Coordination beetwen sphingolipids ans proteins required for primary ciliogenesis

PESCIO LG; SANTACREU BJ; TULINO MS; PAVÁN CH; MONTI JLE; FAVALE NO; STERIN SPEZIALE NB

CABA

Congreso; II Reunión Conjunta de Sociedades de BioCiencias; 2017

SAIB-SAIC-SAI-SAA-SAB-SAB-SAFE-SAFIS-SAH-SAP

The final stage in the process of epithelial cell differentiation is the outgrowth of a primary cilium from the apical surface. We have previously described that extracellular hypertonicity induces the differentiation of MDCK cells by the regulation of the sphingolipid metabolism. The aim of this work was to investigate the ciliogenesis induced by hypertonicity in MDCK cells and the role of glycosphin- golipids in the process. Con uent MDCK cells were cultured under isotonicity (150 mM NaCl, control) or hypertonicity (300 mM NaCl) for 48 h in the presence or absence of D-PDMP, a glucosylceramide synthase inhibitor. The presence of primary cilium was detected in MDCK cells stable expressing α-tubulin GFP subjected to hyperto- nicity. For the immuno uorescence assays, cells were stained with acetylated tubulin to visualize primary cilium, and co-stained with IFT20 or Rab8, proteins required for vesicle-mediated traf cking pathway from the Golgi to the base of the cilium. Isotonicity-cultured cells did not show cilium formation and IFT20 and Rab8 appeared diffusely distributed. Cells cultured under hypertonicity displayed pri- mary cilium and the number of ciliated cells signi cantly decreased when the cells were subjected to hypertonicity in the presence of D-PDMP. Cells cultured under hypertonicity showed IFT20 with a Golgi like distribution and perinuclear Rab8, but when cells were treated with D-PDMP the distribution of these proteins was diffuse. A proteomic analysis performed by mass spectrometry of IFT20 vesicles immune-isolated from cells cultured under hypertonicity re- vealed the presence of galectin 3, a protein with an important role in apical sorting that associates with the primary cilium. In conjunc- tion, these results suggest that hypertonicity induces ciliogenesis by displaying a protein machinery that is correctly assembled in the presence of glycosphingolipids.