Journal: Journal of cell science

Article Title: Clustered PI(4,5)P₂ accumulation and ezrin phosphorylation in response to CLIC5A.

doi: 10.1242/jcs.147744

Figure Lengend Snippet: Fig. 8. CLIC5 deletion reduces ezrin phosphorylation in renal glomeruli and disrupts the ezrin–NHERF2–podocalyxin complex. (A) Glomeruli were isolated from wild-type (+/+) and CLIC5-deficient (2/2) mice. Whole glomeruli were solubilized in Nonidet P40/deoxycholate lysis buffer followed by sedimentation of the detergent-insoluble fraction. Detergent-soluble (Supernatant) and detergent-insoluble (Pellet) fractions were then evaluated by western blot analysis using rabbit anti-CLIC5A, rabbit anti-ezrin and mouse anti-phospho-Thr567 ezrin antibodies. Mouse anti-tubulin was used as the loading control for the supernatants and mouse anti-b-actin for the pellet. CLIC5A was observed in lysates and pellets from wild-type but not CLIC5-deficient mice. The phospho-ERM (P-ERM) abundance was reduced in the detergent-soluble fraction from glomeruli of CLIC5-deficient mice, consistent with previous findings (Wegner et al., 2010). Much less ezrin was observed in the detergent-insoluble fraction of glomeruli from CLIC5-deficient mice compared with that of wild-type mice. (B) Renal cortex from wild-type and CLIC5-deficient mice was homogenized in Nonidet P40/deoxycholate lysis buffer and then separated into soluble (Supernatant) and insoluble (Pellet) fractions followed by western blot analysis. In wild-type mice, substantial NHERF2 was observed in the pellet, whereas NHERF2 remained soluble in CLIC5-deficient mice. (C) Confocal immunofluorescence microscopy for phospho-ERM (rabbit anti-phospho-Thr567 ezrin) and podocalyxin (goat anti- podocalyxin) in TCA-fixed renal glomeruli from wild-type and CLIC5-deficient mice. Nuclei (blue) were visualized with DAPI (49,6-Diamidino-2-Phenylindole). Phospho-ERM is present in glomeruli of both wild-type and CLIC5-deficient mice, but colocalization of phospho-ERM with podocalyxin was substantially reduced in CLIC5-deficient mice compared with that observed in wild-type mice. Scale bar: 10 mm. (D) Schematic model of CLIC5A function in glomerular podocytes. It is postulated that the association of CLIC5A with the inner leaflet of the dorsal plasma membrane serves to promote the generation of clustered PI(4,5)P2. Ezrin binding to PI(4,5)P2 in turn frees the ezrin C-terminus to bind to actin and to promote PKC-dependent ezrin phosphorylation. In the presence of CLIC5A, the active form of ezrin couples the intracellular tail of podocalyxin to actin, a key determinant of the normal apical podocyte architecture.

Article Snippet: Rabbit anti-ezrin and antiPI4P5Ka antibodies were from Cell Signaling (Danvers, MA), rabbit anti-CLIC5 antibodies were from Aviva System Biology (San Diego, CA), mouse anti-b-tubulin and anti-b-actin antibodies were from Millipore (Billerica, MA) and Sigma (Oakville, ON, Canada) respectively.

Techniques: Phospho-proteomics, Isolation, Lysis, Sedimentation, Western Blot, Control, Immunofluorescence, Microscopy, Clinical Proteomics, Membrane, Binding Assay