A Direct CLIC5A/Ezrin Interaction Accounts for CLIC5A Plasma Membrane Localization and CLIC5A-Stimulated Rac1 Activation in Renal Glomeruli
Bibliographic record
Abstract
Background: CLIC5A is part of the Podocalyxin/Ezrin complex at the apical domain of podocyte foot processes. Ezrin binds membrane phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) inducing its activation and C-terminal (T567) phosphorylation. Activated Ezrin bridges membrane-spanning proteins to cortical actin, shaping the cellular architecture. We reported that CLIC5A stimulates Rac1-GTP-dependent PI(4,5)P2 generation and Ezrin activation and that CLIC5A deletion disrupts the foot process architecture (J. Cell Sci. 127:5164, 2014 & Kidney Int. 89:833, 2016). Here, we investigated the mechanism by which CLIC5A interacts with Ezrin. Methods: Direct protein-protein interactions were established by Yeast two-hybrid (Y2H) assay and confirmed by GST pull-down and reciprocal co-immunoprecipitation. Subcellular localization of endogenous proteins was determined by differential detergent fractionation of mouse glomeruli. Ezrin was released from glomerular membranes by stimulating phospholipase C-mediated PI(4,5)P2 hydrolysis with m-3M3FBS. Results: We found that CLIC5A interacts directly with the Ezrin C-terminal domain, but only if Ezrin was in the activated state. The extreme 16 C-terminal amino acids of Ezrin were necessary for the interaction and Ezrin phosphorylation at T567 increased CLIC5A binding. Expression of a C-terminal ezrin fragment (Ezrin 432-586, phosphomimic T567D), but not C-terminally truncated Ezrin 432-570 effectively blocked CLIC5A-stimulated Ezrin and Rac1 activation. In glomeruli 49 +/- 4 % of endogenous CLIC5A was in the cytoplasmic, 33 +/- 3% in membrane and 18 +/- 1 % in the cytoskeleton-associated fraction. As expected, PI(4,5)P2 hydrolysis reduced Ezrin phosphorylation and shifted Ezrin from membrane and cytoskeletal fractions into the soluble pool. Similarly, PI(4,5) P2 hydrolysis shifted CLIC5A from membrane- and cytoskeletal fractions, increasing the soluble component to 72 +/- 13% (p = 0.041, n= 3, mean +/- STD). Conclusions: CLIC5A interacts directly with active Ezrin, and this interaction is required for CLIC5A-dependent Rac1 activation. The release of CLIC5A together with Ezrin from the membrane fraction in response to PI(4,5)P2 hydrolysis suggests that CLIC5A localizes to the plasma membrane because of its direct interaction with Ezrin. Funding: Government Support - Non-U.S.
Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.
How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.001 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".