Myosin-X Silencing in the Trabecular Meshwork Suggests a Role for Tunneling Nanotubes in Outflow Regulation

Investigative Ophthalmology & Visual Science
Ying Ying SunKate E Keller

Abstract

The actin cytoskeleton plays a key role in outflow regulation through the trabecular meshwork (TM). Although actin stress fibers are a target of glaucoma therapies, the role of other actin cellular structures is unclear. Myosin-X (Myo10) is an actin-binding protein that is involved in tunneling nanotube (TNT) and filopodia formation. Here, we inhibited Myo10 pharmacologically or by gene silencing to investigate the role of filopodia/TNTs in the TM. Short hairpin RNA interference (RNAi) silencing lentivirus targeting myosin-X (shMyo10) was generated. Human anterior segments were perfused with shMyo10 or CK-666, an Arp2/3 inhibitor. Confocal microscopy investigated the colocalization of Myo10 with matrix metalloproteinase (MMPs). Western immunoblotting investigated the protein levels of MMPs and extracellular matrix (ECM) proteins. MMP activity and phagocytosis assays were performed. CK-666 and shMyo10-silencing lentivirus caused a significant reduction in outflow rates in anterior segment perfusion culture, an ex vivo method to study intraocular pressure regulation. In human TM cells, Myo10 colocalized with MMP2, MMP14, and cortactin in podosome-like structures, which function as regions of focal ECM degradation. Furthermore, MM...Continue Reading

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Citations

May 1, 2020·BioMed Research International·Holly R Chinnery, Kate E Keller
Nov 2, 2019·Investigative Ophthalmology & Visual Science·Ying Ying SunKate E Keller
May 13, 2020·Neural Regeneration Research·Kate E Keller
Mar 13, 2021·Pharmacological Research : the Official Journal of the Italian Pharmacological Society·Xiao-Tong WangYu-He Yuan
Aug 17, 2020·Progress in Retinal and Eye Research·Ted S AcottMary J Kelley

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Methods Mentioned

BETA
protein assay
confocal microscopy

Software Mentioned

FIJI

Related Concepts

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