The formation of cortical actin arrays in human trabecular meshwork cells in response to cytoskeletal disruption

Abstract

The cytoskeleton of human trabecular meshwork (HTM) cells is known to be altered in glaucoma and has been hypothesized to reduce outflow facility through contracting the HTM tissue. Latrunculin B (Lat-B) and Rho-associated protein kinase (ROCK) inhibitors disrupt the actin cytoskeleton and are in clinical trials as glaucoma therapeutics. We have previously reported a transient increase in HTM cell stiffness peaking at 90 min after Lat-B treatment with a return to pretreatment values after 270 min. We hypothesize that changes in actin morphology correlate with alterations in cell stiffness induced by Lat-B but this is not a general consequence of other cytoskeletal disrupting agents such as Rho kinase inhibitors. We treated HTM cells with 2 µM Lat-B or 100 µM Y-27632 and allowed the cells to recover for 30-270 min. While examining actin morphology in Lat-B treated cells, we observed striking cortical actin arrays (CAAs). The percentage of CAA positive cells (CPCs) was time dependent and exceeded 30% at 90 min and decreased after 270 min. Y-27632 treated cells exhibited few CAAs and no changes in cell stiffness. Together, these data suggest that the increase in cell stiffness after Lat-B treatment is correlated with CAAs.

Keywords: Actin; Cytoskeleton; Latrunculin B; Rho-associated protein kinase; Trabecular Meshwork.

Figure 1

Demonstration of CAA formation in HTM cells. (A) Phalloidin stain of a typical HTM cell in culture, displaying prominent axial stress fibers with. (B) Cortical actin array in an isolated HTM cell stained with phalloidin. (C) Representative image of α-actinin labeling of both the radial and cortical actin structures with enrichment at the junctions (arrowheads). Periodic α-actinin labeling of circumferential actin (bracketed region) was also observed. (D) Three major phenotypes of CAAs. All nuclei are labeled with DAPI (blue). All scale bars are 20 μm.

Figure 2

Induction of CAAs via Lat B treatment. (A) Phalloidin staining of control (DMSO) and Lat B treated cells at 30 min. The application of 2 μM Lat B completely removed phalloidin staining of stress fibers typical of control cells. (B) HTM cells exhibited CAAs during recovery from Lat B, but not control (DMSO) treatments. Phalloidin staining is shown in red; nuclei are labeled with DAPI (blue). Scale bar is 20 μm.

Figure 3

Y-27632 had minimal impact on CAA formation or cell mechanics. (A) Phalloidin staining (red) of control (DPBS) and Y-27632 treated cells at 30 min. 100 μM Y-27632 was sufficient to completely inhibit stress fibers typical of control cells (B) After 90 minutes of recovery, Y-27632 treated cells still had altered morphology, although uniaxial stress fibers had begun to appear. (C) Y-27632 treatment did not induce CAA formation during recovery. (D) Y-27632 treatment did not alter cellular mechanics during recovery. Phalloidin staining is shown in red; nuclei are labeled with DAPI (blue). Scale bars are 20 μm.

Figure 4

Y-27632 had minimal impact on CAA formation at earlier timepoints. (A) Phalloidin staining of HTM cells 15 minutes after Y-27632 washout. The actin cytoskeleton is largely disrupted but visible filaments are forming in some cells. Scale bar is 20 μm. (B) Y-27632 treatment did not induce CAA formation at earlier timepoints during recovery.