Glaucoma is a progressive optic neuropathy and a leading cause of irreversible blindness worldwide [1,2]. In 2020, an estimated 76 million people globally were affected by glaucoma, a number expected to rise to 112 million by 2040 [3]. Primary open-angle glaucoma (POAG) is the most common form of the disease [[4], [5], [6]]. Elevated intraocular pressure (IOP) is the primary risk factor for glaucoma, arising from an imbalance between aqueous humor production in the ciliary body and its drainage through the eye’s conventional outflow pathway [[7], [8], [9], [10], [11], [12]]. The conventional outflow pathway consists of the trabecular meshwork (TM), juxtacanalicular tissue (JCT), and Schlemm’s canal (SC) endothelial cells, along with the adjacent inner-wall basement membrane [12,13]. It is well established that the majority of outflow resistance resides in the JCT region, just a few microns beneath the inner-wall endothelium [9,[14], [15], [16], [17]]. Fig. 1 schematically illustrates the anatomy of the human eye’s anterior chamber and the conventional outflow pathway at the iridocorneal angle, where the TM is located.

Importantly, aqueous humor outflow through the TM is not uniform; it exhibits significant segmental heterogeneity with distinct high-flow (HF) and low-flow (LF) regions [[14], [18]]. Our group and others have identified segmental outflow patterns at both macro- and micro-scales (on the order of 1–5 mm and 50–150 µm) within the conventional outflow pathway [[19], [20], [21], [22], [23], [24], [25], [26], [27]]. These segmental outflow regions can dynamically adapt in response to elevated IOP [23,28]. The HF and LF regions also show molecular differences that are modulated by pressure changes, and their gene expression profiles are altered in glaucoma [[19], [20], [21],27,29,30]. In glaucomatous eyes, there is a shift toward a greater proportion of LF regions and fewer HF regions, suggesting a critical link between segmental outflow patterns and glaucoma pathology [30]. Especially, our recent mechanical tests demonstrated that trabecular tissue from LF regions in glaucomatous eyes is ∼3.5-fold stiffer (higher elastic modulus) than in normal eyes, while HF regions are ∼1.5-fold stiffer than normal [31]. In AFM of fresh human segments, normal eyes showed modest stiffness in the HF region (HF 1X = 3.05 kPa at physiologic IOP, 8.8 mm Hg), which drops after 24 h at double pressure (HF 2 × 24 h = 1.31 kPa at 17.6 mm Hg) before partially recovering by 72 h (2.49 kPa) [21]. Glaucomatous eyes, by contrast, displayed a dramatic regional contrast: HF regions remain soft (1.86 kPa), whereas LF segments elevate to 76.6 kPa, roughly 25-fold above normal HF and LF values [30]. This extreme stiffening shows glaucomatous LF tissue as the principal mechanical barrier for aqueous outflow.

These regional differences indicate a dynamic, bidirectional relationship between TM cells and their surrounding extracellular matrix (ECM) that significantly contribute to outflow resistance [32,33] Indeed, our prior work has shown that TM cells both respond to and remodel their ECM [32,34,35]. The TM’s ECM contains a variety of components, including straight fibrillar collagen as well as irregularly arranged “curly” collagen fibers [17,36]. Cell-ECM adhesion in the TM is mediated by micron-sized protein complexes (focal adhesions), with collagen fibers serving as an important site of mechanical interaction. Through multiple adhesion points within the 3D matrix, TM cells can apply traction forces on the ECM and thereby alter the ECM’s micromechanical properties. Conversely, TM cells sense mechanical changes in their microenvironment via these adhesion sites and convert them into intracellular chemical signals. This process of converting mechanical stimuli into a cellular response, known as mechanotransduction, plays a major role in TM cell behavior [[37], [38], [39]].

The cellular cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments, significantly contribute to regulating cell adhesion, spreading, migration, and force generation in interaction with the ECM [[40], [41], [42], [43]]. These cytoskeleton components also distinctly contribute to cell mechanics. Actin microfilaments (∼7 nm diameter) determine cell shape, drive motility, and generate traction forces through actomyosin contraction [44,45]. Microtubules (∼25 nm diameter) serve as intracellular “bones,” resisting compression and facilitating pushing or pulling via polymerization or depolymerization [44,46,47]. In contrast, intermediate filaments (∼10 nm diameter) form ropelike polymers that provide tensile strength, allowing cells to absorb and distribute stresses without rupturing [44,48]. These networks work together in regulating how cells generate, transmit, and withstand forces, supporting both normal physiology and pathophysiological processes [47].

In the TM, such cytoskeleton-mediated interactions are significantly contributing to maintaining healthy aqueous humor outflow and thus regulating IOP [47]. Consistent with this, our recent studies found that glaucomatous TM/JCT cells exert stronger traction forces than normal cells, which likely contribute to increased outflow resistance [34]. Moreover, our recently developed 2D collagen gel revealed a direct correlation between cytoskeletal organization and collagen fibril orientation [32], emphasizing the role of actomyosin machinery in ECM reorganization [49,50]. Mechanical stress induced by elevated IOP can alter TM cell mechanobiology via cytoskeletal rearrangements. Such biomechanical stimuli also may lead to changes in cell secretion and intracellular signaling that modify the surrounding ECM composition, ultimately affecting IOP homeostasis [51]. The actin cytoskeleton, in particular, is highly responsive to external mechanical forces, as under stress, actin filaments not only rearrange to change cell shape but also trigger alterations in intracellular signaling [12,39,52].

While it is well established that the cytoskeleton maintains cell shape and generates contractile forces, the relative load‑sharing among actin filaments, microtubules, and intermediate filaments in human TM cells has not been quantified. Actin has long been viewed as the principal load‑bearing network, whereas the mechanical roles of microtubules and vimentin remain poorly defined. In TM cells, pharmacological depolymerisation of microtubules triggers a robust contraction only when an intact actomyosin network is present; disruption of actin or myosin activity abolishes the response [53]. This emphasizes the enabling role of actin filaments. Consequently, glaucoma research has focused heavily on actin‑targeted pathways, leaving the contributions of microtubules and intermediate filaments under‑explored. In other cell types, inhibiting myosin II [54], reliably reduces traction forces, whereas microtubule depolymerisation can either enhance or diminish force generation depending on dimensional context. The quantitative consequences of selectively targeting each filament system on 3D traction forces in TM cells, however, remain essentially unexplored.

Our recent work further showed that TM cells in LF regions of glaucomatous eyes exert exceptionally strong traction forces that may lead to local ECM stiffening and contributing to glaucoma pathogenesis [[32], [33], [34], [35]]. Thus, it is vital to understand the mechanistic underpinnings of cellular traction forces, and identify which cytoskeletal components are the key drivers of these forces. Such crucial understandings could inform new therapeutic strategies aimed at targeting the elements that drive pathologic ECM stiffening in glaucoma. In this study, we investigated the individual contributions of actin filaments, microtubules, and intermediate filaments to the traction forces generated by TM cells. We cultured normal, high-flow TM cells on compliant type I collagen gels (4.7 kPa stiffness, confirmed by atomic force microscopy [32]) and selectively inhibited each cytoskeletal component to assess its effect on cell-generated forces. We focused on high-flow region TM cells because these cells are the most compliant and functionally “normal” within the outflow pathway [21,30]. This provided a healthy baseline condition to discern how inhibiting specific cytoskeletal elements affects traction force generation, thereby revealing the primary cytoskeletal drivers of TM cell contractile force. In addition, we examined how collagen fibrils reorganize under the tensile strains induced by each cytoskeletal element knockout. We hypothesize that correlating the measured traction forces with the corresponding collagen fibril reorganization for each knockout condition will yield significant understandings of both cellular biomechanics and cell-ECM interactions. Such information is critical for understanding how cell-ECM dynamics influence aqueous humor outflow resistance and thereby regulate IOP. Here we employ acute, selective filament disruption to determine which cytoskeletal subsystem is indispensable for traction in soft HF TM/JCT cells; this subtraction approach defines the minimum filament target set that must be down-regulated to soften the pathologically stiff LF regions found in glaucoma.