Abstract
Purpose :
The trabecular meshwork (TM) actin cytoskeleton plays a key role in regulating intraocular pressure (IOP). This study aims at understanding the role of cholesterol in the regulation of actin polymerization in TM.
Methods :
Human TM (HTM) cells were treated with SiR-Actin in serum-free media overnight and F-actin changes were recorded using live-cell time-lapse (every 5min) confocal imaging before and during exposure to A) 10mM methyl-β-cyclodextrin (MβCD) (1h) for acute cellular cholesterol removal, B) 100μM cholesterol-saturated MβCD (MβCD-CHOL) (1h) to enrich cholesterol. F-actin changes were calculated, and an unpaired t-test was used for statistical analyses and results were significant if p<0.05 with n= 3-4. HTM cells plated on coverslips were treated as mentioned above, C) with 100μM (24h) atorvastatin -cholesterol synthesis inhibitor, D) 10μM cytochalasin D (CYTD) (1h) -actin polymerization disruptor, E) 10μM CYTD (1h) followed by 100μM MβCD-CHOL (1h). Untreated cells acted as the control. After treatments, cells were fixed and immunofluorescence (IF) was performed for cholesterol using filipin, F-actin by phalloidin, focal adhesion (FA) -vinculin and paxillin-localization. Further F/G-actin in vitro assay was performed to quantify the actin polymerization changes under the conditions A–E compared to control.
Results :
Imaging analysis showed cholesterol removal significantly decreased F-actin from 20min sustaining up to 1h (p<0.05, n=3) and decreased FA distribution. Cholesterol enrichment significantly increased F-actin from 10min sustaining up to 1h (p<0.05, n=4) and reorganized FA. IF results showed that atorvastatin decreased F-actin and FA distribution. CYTD disrupted F-actin networks leaving remnant filament ends and decreased FA but cholesterol enrichment increased F-actin branches formed at ~70° angle indicating Arp2/3 activation. These results were confirmed by quantitative F/G-actin ratio decrease and increase upon cholesterol removal/synthesis inhibition and enrichment, respectively. Interestingly, F/G-actin ratio increased under CYTD treatment indicating that CYTD disrupted longer actin filaments but helped polymerization of newly formed actin filaments.
Conclusions :
This systematic evaluation of cholesterol modulation on TM actin polymerization identifies the significance of maintaining membrane and cellular cholesterol levels for achieving IOP homeostasis.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.