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Kate E Keller, John Bradley, Ying Ying Sun, Yong-feng Yang, Ted S Acott; The role of Myosin-X in tunneling nanotube formation by trabecular meshwork cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4691. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Tunneling nanotubes (TNTs) are specialized actin-based filopodia that allow unidirectional transfer of molecular cargo between cells through tubular conduits. Our previous work using live-cell imaging established that cellular vesicles and mitochondria are directly transferred between trabecular meshwork (TM) cells via TNTs. Myosin-X (Myo10) is a critical regulator of TNT formation. In this study, we investigate Myo10 distribution in TM cells and normal and glaucomatous TM tissue and measure the effects of disruption of filopodia formation on outflow in perfusion culture.
Primary cultured TM cells and normal and glaucomatous human cadaver TM tissue were stained with anti-Myo10 antibodies and subject to confocal microscopy. shRNA lentivirus targeting Myo10 was generated and knockdown of Myo10 mRNA and protein was measured using quantitative RT-PCR and Western immunoblotting. Myo10 shRNA lentivirus and CK-666, an Arp2/3 inhibitor, were applied to human anterior segments in perfusion culture and outflow rates were measured for a further 72 hours.
Myo10 labeled punctate dots at the tips of filopodia in cultured TM cells. In glaucomatous TM tissue, the expression pattern of Myo10 was severely disrupted compared to normal age-matched TM tissue. shMyo10 lentivirus reduced Myo10 mRNA expression and protein levels in TM cells. Application of shMyo10 lentivirus and CK-666 to human anterior segments significantly reduced outflow in perfusion culture.
TNTs are a novel method by which TM cells can directly communicate with each other. Disruption of filopodia/TNT formation by reducing Myo10 levels increases outflow resistance. Investigating TNT formation by TM cells not only provides an important new understanding of how the actin cytoskeleton participates in intraocular pressure regulation, but also how cells can communicate in a fluid environment.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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