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Kate Keller, Ying Ying Sun, Yong-feng Yang; Tunneling nanotube formation by normal and glaucomatous trabecular meshwork cells in response to mechanical stretch. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2331.
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© ARVO (1962-2015); The Authors (2016-present)
Tunneling nanotubes (TNTs) are a novel actin-based method of cellular communication. TNTs are formed by normal (NTM) and glaucomatous trabecular meshwork (GTM) cells. Mechanical stretching, a proxy for elevated intraocular pressure (IOP), alters the actin cytoskeleton and induces focal extracellular matrix (ECM) remodeling at podosome and invadopodia-like structures (PILS). Here, we investigate cellular communication via TNTs in NTM and GTM in response to mechanical stretch. In addition, we investigate the effect of mechanical stretch on myosin-X (Myo10), a regulator of TNT formation, and its association with PILS proteins.
Primary NTM and GTM cells were characterized by standard methods. A vesicle transfer assay was used to quantitate communication via TNTs by TM cells with and without mechanical stretch. Western blots were performed to quantitate the levels of Myo10 after mechanical stretching of TM cells. Colocalization of Myo10 and PILS proteins was measured using Imaris software analysis of confocal images. A Duolink assay was used to quantitate the colocalization of Myo10 and PILS proteins in response to mechanical stretch. ANOVA was used to test significance.
NTM and GTM cells were mechanically stretched for 24 hours. Both NTM and GTM reduced vesicle transfer via TNTs by 23% and 20%, respectively (N=28-35; p≤0.01). Myo10 protein levels were increased by 6% in stretched GTM cells compared to NTM cells (n=10; p=0.04). PILS proteins include matrix metalloproteinases (MMPs) and integrins. Myo10 colocalized strongly with integrin-β1 (Pearson’s value = 0.4) in both NTM and GTM cells, as well as MMP2 and MMP14. A Duolink assay showed that MMP2 and MMP14 were significantly more associated in mechanically stretched NTM cells (n=3; p=0.0001). Similarly, Myo10 and integrin-αVβ3 became significantly more associated (p=0.003), but Myo10-integrin-α5β1 interactions were unaffected.
Colocalization of the actin-binding protein Myo10 with PILS proteins suggests coordination of the actin cytoskeleton with specific proteins of the ECM degradation complex in response to mechanical stretch. Our vesicle transfer results suggest that actin-rich TNTs are reduced in both NTM and GTM cells in response to stretch. This suggests that communication via TNTs may be more important for normal homeostasis of TM cells than for relaying signals related to changes in IOP.
This is a 2020 ARVO Annual Meeting abstract.
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