Abstract
Purpose :
The role of Wnt signaling in human trabecular meshwork (HTM) cell contractility remains unknown. Dexamethasone (Dex) has been proposed to induce Cross-Linked Actin Networks (CLANs) through the Wnt signaling pathway which can generate contractile forces. Additionally, observed cell heterogeneity in primary HTM cultures call for individual cell contractility measurements. Here, we present a novel assay known as FLECS, fluorescently labelled elastomeric contractible surfaces, developed by researchers at the UCLA bioengineering department (PMC5775112) to measure the contractility of thousands of cells at the single-cell level and asses the role of Wnt signaling in primary HTM cell contractility.
Methods :
To assess the role of Wnt signaling in HTM cell contractility using the FLECS assay, primary HTM cells were treated for five days with DMSO vehicle, Dexamethasone (Dex) (0.1µM), or Dex in the presence of the Wnt signaling inhibitor 3235-0367 (3µM) and transferred to a 24-well FLECS plate. Each well contained precise arrays of ‘X’ shaped fluorescent adhesive micropatterns embedded into soft substrates with controlled elasticity by using a unique microfabrication process. Single-cells adhered to, spread over, and applied contractile forces onto the micropatterns, resulting in observable displacements which may be quantified using computational algorithms.
Results :
Treatment with Dex resulted in strongly contracting HTM cells compared to the vehicle treatment as observed by the inward displacement of the terminals of a fluorescently labeled ‘X’ pattern. Treatment with Dex in the presence of Wnt signaling inhibitor 3235-0367 resulted in weakly contracting HTM cells compared to the Dex treatment only.
Conclusions :
Wnt signaling inhibition was associated with a reduction in primary HTM cell contraction. Additionally, the novel high-throughput FLECS contraction assay has the power to analyze contractility at the individual cell level and appears promising to study the role of trabecular meshwork contractility in a heterogeneous population of cells. Future experiments will quantify the degree of contraction of individual cells.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.