Physiologically induced restructuring of focal adhesions causes mobilization of vinculin by a vesicular endocytic recycling pathway

AbstractIn epithelial cells, vinculin is enriched in cell adhesion structures but is in equilibrium with a large cytosolic pool. It is accepted that when cells adhere to the extracellular matrix, a part of the soluble cytosolic pool of vinculin is recruited to specialized sites on the plasma membrane called focal adhesions (FAs) by binding to plasma membrane phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2). We have previously shown that bradykinin (BK) induces both a reversible dissipation of vinculin from FAs, by the phospholipase C (PLC)-mediated hydrolysis of PtdIns(4,5)P2, and the concomitant internalization of vinculin. Here, by using an immunomagnetic method, we isolated vinculin-containing vesicles induced by BK stimulation. By analyzing the presence of proteins involved in vesicle traffic, we suggest that vinculin can be delivered in the site of FA reassembly by a vesicular endocytic recycling pathway. We also observed the formation of vesicle-like structures containing vinculin in the cytosol of cells treated with lipid membrane-affecting agents, which caused dissipation of FAs due to their deleterious effect on membrane microdomains where FAs are inserted. However, these vesicles did not contain markers of the recycling endosomal compartment. Vinculin localization in vesicles has not been reported before, and this finding challenges the prevailing model of vinculin distribution in the cytosol. We conclude that the endocytic recycling pathway of vinculin could represent a physiological mechanism to reuse the internalized vinculin to reassembly new FAs, which occurs after long time of BK stimulation, but not after treatment with membrane-affecting agents

Esfingosina 1 fosfato como regulador de la dínamica de lipid droplets

Los efingolípidos son lípidos complejos que participan en variados procesos celulares. En el laboratorio hemos descripto la participación de estos en la diferenciación celular del túbulo colector renal. Mas aun, también actúan en fenómenos a nivel tisular modulando su arquitectura, al desencadenar procesos que regulan su homeostasis, como lo es la extrusión celular.Fil: Santacreu, Bruno J.. Consejo Nacional de Investigaciones Científicas y TécnicasFil: Romero, Daniela J.. Consejo Nacional de Investigaciones Científicas y TécnicasFil: Tarallo, Estefania. Consejo Nacional de Investigaciones Científicas y TécnicasFil: Sterin de Speziale, Norma. Consejo Nacional de Investigaciones Científicas y TécnicasFil: Favale, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnica

La coordinación entre esfingolípidos y fosfoinosítidos es esencial para la diferenciación de las células tubulares renales

La diferenciación de las células epiteliales se produce por maduración de la membrana apical y desarrollo de cilio primario. Resultados previos de nuestro laboratorio demostraron que la hipertonicidad extracelular induce la diferenciación de las células MDCK, una línea celular derivada de túbulos colectores renales caninos, y que en este proceso son esenciales los esfingolípidos. Por otro lado, los fosfoinosítidos, otros lípidos bioactivos, son considerados determinantes de la polaridad apico - basal, dada su ubicación en dominios específicos de membrana en células epiteliales polarizadas: con PI(4,5)P2 localizado en el dominio apical y PI(3,4,5)P3 en el basolateral. Esta ubicación específica es la que determina el reclutamiento de complejos importantes de polaridad, como el Par3/Par6/aPKC.Fil: Pescio Lucila G.. Universidad de Buenos AiresFil: Romero, Daniela J.. Universidad de Buenos AiresFil: Santacreu, Bruno J.. Universidad de Buenos AiresFil: Francisco, María N..Fil: Favale, Nicolás O.. Universidad de Buenos AiresFil: Sterin-Speziale, Norma B.. Universidad de Buenos Aire

Novel cellular mechanism that mediates the collecting duct formation during postnatal renal development

We have previously demonstrated that kidney embryonic structures are present in rats, and are still developing until postnatal Day 20. Consequently, at postnatal Day 10, the rat renal papilla contains newly formed collecting duct (CD) cells and others in a more mature stage. Performing primary cultures, combined with immunocytochemical and time-lapse analysis, we investigate the cellular mechanisms that mediate the postnatal CD formation. CD cells acquired a greater degree of differentiation, as we observed that they gradually lose the ability to bind BSL-I lectin, and acquire the capacity to bind Dolichos biflorus. Because CD cells retain the same behavior in culture than in vivo, and by using DBA and BSL-I as markers of cellular lineage besides specific markers of epithelial/mesenchymal phenotype, the experimental results strongly suggest the existence of mesenchymal cell insertion into the epithelial CD sheet. We propose such a mechanism as an alternative strategy for CD growing and development.Fil: Guaytima, Edith del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Rioja. Departamento de Ciencias de la Salud y Educación. Instituto de Investigaciones en Ciencias de la Salud Humana; ArgentinaFil: Brandán, Yamila Romina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Rioja. Departamento de Ciencias de la Salud y Educación. Instituto de Investigaciones en Ciencias de la Salud Humana; ArgentinaFil: Favale, Nicolas Octavio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Sterin Speziale, Norma B.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Márquez, María Gabriela. Universidad Nacional de La Rioja. Departamento de Ciencias de la Salud y Educación. Instituto de Investigaciones en Ciencias de la Salud Humana; Argentin

The Expression of Sphingosine Kinase-1 in Head and Neck Carcinoma

Sphingosine kinase-1 (SPHK1) modulates the proliferation, apoptosis and differentiation of keratinocytes through the regulation of ceramide and sphingosine-1-phosphate levels. However, studies on the expression of SPHK1 in human head and neck squamous cell carcinoma (HNSCC) specimens are lacking. Therefore, the aim of the present work was to evaluate SPHK1 expression in human primary HNSCCs and to correlate the results with clinical and anatomopathological parameters. We investigated the expression of this protein by immunohistochemistry performed in tissue microarrays of HNSCC and in an independent cohort of 37 paraffin-embedded specimens. SPHK1 expression was further validated by real-time PCR performed on laser capture-microdissected tissue samples. The positive rate of SPHK1 protein in the cancerous tissues was significantly higher (74%) than that in the nontumor oral tissues (23%), and malignant tissues showed stronger immunoreactivity for SPHK1 than normal matching samples. These results were confirmed by real-time PCR quantification of SPHK1 mRNA. Interestingly, the positive expression of SPHK1 was associated with shorter patient survival time (Kaplan-Meier survival curves) and with the loss of p21 expression. Taken together, these results demonstrate that SPHK1 is upregulated in HNSCC and provide clues of the role SPHK1 might play in tumor progression

Tyrosine isomers mediate the classical phenomenon of concomitant tumor resistance

Concomitant tumor resistance (CR) is a phenomenon originally described in 1906 in which a tumor-bearing host is resistant to the growth of secondary tumor implants and metastasis. Although recent studies have indicated that T-cell-dependent processes mediate CR in hosts bearing immunogenic small tumors, manifestations of CR induced by immunogenic and nonimmunogenic large tumors have been associated with an elusive serum factor. In this study, we identify this serum factor as tyrosine in its meta and ortho isoforms. In three different murine models of cancer that generate CR, both meta-tyrosine and ortho-tyrosine inhibited tumor growth. In addition, we showed that both isoforms of tyrosine blocked metastasis in a fourth model that does not generate CR but is sensitive to CR induced by other tumors. Mechanistic studies showed that the antitumor effects of the tyrosine isoforms were mediated, in part, by early inhibition of mitogen-activated protein/extracellular signal-regulated kinase pathway and inactivation of STAT3, potentially driving tumor cells into a state of dormancy. By revealing a molecular basis for the classical phenomenon of CR, our findings may stimulate new generalized approaches to limit the development of metastases that arise after resection of primary tumors, an issue of pivotal importance to oncologists and their patients. ©2011 AACR.Fil: Ruggiero, Raul Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Bruzzo Iraola, Juan. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Chiarella, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Di Gianni, Pedro. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Isturiz, Martín Amadeo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Linskens, Susana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Speziale, Norma. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Meiss, Roberto P.. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Bustuoabad, Oscar David. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Pasqualini, Christiane D.. Academia Nacional de Medicina de Buenos Aires; Argentin