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Kidney Week

Abstract: SA-OR088

Polycystin 1 Regulates Actomyosin Contraction and the Cellular Response to Extracellular Stiffness

Session Information

Category: Genetic Diseases of the Kidneys

  • 1001 Genetic Diseases of the Kidneys: Cystic

Authors

  • Nigro, Elisa Agnese, San Raffaele Scientific Institute, Milan, Italy
  • Distefano, Gianfranco, San Raffaele Scientific Institute, Milan, Italy
  • Chiaravalli, Marco, San Raffaele Scientific Institute, Milan, Italy
  • Matafora, Vittoria, IFOM-FIRC Institute of Molecular Oncology, Milano, Italy
  • Castelli, Maddalena, San Raffaele Scientific Institute, Milan, Italy
  • Pesenti Gritti, Angela, San Raffaele Scientific Institute, Milan, Italy
  • Bachi, Angela, IFOM-FIRC Institute of Molecular Oncology, Milano, Italy
  • Boletta, Alessandra, San Raffaele Scientific Institute, Milan, Italy
Background

The Polycystins (PC-1 and PC-2) are the products of the genes mutated in Autosomal Dominant Polycystic Kidney Disease. PC-1 is a receptor with a large extracellular extendible domain postulated to act as a mechanosensor. It associates with PC-2 to form a receptor/channel complex possibly activated by the mechanical stimulus of ciliary bending and resulting in calcium influx. This model has recently been challenged, leaving the open question of what mechanical stimuli activate the polycystins.

Methods

To identify interactors of PC-1, we immunoprecipitated (IP) endogenous tagged PC-1 in mouse embryonic fibroblasts derived from Pkd1HA/HA or Pkd1WT/WT embryos, followed by liquid chromatography mass-spectrometry (LC-MS). We used SILAC (Stable Isotopes Labeling by Aminoacids in Cell culture) followed by either in-gel proteins digestion or protein digestion directly on the beads prior to LC-MS. Each assay was performed in two experiments.

Results

We identified 316 proteins, 30 of them significantly enriched in the IP versus the control in both SILAC studies. Both approaches yielded similar results. The list of binding partners all pointed to a central role for PC-1 in regulation of the actomyosin contraction machinery. Using Pkd1 gain and loss of function cellular models, we found that PC-1 reduces myosin light chain phosphorylation (pMLC), the main regulator of actomyosin contraction. Furthermore, we confirmed that PC-1 inhibits cell contractility using a collagen disc contractility assay. Given the role of the actomyosin pathway in the response of cells to mechanical stimuli, we used hydrogels or graded epithelial density conditions to mimic different extracellular rigidity states, ranging from 0.4 to 40 kPa. We found that PC-1 negatively regulates Yes-Associated Protein (YAP) activation in response to extracellular stiffness. Finally, in an orthologous murine model of PKD we found increased pMLC, and enhanced YAP nuclear translocation and transcriptional activity, both reversed by the ROCK-inhibitor Fasudil. Notably, treatment with this compound ameliorates disease progression.

Conclusion

Our data suggest a possible direct role for PC-1 as a mechanosensor able to negatively regulate the cellular response to extracellular stiffness.