Abstract
Purpose :
Actin cytoskeletal organization and cell adhesive interactions of the trabecular meshwork (TM) are recognized to influence aqueous humor outflow and intraocular pressure in humans. However, the molecular mechanisms regulating such cellular characteristics of TM cells remain poorly understood. This study describes for the first time the role of septins, conserved GTP-binding cytoskeletal proteins that polymerize into filaments, in the regulation of actin cytoskeletal organization in human TM cells.
Methods :
The presence and distribution of septin isoforms in human TM cells were evaluated by mass spectrometry, immunoblot, and immunofluorescence analyses. The septin interactome of human TM cells was determined using SEPT9 antibody and proteomics analyses. Septin levels in dexamethasone and TGF-β2 treated TM cells were evaluated by immunoblotting. The effects of actin cytoskeleton and microtubule depolymerizing agents as well as inhibitors of septin and Rho kinase on septin and actin cytoskeletal organization were studied in TM cells by immunofluorescence.
Results :
Proteomics and immunoblot analyses readily identified the presence of various septin isoforms (Septin 2, 7, 9, 10, 11, 8 and 5) in human TM cells. Several of these septins revealed filamentous organization, co-localization with actin and microtubule networks, as well as distribution to the TM cell cortical, nuclear, and membrane regions. Dexamethasone and TGF-β2 treatments significantly increased SEPT9 and 11 levels in TM cells. Analysis of the human TM cell SEPT9 interactome identified regulatory proteins of the Rho GTPase, actin cytoskeleton, and cell adhesion pathways, in addition to the expected presence of several septins. Pharmacological inhibition of septin oligomerization decreased septin filaments and actin stress fibers in TM cells. While treatment with a Rho kinase inhibitor and latrunculin decreased septin filaments, nocodazole led to an increase in septin filament organization in human TM cells.
Conclusions :
This study reveals not only the presence of septin cytoskeleton but also its direct role in regulation of actin cytoskeletal organization, cell shape, and adhesion in TM cells. Based on the effects of the septin inhibitor on actin cytoskeletal organization in TM cells, we posit that septins are potential therapeutic targets for lowering intraocular pressure in glaucoma.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.