Cross-linked actin networks (CLANs) affect stiffness and/or actin dynamics in transgenic transformed and primary human trabecular meshwork cells.

Cross-linked actin networks (CLANs) in trabecular meshwork (TM) cells may contribute to increased IOP by altering TM cell function and stiffness. However, there is a lack of direct evidence. Here, we developed transformed TM cells that form spontaneous fluorescently labelled CLANs. The stable cells were constructed by transducing transformed glaucomatous TM (GTM3) cells with the pLenti-LifeAct-EGFP-BlastR lentiviral vector and selection with blasticidin. The stiffness of the GTM3-LifeAct-GFP cells were studied using atomic force microscopy. Elastic moduli of CLANs in primary human TM cells treated with/without dexamethasone/TGFβ2 were also measured to validate findings in GTM3-LifeAct-GFP cells. Live-cell imaging was performed on GTM3-LifeAct-GFP cells treated with 1 μM latrunculin B or pHrodo bioparticles to determine actin stability and phagocytosis, respectively. The GTM3-LifeAct-GFP cells formed spontaneous CLANs without the induction of TGFβ2 or dexamethasone. The CLAN containing cells showed elevated cell stiffness, resistance to latrunculin B-induced actin depolymerization, as well as compromised phagocytosis, compared to the cells without CLANs. Primary human TM cells with dexamethasone or TGFβ2-induced CLANs were also stiffer and less phagocytic. The GTM3-LifeAct-GFP cells are a novel tool for studying the mechanobiology and pathology of CLANs in the TM. Initial characterization of these cells showed that CLANs contribute to at least some glaucomatous phenotypes of TM cells.