Restructuring of focal contacts by bradykinin in rat renal papilla

GABRIELA MÁRQUEZ; SERRANO DIEGO; LAURA GAGLIANO; NORMA STERIN-SPEZIALE

Bariloche , Rio Negro

Congreso; XXXIX REUNIÓN DE LA SOCIEDAD ARGENTINA DE INVESTIGACIONES BIOQUÍMICAS Y BIOLOGÍA MOLECULAR (SAIB), XXXII REUNION ANUAL DE LA SOCIEDAD ARGENTINA DE BIOFISICA y Protein Symposium; 2003

XXXIX REUNIÓN DE LA SOCIEDAD ARGENTINA DE INVESTIGACIONES BIOQUÍMICAS Y BIOLOGÍA MOLECULAR (SAIB)

Focal contacts (FC) are dynamic cytoskeletal structures where the cell membrane attaches to the extracellular matrix. In previous works we found that FC proteins vinculin (V), talin (T) and paxilin (P) are associated with DRMs -lipid?detergent (Triton X-100)?resistant membrane domains in rat renal papilla; and that bradykinin (BK) can regulate the assembly of FC altering the interaction between PIP2 and V. In the present work, we have investigated in adult rat renal papilla: a) the interaction between T and P with V in DMRs, by ultracentrifugation, immunoprecipitation with an antibody (Ab) to V and subsequent immunoblotting with Ab to T and P, b) the dynamic of binding between T and P with V in DMRs, and the movilization of V from FC by incubating papilla slices with BK during 1, 5 and 10 minutes, and c) the ?in vivo? restructuring of FC by BK in a primary culture of renal papillary collecting duct (CD) cells, by immunocytochemistry and confocal microscopy. Results: a) T and P co-immunoprecipitate with V in DMRs but not in Triton X-100 soluble fraction, indicating an interaction between them only in FC. b) BK induces a decrease in the association of T and P with V after 10 minutes in DRMs. c) In control cells, intense V staining was localized to FC, with a less intense and diffuse V satining in the perinuclear zone. When cells were treated with BK during 1 minutes, V staining was almost removed from FC and concentrated in the perinuclear region, although cells remained attached. This correlates with a dissipation of V from DMRs to the Triton X-100 soluble fraction observed by immunoblotting. After 5 minutes, it was observed a dissipation of V staining from the perinuclear region to FC, and at 15 minutes V staining pattern resemble the ones observed in control cells. the redistribution of V and PIP2 from FC to the Triton X-100 insoluble fraction after 1 minutes. Taking into account, that T and P remain associated to V and V remain localized to FC after BK treatment, we can suggest that BK induce a restructuration of FC rather than a dissipation.