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Kate E Keller, Yong-Feng Yang, Justin Nguyen, Ying Ying Sun; The effect of substratum on trabecular meshwork cell morphology and cellular communication by tunneling nanotubes. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4720.
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© ARVO (1962-2015); The Authors (2016-present)
Trabecular meshwork (TM) cells form a continuous monolayer on the beams of the corneoscleral meshwork, but cells in the juxtacanalicular (JCT) region are sparse and interact via long cellular extensions. Some of these cell processes may form tunneling nanotubes (TNTs), which are actin-based structures that directly communicate signals and mitochondria via a tubular conduit. Studies with other cell types have demonstrated that extracellular matrix (ECM) influences TNT formation. ECM composition differs in the corneoscleral meshwork and JCT as well as in glaucoma versus normal TM. Here, we cultured TM cells on different ECM molecules, or on substrates that mimic the stiffness of normal and glaucomatous TM, to reveal factors that influence TNT formation.
Human TM cells were cultured on collagen I, collagen IV, elastin, laminin, and Pronectin, or on substrates of defined stiffness (4, 12, and 50 kPa) for 24 hours. Actin was labeled with Spirochrome SiR-actin and cell membranes were immunolabeled with CD44. Z-stack images were acquired by confocal microscopy. Imaris software (Bitplane) was used to calculate the area and volume of cells. To measure cellular communication via TNTs, TM cells were labeled separately with fluorescent Vybrant dyes, DiO and DiD, or MitoTracker and the number of cells containing vesicles/mitochondria of the opposite color were counted.
TM cells cultured on elastin were approximately 24% larger in area and volume (8,444 µm2, 11,077 µm3; n=42) than those cultured on collagen type IV (6403 µm2, 8497 µm3; n=59; p<0.05). TM cells cultured on elastin and collagen type I showed formation of prominent thick actin stress fibers, whereas stress fibers were largely absent when TM cells were cultured on collagen type IV. Seventy percent of TM cells cultured on laminin contained transferred mitochondria, whereas only 50% of cells cultured on collagen type IV had transferred mitochondria. As stiffness increased, the percentage of cells containing vesicles of the opposite color significantly increased from 35% (4kPa) to 51% (50 kPa; n=19 images; p<0.05).
Our data clearly demonstrate that ECM molecules and substrate stiffness affects TM cellular morphology and cellular communication via TNTs. Because ECM composition and stiffness varies in glaucoma TM, these results may reveal factors that maintain the health of endogenous TM cells in situ.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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