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Abstract: FR-PO966

Structure-Function Analysis of Inversin in Proximal Ciliary Patterning and Ciliary Transport

Session Information

Category: Genetic Diseases of the Kidney

  • 1001 Genetic Diseases of the Kidney: Cystic


  • Xu, Lillian, Princeton University, Crozet, Virginia, United States
  • Bianchi, Giada Giada Bianchi, Dana Farber Cancer Institute, Boston, Massachusetts, United States
  • Czarnecki, Peter G., Brigham and Women's Hospital, Boston, Massachusetts, United States
  • Shah, Jagesh V., Harvard Medical School, Boston, Massachusetts, United States

The proximal end of the primary cilium contains the inversin compartment (IC), comprised of four interacting ciliopathy proteins, inversin (INVS), NEK8, ANKS6 and NPHP3. Mutations in these proteins give rise to a multiorgan malformation syndrome, with L-/R-asymmetry perturbations, congenital heart defects and polycystic kidney disease. The most upstream factor in the IC is inversin, which is necessary for IC localization of the other proteins. The goal of this study is (1) to identify the structural determinants of inversin, necessary and/or sufficient for IC reconstitution and (2) to analyze ciliary length and intraflagellar transport (IFT) phenotypes in presence or absence of IC proteins.


CRISPR/Cas9-mediated gene knockout
Lentiviral and retroviral transduction
Immunofluorescence microscopy
Live fluorescence cell imaging
Kymograph analysis


Immunofluorescence for FLAG-antigen revealed proximal ciliary localization only of FLAG-tagged full-length inversin and C-terminal truncations (INVS[745-1065] and INVS[554-1065]), but not N-terminal truncations INVS[1-553] and INVS[1-744]. Furthermore, only full-length inversin, and none of the partial add-backs were capable to localize NEK8 and ANKS6 to the IC. These results suggest that the ciliary signal sequence of inversin lies in its C-terminal domain, but the full-length protein, including the N-terminal ANK-repeat domain is required to assemble the other proteins in the IC. Moreover, we analyzed ciliary length and IFT-B particle velocities in CRISPR/Cas9-knockouts and full-length add-backs of IC proteins. Loss of inversin led to a >30% decrease in ciliary length and of IFT-B particle velocity, while a lentiviral full-length INVS add-back completely rescued both knockout phenotypes. An identical phenotype was observed with NPHP3, while NEK8 knockout revealed ~30% ciliary length increase and acceleration in IFT-B transport velocity.


As ciliary lengthening control and IFT particle velocity are physiological readouts for ciliary function, our results indicate that inversin is necessary and sufficient for ciliary homeostasis. The similar behavior of inversin and NPHP3 knockout phenotypes suggests that inversin may function through NPHP3, while NEK8 may play an independent role as a kinase.


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