Differential Cytoskeletal Control of Cell-Collagen Mechanics in High- and Low-Flow Glaucomatous Trabecular Meshwork Cells

We quantified cytoskeletal regulation of cell-collagen mechanics in glaucomatous trabecular meshwork (TM) cells from tracer-identified high-flow (HF) and low-flow (LF) regions using 3D traction force microscopy coupled to collagen kinematics. Matched HF and LF cultures were derived from a single glaucomatous donor eye (n = 3 gels per condition per region) and seeded on fibrillar type I collagen gels (elastic modulus 27.7 kPa) containing 0.2 μm FluoSpheres. Cells were imaged hourly for 12 h after latrunculin B (0.5 μM), no codazole (1 μM), or withaferin A (2 μM), with region-matched vehicle controls. In controls, reconstructed mean traction increased from 4 to 12 h in both HF (36.2-44.8 kPa) and LF (23.6-40.6 kPa). Latrunculin B reduced traction early with partial recovery, while nocodazole timepoint means differed between HF and LF at 12 h (HF: 64.1 kPa, 3.96% strain; LF: 17.3 kPa, 0.98% strain) and shifted divergence toward convergence. Withaferin A reduced traction, strain, and curl at 12 h in both HF and LF and differed from control in LF collagen orientation angle dynamics (p = 0.021). Mixed-effects models with Bonferroni correction did not detect treatment-control differences in the rate of change over time for traction, strain, divergence, or curl after accounting for HF/LF; within LF, latrunculin B decreased divergence (p = 0.018) and withaferin A differed in strain change over time (p = 0.045). This segment-resolved assay supports comparative evaluation of cytoskeletal perturbations and motivates validation across additional donor-matched HF/LF pairs.