Technical Reference #3139

3139.

Rab10 associates with primary cilia and the exocyst complex in renal epithelial cells Clifford M. Babbey; Robert L. Bacallao; and Kenneth W. Dunn, Indiana University Medical Center, American Journal of Physiology - Renal Physiology, 299(3139), (2010)
Link To Paper

Abstract:
Rab10 a mammalian homolog of the yeast Sec4p protein has previously been associated with endocytic recycling and biosynthetic membrane transport in cultured epithelia and with Glut4 translocation in adipocytes. Here we report that Rab10 associates with primary cilia in renal epithelia in culture and in vivo. In addition we find that Rab10 also colocalizes with exocyst proteins at the base of nascent cilia and physically interacts with the exocyst complex as detected with anti-Sec8 antibodies. These data suggest that membrane transport to the primary cilum may be mediated by interactions

Keywords:
primary cilium; Sec8

Materials & Methods:
Cell culture. Studies were conducted using PTR cells MDCK strain II cells transfected with both the human TfR and the rabbit polymeric immunoglobulin receptor (pIgR) as previously described (3 5 70 71). PTR cells were grown in MEM (Life Technologies Grand Island NY) with 8% FBS 1% L-glutamine streptomycin and 0.05% hygromycin (Calbiochem San Diego CA). Cells were passed every 3 to 4 days and growth medium was changed daily. New cultures of cells were thawed every 4–5 wk. For fluorescence experiments cells were plated either on coverslip-bottomed 35-mm dishes (Mattek Ashland MA) or plated at confluence on the bottoms of collagen-coated Millipore CM 12-mm filters and cultured for 4–5 days before experiments as described previously (3 5 70 71).

Microscopic Technique
Fluorescence experiments, Inverted microscope

Cell Type(s)
PTR cells; MDCK strain II cells