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Darryl R Overby, Alice Spenlehauer, Andrea Cairoli, Joseph M Sherwood, Amir Vahabikashi, W Daniel Stamer, Chiu Fan Lee; Actomyosin contractility and the vimentin cytoskeleton influence giant vacuole life-cycle in Schlemm’s canal endothelial cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5122.
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Giant vacuoles (GVs) are pressure-dependent cellular outpouchings that impose significant stretch on the inner wall endothelium of Schlemm’s canal (SC). Micron-sized pores, which provide pathways for flow across an otherwise continuous endothelium, are frequently associated with GVs and form in response to stretch. GVs, by influencing pores, may thereby regulate outflow and hence IOP. We investigated the hypothesis that actomyosin contractility and vimentin intermediate filaments control the life-cycle of GVs.
Human SC cells were seeded at confluency on track-etched filters and cultured for 24 hrs. For live-imaging, cells were stained with fluorescent dye (calcein-AM) and treated with the rho-kinase inhibitor, Y-27632 (25 µM) or vehicle (n = 4 or 5 filters). Cells were perfused in the basal-to-apical direction at 2 mmHg, followed by 6 mmHg using a modified version of iPerfusion mounted on a fluorescence microscope. GVs were tracked using an active contour algorithm. For cytoskeletal imaging, SC cells were perfusion-fixed and imaged by confocal microscopy after labelling the nucleus, actin and vimentin intermediate filaments.
We tracked 102 GVs in vehicle and Y-27632-treated filters (n = 60 and 42). Y-27632, which reduces actomyosin contractility, increased the fraction of GVs forming at 2 mmHg from 30% to 62% (p = 0.001, Chi-squared test). Y-27632 tended to increase the maximum GV size at 6 mmHg relative to vehicle-treated controls (p = 0.06). After reaching a maximum size, a fraction of GVs collapsed and disappeared entirely. Y-27632 reduced the fraction collapsing from 68% to 22% (p = 0.001). Confocal microscopy revealed a peripheral band of actin surrounding some GVs and a dense vimentin network enveloping the GV wall (Figure).
Live-imaging revealed GVs as dynamic structures that form, grow and collapse over a life-cycle of minutes. Actomyosin contractility controls the threshold pressure for GV formation, influences GV size, and allows GVs to collapse despite an opposing pressure drop. Organized actin and vimentin structures within the GV wall implicate the cytoskeleton as a key regulator of the GV life-cycle.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
The cytoarchitecture of a GV, showing the nucleus (blue), vimentin (green) and actin (red). Note the actin band (arrowhead) and vimentin network (asterisk) surrounding the GV.
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