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Kidney Week

Abstract: FR-PO584

Primary Cilia Change Protein Composition in Response to Glutamine Deprivation

Session Information

Category: Genetic Diseases of the Kidneys

  • 1201 Genetic Diseases of the Kidneys: Cystic

Authors

  • Nielsen, Anne Kallehauge, IRCCS Ospedale San Raffaele, Milano, Lombardia, Italy
  • Steidl, Maria Elena, IRCCS Ospedale San Raffaele, Milano, Lombardia, Italy
  • Aslanyan, Mariam, Radboud Universiteit, Nijmegen, Gelderland, Netherlands
  • van Beersum, Sylvia E C, Radboud Universiteit, Nijmegen, Gelderland, Netherlands
  • Spies, Daniel, IRCCS Ospedale San Raffaele, Milano, Lombardia, Italy
  • Pedersen, Lotte B., Kobenhavns Universitet, Kobenhavn, Denmark
  • Boldt, Karsten, Universitatsklinikum Tubingen, Tubingen, Germany
  • Roepman, Ronald, Radboud Universiteit, Nijmegen, Gelderland, Netherlands
  • Boletta, Alessandra, IRCCS Ospedale San Raffaele, Milano, Lombardia, Italy
Background

ADPKD is a genetic disorder in majority of cases caused by mutations in PKD1 and PKD21. Genes encode for Polycystin 1 and 2 which form a receptor-complex located to primary cilia. Primary cilia are sensory organelles that protrude from the cell membrane. From this extroverted position it responds to changes in the cellular environment and convey various signaling pathways to control cell function, such as cell maintenance. Our lab previously published that loss-of-function of Pkd1 leads to metabolic reprogramming. Recently we published the importance of glutamine availability during nutrients-stress conditions on cilia length. Demonstrating that primary cilia respond to nutrients availability and particularly to glutamine. We also showed that the glutamine response of cilia requires the presence of Asparagine Synthetase (ASNS), which localizes to the cilia-centrosome complex.

Methods

To follow up on these findings we set up a large-scale proteomics study using mIMCD3 cells expressing a ciliary target fused to the proximity labelling tag, BioID. We affinity purified biotinylated proteins specific to the cilium by taking advantage of the high affinity of streptavidin coupled to beads. IP samples were analyzed with LC-MS. We generated an expression vector with ASNS fused to mNeonGreen fluorescent-tag and generated stable transfectants in IMCD3s.

Results

A first LC-MS analysis of proteins exclusively observed in cilia of cells grown in medium with or without glutamine revealed a total of 22 proteins significantly changing with removal of glutamine. A few proteins were found to change both in expression levels and in ciliary abundance. Among these, ASNS was upregulated with removal of glutamine in both the cytoplasm, and in the cilium. In line with these findings live imaging and IF performed with mNG-ASNS IMCD3s showed ASNS localizing not only at the base of the cilium, as we reported, but also within the axoneme. To accumulate ASNS in the cilium we treated mNG-ASNS cells with CilioBrevin D which inhibits dynein proteins and found that ASNS accumulates at the tip of the cilium.

Conclusion

Our data demonstrate that primary cilia respond to glutamine removal by changing their protein composition. And that the enzyme ASNS is among the proteins responding to glutamine availability.