The role of α-actinin and RhoA in the mechanical properties of Schlemm's canal cells

Abstract

Purpose : Schlemm’s canal (SC) cells isolated from glaucomatous donor eyes are stiffer than cells isolated from normal donor eyes (Overby et al. 2014). Increased stiffness of glaucomatous SC cells leads to a decrease in pore-forming ability, likely contributing to increased outflow resistance in ocular hypertension. To explore ways of directly testing the impact of stiffness on SC cell physiology in a controlled fashion, we constructed two adenoviruses encoding cytoskeletal modulators and measured their ability to alter SC cells mechanics.

Methods : Two adenoviruses were engineered, one encoding α-actinin (tagged with a GFP reporter) and the other constitutively active RhoA (CA-RhoA), both driven by an ubiquitin promoter. Primary cultures of SC cells (n_normal=4, n_glaucoma=1) were transduced with adenovirus (MOI=100). Non-transduced cells were used as the first control and an adenovirus construct containing ubiquitin promoter and GFP reporter with no intervening sequence was used as the second control. Atomic force microscope (AFM) was used to measure cortical and subcortical stiffness in cells (15-20 cells per group) as described previously (Vargas-Pinto et al. 2013). Traction force microscopy (TFM) was used to assess traction forces on cell substrate. We used Western blot analysis to determine recombinant and endogenous protein expression and confocal microscopy to visualize F-actin distribution.

Results : AFM and confocal imaging results showed that doubling expression of α-actinin or CA-RhoA in SC cells increased cortical actin content, with a corresponding elevation in cortical stiffness (Fig. 1: p<0.03), but does not change the subcortical stiffness (p>0.15). TFM studies indicate that overexpression of RhoA (Fig. 2: p<0.05), but not α-actinin (p>0.7) increased traction forces in SC cells.

Conclusions : Overexpression of either α-actinin or CA-RhoA significantly increased cortical actin content and stiffness of human SC cells in culture. Moreover, CA-RhoA overexpression increased traction forces in cultured SC cells. Future studies will determine the impact of virus-directed increase of SC cell stiffness on pore formation in vitro and outflow resistance in situ.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

 

Figure 1: Young’s modulus (mean±SE) for cortical stiffness of the five SC cell strains examined.

 

Figure 2: TFM measurements (mean+SD) for SC cells.