Importance of the Sphingolipids in the Cilium Formation Of MDCK Cells

PESCIO LG; FAVALE NO; STERIN-SPEZIALE NB

Rosario

Congreso; Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; 2014

SAIB

The final stage in the differentiation of epithelial cells is the outgrowth of a primary cilium from the apical surface. The mechanisms underlying cilia formation are poorly understood. We have demonstrated that glycosphingolipid synthesis is essential in MDCK cell differentiation induced by hypertonicity. The aim of this study was to evaluate the role of different sphingolipids in primary cilium formation. Confluent MDCK cells were cultured under isotonicity or subjected to hypertonic media for 72h in the presence or absence of D-PDMP, t-DHS and D609, inhibitors of glucosylceramide synthase, sphingosine 1-phosphate and sphingomyelin synthase, respectively. The primary cilium formation was analyzed by immunofluorescence for acetylated tubulin to reveal the cilium, and for Zo1, a tight junction marker. Cells cultured under isotonicity failed in developing primary cilium, whereas cells subjected to hypertonicity showed cilium formation and this process was impaired by D-PDMP. D609 induced a fibroblastic-like phenotype, with no cilium development. On the other hand, t-DHS did not affect the cilium formation, but induced a dissipation of Zo1 in specific areas. These results demonstrate that, even under hypertonicity, MDCK cells cannot fully differentiate in the presence of D-PDMP and D609, indicating that glucosylceramide and sphingomyelin are essential for primary cilium formation.