ZFIN ID: ZDB-PUB-190303-5
The exocyst acting through the primary cilium is necessary for renal ciliogenesis, cystogenesis, and tubulogenesis
Zuo, X., Lobo, G., Fulmer, D., Guo, L., Dang, Y., Su, Y., Ilatovskaya, D.V., Nihalani, D., Rohrer, B., Body, S.C., Norris, R.A., Lipschutz, J.H.
Date: 2019
Source: The Journal of biological chemistry   294(17): 6710-6718 (Journal)
Registered Authors: Lobo, Glenn
Keywords: cell biology, cilia, ciliary targeting sequence, development, exocyst, exocytosis, primary cilia, protein trafficking, trafficking, vesicle
MeSH Terms:
  • Animals
  • Cilia/physiology*
  • Cysts/pathology*
  • DNA, Complementary/genetics
  • Dogs
  • Gene Knockdown Techniques
  • Humans
  • Kidney/metabolism*
  • Kidney Diseases/pathology
  • Kidney Tubules/growth & development*
  • Madin Darby Canine Kidney Cells
  • Mutagenesis, Site-Directed
  • Protein Binding
  • Protein Transport
  • RNA, Messenger/metabolism
  • Vesicular Transport Proteins/genetics
  • Vesicular Transport Proteins/metabolism*
  • Zebrafish
PubMed: 30824539 Full text @ J. Biol. Chem.
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ABSTRACT
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 exocyst complex component 5 (EXOC5), a central exocyst 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 (HGF) 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.
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