Abstract
Purpose :
Cross-linked actin networks (CLANs) in trabecular meshwork (TM) cells may contribute to increased IOP by altering TM cell function and stiffness. CLANs have been observed in TM cells and tissues, but little is known about their formation or dynamics. We developed a transformed TM cell line that forms spontaneous, fluorescently labeled CLANs. Using live cell imaging, we characterized the actin dynamics of transformed HTM cells treated with latrunculin B.
Methods :
A stable cell line was constructed by transducing transformed glaucomatous TM (GTM3) cells with the pLenti-Lifeact-EGFP-BlastR lentiviral vector. CLANs in cells were further increased upon treatment with different concentrations of blasticidin. The transduced GTM3 cells were treated with 1uM latrunculin B for 2 hrs, and time lapse fluorescent images were recorded in 1 min intervals during treatment and 2 hrs after removal of the agent.
Results :
The transformed human TM cell line with stably transduced Lifeact-GFP expression cassette demonstrated the morphology of actin stress fibers and the spontaneous formation of CLANs using fluorescent microscopy. The number of CLANs in cell cultures were increased when treated with high concentrations of blasticidin at 10ug/ml (p<0.05, N=3) and 20ug/ml (p<0.05, N=3). Using live cell imaging, we found that CLANs were more resistant to actin depolymerization agent latrunculin B, as compared to actin stress fibers.
Conclusions :
Live cell imaging of a CLAN forming transformed human TM cell line suggests that CLANs are a stable structure and likely play an important role in elevated outflow resistance and glaucoma.
This is a 2021 ARVO Annual Meeting abstract.