June 2013
Volume 54, Issue 15
ARVO Annual Meeting Abstract  |   June 2013
Live Imaging of Actin Structure in Human Trabecular Meshwork
Author Affiliations & Notes
  • Jeremy Hwang
    Ophthalmology, University of Southern California, Los Angeles, CA
  • Jose Gonzalez
    Ophthalmology, University of Southern California, Los Angeles, CA
  • James Tan
    Ophthalmology, University of Southern California, Los Angeles, CA
  • Footnotes
    Commercial Relationships Jeremy Hwang, None; Jose Gonzalez, None; James Tan, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 3528. doi:
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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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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: 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).

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: 735 trabecular meshwork • 411 adenovirus • 551 imaging/image analysis: non-clinical  

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