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Jeremy Hwang, Jose Gonzalez, James Tan; Live Imaging of Actin Structure in Human Trabecular Meshwork. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3528.
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To characterize the in-situ organization of live filamentous actin (F-actin) in viable ex-vivo human trabecular meshwork (TM) using 2-photon excitation fluorescence microscopy (TPEF).
Human corneoscleral donor rim tissue was cut into 30 degree wedges and transduced with adenovirus carrying a red fluorescence-conjugated F-actin marker (LifeAct). Tissues were incubated with virus in serum-free DME for 2h at 37°C and 8% CO2 and then overnight in complete medium. Live tissue was then analyzed with TPEF in situ at 24, 48 and 72h post-transduction. Some tissues were co-incubated with Calcein AM, a cytosolic intravital dye used to identify living cells. Some tissues (untreated and transduced) were fixed and labeled with Alexa-488-conjugated phalloidin for comparison.
In contrast to phalloidin-labeled fixed tissue, where cellular F-actin labeling was cortical at membrane edges and punctate in cytosolic and perinuclear regions, LifeAct-transduced TM cells showed a more diffuse intracellular F-actin pattern. Similar to phalloidin-labeled F-actin, LifeAct fluorescence was observed wrapped around or stretched across trabecular beams. LifeAct colocalized with F-actin. LifeAct expressing cells co-labeled with Calcein indicating viability. In these co-labeled cells, LifeAct had a more cortical distribution compared with the diffusely cytosolic distribution of Calcein. LifeAct expression was seen in the uveal meshwork and the superficial corneoscleral meshwork. Expression was patchy, likely due to a dependence on transduction efficiency, cellular viability and the functional barrier properties of the TM.
Changes in actin structure in the TM alter aqueous outflow facility and intraocular pressure. Modulating actin structure thus has therapeutic implications for glaucoma. We characterize a new system for studying actin dynamics in viable human TM tissue. A combined approach involving TPEF, LifeAct and phalloidin labeling may be useful for analyzing the effect of potential glaucoma therapies.
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