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

BRANDAN YAMILA; GUAYTIMA EDITH; MARIA BELEN DE LA VEGA; FAVALE NICOLAS; STERIN SPEZIALE NORMA; MARÍA GABRIELA MÁRQUEZ

Mendoza

Congreso; XLVIII REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INVESTIGACIONES BIOQUÍMICAS Y DE BIOLOGÍA MOLECULAR (SAIB); 2012

SOCIEDAD ARGENTINA DE INVESTIGACIONES BIOQUÍMICAS Y DE 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 X114 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 vesiclemembrane associated protein, which is orientated to the citosol, and probably associated with the membranes via electrostatic interactions.