Actin-microtubule synergy dominates force transmission and collagen strain in human trabecular meshwork

Trabecular meshwork (TM) cells contribute to regulating intraocular pressure by generating contractile or traction forces on their collagen-rich extracellular matrix (ECM). The relative mechanical contributions of the three cytoskeletal filament systems, however, remain incompletely defined. We cultured normal human high-flow TM cells on type I collagen gels (elastic modulus 4.7 kPa) and used three-dimensional traction force microscopy coupled with fibril strain mapping to study cytoskeletal hierarchy. Cells were treated with Latrunculin B (actin depolymerization), Nocodazole (microtubule depolymerization) or Withaferin A (intermediate filament disassembly), and imaged every 30 min for 12 h. Actin-, microtubule-, and intermediate filament disruption lowered mean traction stress by ∼8-, 3.5- and 2.1-fold, respectively; collagen-fibril tensile strain decreased by ∼7.6-, 3.3- and 2-fold. Mixed-effects analysis confirmed significant reductions for actin and microtubule inhibition (p ≤ 0.01), whereas intermediate filament loss was neutral. Confocal-based orientation mapping and scanning electron microscopy showed no change in filament alignment or collagen ultrastructure at 12 h, likely indicating that force attenuation precedes structural remodeling. A ∼10 kPa (∼80 %) drop at the cell-matrix interface is theoretically sufficient to alter segmental outflow resistance. These data establish actomyosin contraction as the dominant driver of TM traction, supported by microtubules acting as compressive struts, while intermediate filaments are dispensable over the first 12 h. This quantitative hierarchy can inform cytoskeleton-guided biomaterial design and therapeutic strategies aimed at restoring TM biomechanics in glaucoma. STATEMENT OF SIGNIFICANCE: In glaucoma, fluid leaves the eye through the trabecular meshwork (TM), yet we still do not know which of the three cytoskeletal filament systems, actin, microtubules, or intermediate filaments, drives the contractile forces that set outflow resistance. We combine 3D traction force microscopy with collagen fibril strain mapping to deliver the first quantitative "mechanical hierarchy" for human TM cells. Selective inhibition shows that actin generates, and microtubules sustain, ∼80 % of TM traction, whereas vimentin filaments are largely dispensable over 12 h. These understandings link cell mechanics directly to matrix stiffening, establishing a framework for biomaterial design and pinpointing cytoskeletal targets that could normalize eye pressure. The work therefore advances both fundamental mechanobiology and glaucoma-focused biomaterials research.