Role of LIM-Nebulette in Podocyte Mechanoresponse

Background: Podocytes are mechanosensitive cells critical to the glomerular filtration barrier, constantly exposed to mechanical forces. Understanding their mechanosensitivity is essential for addressing foot process effacement and glomerular dysfunction. LIM-nebulette, an actin-binding protein, supports cytoskeletal resilience and influences calcium dynamics. Here, we explore LIM-nebulette’s role in podocyte mechanosensitivity under biomechanical stress. Methods: We developed an automated biaxial cyclic stretcher for standard incubators with adjustable strain, frequency, and duration. Immortalized human podocytes expressing mClover-LIM-nebulette and mScarlet-LifeAct underwent 10% strain under three conditions: control, stretched, and 10 mM blebbistatin (PAB). Samples were analyzed by confocal microscopy. Additionally, wild-type and shRNA LIM-nebulette knockdown podocytes were stretched for 5, 15, 30, 60 minutes, and 24 hours, with nebulette expression evaluated by qPCR after 24 hours. Live-cell TIRF imaging was separately conducted using 1 uM cytochalasin D and 200 uM PAB. Results: Stretched cells initially decreased in area but recovered over time, with changes in LIM-nebulette localization. After 30 minutes of stretch, nebulette shifted to the periphery, whereas in control and PAB-treated cells, it remained near the nucleus and perinuclear regions, respectively. Nebulette localized to focal adhesions (FAs) and stress fibers (SFs), disrupted by cytochalasin D within 10 minutes. At 60 minutes post-washout, nebulette reappeared in wave-like patterns with SF reformation. PAB treatment reduced nebulette at FAs but not SFs; washout restored contraction and FA localization. Thus, podocytes dynamically adapt. Conclusion: Podocytes dynamically adapt to external mechanical forces, recovering after prolonged cyclic stretch. Increased nebulette intensity at the cell periphery, stress fibers, and focal adhesions indicates its role in stress response and actin stabilization, while myosin inhibition suggests nebulette mobilization relies on intracellular contraction. Funding: NIDDK SupportMyosin IIA inhibition (A) Control (B) Blebbistatin , Washout (C) 30 and (D) 60 min.