Characterization of vesicles containing vinculin in rat renal papillary collecting duct cells

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

Ciudad de Mendoza, Mendoza

Congreso; XLVIII Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2012

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

We have showed that bradykinin (BK) induces a dissipation of vinculin-stained focal adhesions (FA) - structures of cell attachment to the extracellular matrix- with formation of vesicles containing vinculin, PIP2, and markers of the recycling endosomal compartment. Now, we performed a biochemical characterization of these vesicles. For the analysis of membrane association properties of vinculin in these vesicles, microsomes isolated from BK-treated renal collecting duct cells were incubated with increasing amounts of KCl, Na2CO3 pH 10, or Triton X-114. To verify the membrane orientation of vinculin, microsomes were subjeted to tripsin digestion with or without Triton X-100. After these treatments, the presence of vinculin was analyzed by Western blot. To study the phospholipid composition, vinculin containing vesicles isolated by immunomagnetic method were analysis by thin layer chromatography (TLC). Most of the vinculin was released with alkaline and high salt solutions, and segregated into the aqueous phase on Triton X-114 solubilization and phase separation. Vinculin was sensitive to protease in the absence of detergent. The TLC revealed the presence of phosphatidylcholine and sphingomyelin. We concluded that vinculin is a peripheral vesicle-membrane associated protein, which is orientated to the citosol, and problably associated with the membranes via electrostatic interactions.