The exocyst is a highly conserved protein complex found in most eukaryotic cells and is associated with many functions, including protein translocation in the endoplasmic reticulum, vesicular basolateral targeting, and ciliogenesis in the kidney. To investigate the exocyst functions, here we exchanged proline for alanine in the highly conserved VXPX ciliary targeting motif of EXOC5 (exocyst complex component 5), a central exocyst gene/protein, and generated stable EXOC5 ciliary targeting sequence-mutated (EXOC5CTS-m) Madin-Darby canine kidney (MDCK) cells. The EXOC5CTS-m protein was stable and could bind other members of the exocyst complex. Culturing stable control, EXOC5-overexpressing (OE), Exoc5-knockdown (KD), and EXOC5CTS-m MDCK cells on Transwell filters, we found that primary ciliogenesis is increased in EXOC5 OE cells and inhibited in Exoc5-KD and EXOC5CTS-m cells. Growing cells in collagen gels until the cyst stage, we noted that EXOC5-OE cells form mature cysts with single lumens more rapidly than control cysts, whereas Exoc5-KD and EXOC5CTS-m MDCK cells failed to form mature cysts. Adding hepatocyte growth factor to induce tubulogenesis, we observed that EXOC5-OE cell cysts form tubules more efficiently than control MDCK cell cysts, EXOC5CTS-m MDCK cell cysts form significantly fewer tubules than control cell cysts, and Exoc5-KD cysts did not undergo tubulogenesis. Finally, we show that EXOC5 mRNA almost completely rescues the ciliary phenotypes in exoc5-mutant zebrafish, unlike the EXOC5CTS-m mRNA, which could not efficiently rescue the phenotypes. Taken together, these results indicate that the exocyst, acting through the primary cilium, is necessary for renal ciliogenesis, cystogenesis, and tubulogenesis.
Bibliographical noteFunding Information:
This work was supported in part by Veterans Affairs Merit Award I01 BX000820 (to J. H. L.) and Grants RX000444 and BX003050 (to B. R.); National Institutes of Health Grants P30DK074038 (to J. H. L.), R21EY025034 (to G. P. L.), R01HL131546 to (R. A. N.), P20GM103444 (to R. A. N.), R01HL127692 (to R. A. N.), R01DK087956 (to D. N.), R01EY019320 (to B. R.), R00DK105160 (to D. V. I.), and T32HL007260 and F31HL142159 (to D. B. F.); Polycystic Kidney Disease Foundation Grant 221G18a (to D. V. I.); a grant from the SC SmartState Centers of Excellence Endowment (to B. R., and D. N.); and American Heart Association AWRP Winter 2017 Collaborative Sciences Award (to R. A. N., J. H. L., and S. B.) and Grant 18PRE34080172 (to L. G.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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