Alteration of lipid rafts evokes the formation of non recycling -vinculin containing vesicles

YAMILA ROMINA BRANDÁN; EDITH DEL VALLE GUAYTIMA; MARÍA BELÉN DE LA VEGA PAEZ; NORMA B. STERIN-SPEZIALE; MARÍA GABRIELA MÁRQUEZ

Buenos Aires

Congreso; "Molecular mechanisms in cell signaling and gene expresion"- Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2013

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB)

We reported that bradykinin (BK) induces a recycling of vinculin from focal adhesion (FAs) through the recycling compartment, denoted by the appearance of vinculin/ Rab5/ Rab11/ transferrin receptor positive vesicles in the cytosol. Now, we investigated whether the formation of these vesicles is a BK-specific or a general phenomenon. Since FA complexes are located in specific membrane microdomains (raft), we treated cultured renal papillary collecting duct cells with the lipid membrane-affecting agents cyclodextrin (CD) and neomycin (Neo), which caused dissipation of vinculin-stained FAs due to their deleterious effect on membrane microdomains, where FAs are inserted. After CD and Neo treatment of cultured cells, we observed the presence of vesicle-like structures containing vinculin in the cytosol by immunofluorescence. We treated papillary slices with CD and Neo and isolated vinculin-containing vesicles by an immunomagnetic method. Western blot analysis showed that these vesicles do not contain markers of the recycling endosomal compartment. We conclude that the endocytic recycling pathway of vinculin is not a general phenomenon, and could be a physiological mechanism to reuse the internalized vinculin to reassembly new FA which occur after long time of BK stimulation, but not after CD and Neo treatment.