July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Actomyosin contractility and the vimentin cytoskeleton influence giant vacuole life-cycle in Schlemm’s canal endothelial cells
Author Affiliations & Notes
  • Darryl R Overby
    Bioengineering, Imperial College London, London, United Kingdom
  • Alice Spenlehauer
    Bioengineering, Imperial College London, London, United Kingdom
  • Andrea Cairoli
    Bioengineering, Imperial College London, London, United Kingdom
  • Joseph M Sherwood
    Bioengineering, Imperial College London, London, United Kingdom
  • Amir Vahabikashi
    Cell and Molecular Biology, Northwestern University, Evanston, Illinois, United States
  • W Daniel Stamer
    Ophthalmology, Duke University, Durham, North Carolina, United States
  • Chiu Fan Lee
    Bioengineering, Imperial College London, London, United Kingdom
  • Footnotes
    Commercial Relationships   Darryl Overby, None; Alice Spenlehauer, None; Andrea Cairoli, None; Joseph Sherwood, iPerfusion (F); Amir Vahabikashi, None; W Daniel Stamer, None; Chiu Fan Lee, None
  • Footnotes
    Support  NIH Grants EY019696 and EY022359 and a PhD studentship from the BBSRC
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 5122. doi:
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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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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : 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.

Methods : 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.

Results : 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).

Conclusions : 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.

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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