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Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes
Published on: December 20, 2014
Polycystin-1 regulates ARHGAP35-dependent centrosomal RhoA activation and ROCK signaling
Insights
Mutations in polycystic kidney disease 1 (PKD1) disrupt centrosomal RhoA signaling, leading to kidney cyst formation. Targeting the RhoA/ROCK pathway with hydroxyfasudil effectively inhibited cyst expansion in models of autosomal dominant polycystic kidney disease (ADPKD).
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Autosomal dominant polycystic kidney disease (ADPKD) affects 80-85% of patients due to PKD1 mutations.
- PKD1 mutations are linked to cellular phenotypes including actin cytoskeleton disorganization and dysregulated signaling.
Purpose of the Study:
- To investigate if PKD1 mutations cause actin dynamics changes via dysregulated centrosomal RhoA signaling.
- To identify Rho GTPase-activating proteins (RhoGAPs) involved in centrosomal RhoA regulation and ciliation.
Main Methods:
- Examined actin cytoskeleton organization and RhoA/ROCK signaling in PKD1-mutant cells.
- Used cilia length as a readout for centrosomal RhoA activity.
- Employed proximity ligation assays and immunofluorescence to assess ARHGAP35 levels.
- Tested the ROCK inhibitor hydroxyfasudil in 3D cyst cultures and an inducible Pkd1 mouse model.
Main Results:
- PKD1-mutant cells showed disorganized actin, increased RhoA, and ROCK signaling.
- ARHGAP5, -29, and -35 were identified as key ciliation regulators.
- Centrosomal ARHGAP35 was significantly decreased in PKD1-null cells.
- Hydroxyfasudil reduced cyst expansion in both human cell and mouse models.
Conclusions:
- A novel interaction between polycystin-1 (PC1) and ARHGAP35 in regulating centrosomal RhoA/ROCK signaling is proposed.
- The RhoA/ROCK pathway is crucial for ADPKD pathogenesis.
- Targeting the RhoA/ROCK pathway offers a potential therapeutic strategy for inhibiting cyst initiation in ADPKD.
Abstract:
Mutations in PKD1 (encoding for polycystin-1 [PC1]) are found in 80%-85% of patients with autosomal dominant polycystic kidney disease (ADPKD). We tested the hypothesis that changes in actin dynamics result from PKD1 mutations through dysregulation of compartmentalized centrosomal RhoA signaling mediated by specific RhoGAP (ARHGAP) proteins resulting in the complex cellular cystic phenotype. Initial studies revealed that the actin cytoskeleton was highly disorganized in cystic cells derived from patients with PKD1 and was associated with an increase in total and centrosomal active RhoA and ROCK signaling. Using cilia length as a phenotypic readout for centrosomal RhoA activity, we identified ARHGAP5, -29, and -35 as essential regulators of ciliation in normal human renal tubular cells. Importantly, a specific decrease in centrosomal ARHGAP35 was observed in PKD1-null cells using a centrosome-targeted proximity ligation assay and by dual immunofluorescence labeling. Finally, the ROCK inhibitor hydroxyfasudil reduced cyst expansion in both human PKD1 3D cyst assays and an inducible Pkd1 mouse model. In summary, we report a potentially novel interaction between PC1 and ARHGAP35 in the regulation of centrosomal RhoA activation and ROCK signaling. Targeting the RhoA/ROCK pathway inhibited cyst formation in vitro and in vivo, indicating its relevance to ADPKD pathogenesis and for developing new therapies to inhibit cyst initiation.
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