein

The centromere of mitotic chromosomes organizes the kinetochore, a proteinaceous matrix that interfaces with spindle microtubules at one plane and with the centromeric DNA at the other. Antibodies present in the sera from patients with CREST scleroderma recognize several proteins localized to the centromere. We have studied the ability of the two main human centromere proteins CENP-A (18 kD) and CENP-B (80 kD) to bind tubulin, in order to correlate with one of the putative functional roles in spindle microtubule attachment. CENP-A was partially solubilized from nuclear extracts by high salt treatment and then purified by reverse phase HPLC. CENP-B was obtained by gel electrophoresis and electroelution from nuclear insoluble extracts. CENP-B binds to tubulin while no significant interaction was found for CENP-A. CENP-B binds to the C-terminal region of tubulin, a characteristic similar to that found for other better characterized microtubule-associated proteins.Peer reviewe

Increase in cytoplasmic casein kinase II-type activity accompanies neurite outgrowth after DNA synthesis inhibition in NIA-103 neuroblastoma cells

Whereas cells from most clonal lines derived from the murine neuroblastoma C1300 tumor can be induced to differentiate by serum withdrawal from culture medium, the NIA-103 clonal cell line has been considered unable to extend axon-like processes (neurites). Neurite growth depends on microtubule protein assembly, and although NIA-103 cells have essentially the same amounts of microtubule-associated protein (MAP)-1B and the neuronal-specific class β3-tubulin isoform as other neuroblastoma cell lines, these proteins are not phosphorylated in NIA-103 cells on serum withdrawal. The lack of microtubule protein phosphorylation may be due to the different sorting between the nucleus and the cytoplasm of the casein kinase II-related enzyme that is possibly involved in the modification of microtubule proteins. It is interesting that addition of DNA synthesis inhibitors to serum-starved NIA-103 cell cultures induces an increase in the level of cytosolic casein kinase II, an augmented in situ phosphorylation of MAP-1B, and the extension of neurites. Thus, the level of cytoplasmic casein kinase II appears to be controlled by the growth status of neuroblastoma cells. The shift to an increased cytoplasmic concentration of casein kinase II in nonproliferating, differentiating neuroblastoma cells is consistent with its putative role in the regulation of the cytoskeletal rearrangements underlying neuronal morphogenesis and plasticity.Peer reviewe

Tissue-type plasminogen activator (tPA) is the main plasminogen activator associated with isolated rat nerve growth cones

Different studies in tissue culture have shown the involvement of plasminogen activators (PAs) in nerve growth-cone migration. We have studied PA activity associated with isolated rat brain growth cones. Fibrin-agarose zymographies show that tissue-type PA (tPA) is the main PA associated with these structures. After fractionation of growth cones, a slightly higher tPA activity was found associated with the particulate fraction. The present findings support the requirement of this protease for neurite growth.This work was supported by Spanish CICYT and Fundacion Ramon Areces.Peer reviewe

A 205 kDa protein from non-neuronal cells in culture contains tubulin binding epitopes

Microtubule-associated proteins (MAPs) interact with tubulin in vitro and in vivo. Despite that there is a large amount of information on the roles of these proteins in neurons, the data on non-neuronal MAPs or MAPs-related proteins is scarce. There is an increasing number of microtubule-interacting proteins that have been identified in different cultured cell lines, and some of them share common functional epitopes with the most well-known MAPs, MAP-2 and tau. In a search for tubulin-interacting proteins in non-neuronal cells we identified a 205 kDa protein in the monkey kidney Vero cells in culture, on the basis of immunological studies and affinity chromatography. This protein interacts with the C-terminal moiety of β-tubulin and cosediments with taxol assembled microtubules, but it was not recovered after successive cycles of assembly and disassembly. The presence of neuronal MAPs such as MAP-1, MAP-2 and tau was not detected in these cells. Interestingly, the studies showed that

Distribution and characteristics of βII tubulin-enriched microtubules in interphase cells

We have used a polyclonal antibody (Ab196) that specifically recognizes the βII tubulin isotype to examine the subcellular distribution and properties of microtubules enriched in this isotype. Antibody specificity was tested by a method that involves the analysis of its interaction with individual β isotypes. Using photoimaging analysis, we observed βII tubulin-enriched microtubules in the perinuclear region, as well as in the microtubules close to the periphery of interphase cells. The observed sorting of βII-enriched microtubules together with the reported increased levels of βII tubulin in taxol-resistant cells (M. Haberet al.,1995,J. Biol. Chem.270, 31269–31275) prompted us to study the behavior of microtubules enriched in this isotype after different depolymerizing treatments. After cold or nocodazol treatments, βII-enriched microtubules anchored at the centrosome and at the cell periphery were observed. In addition, cold-resistant microtubules were marked mainly by the specific anti-βII tubulin antibody but not by anti-acetylated α tubulin, suggesting the presence of different stable microtubule subsets enriched in particular tubulin isoforms.This work was supported by the Spanish CICYT, a European Union Grant, and an institutional grant from the Fundacion Ramon Areces. The Department of Immunology and Oncology was foundedand is supported by the CSIC and Pharmacia and Upjohn. M.A.P. was supported by an AECI-ICI fellowshipPeer reviewe

A Single Amino Acid Substitution in the Capsid of Foot-and-Mouth Disease Virus Can Increase Acid Lability and Confer Resistance to Acid-Dependent Uncoating Inhibition▿ ¶

The acid-dependent disassembly of foot-and-mouth disease virus (FMDV) is required for viral RNA release from endosomes to initiate replication. Although the FMDV capsid disassembles at acid pH, mutants escaping inhibition by NH4Cl of endosomal acidification were found to constitute about 10% of the viruses recovered from BHK-21 cells infected with FMDV C-S8c1. For three of these mutants, the degree of NH4Cl resistance correlated with the sensitivity of the virion to acid-induced inactivation of its infectivity. Capsid sequencing revealed the presence in each of these mutants of a different amino acid substitution (VP3 A123T, VP3 A118V, and VP2 D106G) that affected a highly conserved residue among FMDVs located close to the capsid interpentameric interfaces. These residues may be involved in the modulation of the acid-induced dissociation of the FMDV capsid. The substitution VP3 A118V present in mutant c2 was sufficient to confer full resistance to NH4Cl and concanamycin A (a V-ATPase inhibitor that blocks endosomal acidification) as well as to increase the acid sensitivity of the virion to an extent similar to that exhibited by mutant c2 relative to the sensitivity of the parental virus C-S8c1. In addition, the increased propensity to dissociation into pentameric subunits of virions bearing substitution VP3 A118V indicates that this replacement also facilitates the dissociation of the FMDV capsid

Productive entry of type C foot-and-mouth disease virus into susceptible cultured cells requires clathrin and is dependent on the presence of plasma membrane cholesterol

AbstractWe have characterized the entry leading to productive infection of a type C FMDV in two cell lines widely used for virus growth, BHK-21 and IBRS-2. Inhibition of clathrin-mediated endocytosis by sucrose treatment decreased both cell entry and virus multiplication. Evidence of a direct requirement of clathrin for productive viral entry was obtained using BHK21-tTA/anti-CHC cells, which showed a significant reduction of viral entry and infection when the synthesis and functionality of clathrin heavy chain was inhibited (Tet− cells). This was also observed for vesicular stomatitis virus (VSV) productive entry. The effect of NH4Cl and concanamycin A on FMDV entry and infection was consistent with the requirement of acidic compartments for decapsidation and virus replication. As expected from its higher stability at acidic pH, this requirement was higher for VSV. Since BHK-21 and IBRS-2 cells expressed caveolin-1, we explored the effect on productive virus entry of drugs that interfere with caveolae-mediated endocytosis. Treatment with nystatin did not reduce entry and infection of FMDV or VSV, while cholesterol depletion with MβCD significantly inhibited both steps of the FMDV cycle, indicating that plasma membrane cholesterol is required for virus productive entry

Internalization of Swine Vesicular Disease Virus into Cultured Cells: a Comparative Study with Foot-and-Mouth Disease Virus▿ †

We performed a comparative analysis of the internalization mechanisms used by three viruses causing important vesicular diseases in animals. Swine vesicular disease virus (SVDV) internalization was inhibited by treatments that affected clathrin-mediated endocytosis and required traffic through an endosomal compartment. SVDV particles were found in clathrin-coated pits by electron microscopy and colocalized with markers of early endosomes by confocal microscopy. SVDV infectivity was significantly inhibited by drugs that raised endosomal pH. When compared to foot-and-mouth disease virus (FMDV), which uses clathrin-mediated endocytosis, the early step of SVDV was dependent on the integrity of microtubules. SVDV-productive endocytosis was more sensitive to plasma membrane cholesterol extraction than that of FMDV, and differential cell signaling requirements for virus infection were also found. Vesicular stomatitis virus, a model virus internalized by clathrin-mediated endocytosis, was included as a control of drug treatments. These results suggest that different clathrin-mediated routes are responsible for the internalization of these viruses