Sphingomyelin synthesis is implicated in the maintenance of cell-cell adhesion structures

BRANDAN YAMILA; EDITH GUAYTIMA; STERIN-SPEZIALE N; M GABRIELA MARQUEZ

Puerto Iguazu-Misiones

Congreso; 56th INTERNATIONAL CONFERENCE ON THE BIOSCIENCE OF LIPIDS (ICBL); 2015

INTERNATIONAL CONFERENCE ON THE BIOSCIENCE OF LIPIDS (ICBL)

Adherens junctions (AJ) and focal adhesions (FA) are structures that mediate tissue organization at two different levels of plasma membrane. Vinculin is a cytoskeletal protein associated with both structures, interacting with α-catenin recruits F-actin to the AJs. Taking into account that we have previously demonstrated that both adhesion structures are located in sphingomyelin (SM) enriched membrane microdomain, we investigated the influence of SM synthesis in AJ and FA integrity. To this end, primary cultures of renal papillary collecting duct cells were performed. When cultured, these cells preserve the fully differentiated epithelial phenotype as reflected by the presence of primary cilium. Cells were incubated for 24 hs with increasing concentration of D609, a SM synthase1 inhibitor. Knock down experiments silencing SM synthase 1 and 2 were also performed. Cell adhesion structures were analyzed by confocal immunofluorescence with anti-vinculin and anti α-catenin antibodies, and AJ proteins were analyzed by Western blot. D609 treatment showed cells with irregular shape and a progressive AJ impairment reflected by alteration in the distribution of both proteins. At higher D609 concentration, cell-cell contacts were lost but retained the spread morphology by increasing the number of FA. The silencing of SM synthase1, but not SM synthase 2, induced an irregular distribution of α-catenin and loss of AJ integrity. D609 evoked AJ impairment appeared reversible since cell-cell adhesion restoration was observed after reincubation in the absence of D609 for 24 hs. These results suggest that the activity of SM synthase1 is essential for the maintenance of collecting duct tissue organization.