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

Abstract

Purpose: 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.

Methods: 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.

Results: 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, MMP activity, thrombospondin-1 and SPARC protein levels were significantly reduced in the media of CK-666-treated and shMyo10-silenced TM cells. However, neither Myo10 silencing or CK-666 treatment significantly affected phagocytic uptake.

Conclusions: Inhibiting filopodia/TNTs caused opposite effects on outflow compared with inhibiting stress fibers. Moreover, Myo10 may also play a role in focal ECM degradation in TM cells. Our results provide additional insight into the function of actin supramolecular assemblies and actin-binding proteins in outflow regulation.

Figure 1

Generation of shRNA Myo10-silencing lentivirus (shMyo10) silencing lentivirus. Efficacy of Myo10 knockdown in human TM cells by infection with shMyo10-silencing lentivirus was tested by (A) quantitative RT-PCR using Myo10-specific primer sets. *P = 0.0001; n = 4 from 4 biological replicates. (B) Western immunoblotting of Myo10 with tubulin as a loading control and (C) densitometry of Myo10 knockdown. **P = 0.001; n = 7 from 5 biological replicates. Immunofluorescence of Myo10 in (D) shCtrl and (E) shMyo10-silenced human TM cells. Insets show DAPI staining. Scale bar: 20 μm.

Figure 2

Human anterior segment perfusion culture. Following baseline stabilization, treatments were applied at time point 0 as follows: (A) a bolus of 100 μM CK-666 (n = 4) or 0.04% DMSO vehicle control (n = 3), or (B) shRNA Myo10-silencing lentivirus (shMyo10; n = 7) or shControl (shCtrl; n = 3). Flow rate data at each time point were normalized to the average flow rate before treatment. Data from individual eyes were then averaged. Error bars are the SEM. *P < 0.01, **P < 0.05 by ANOVA. (C) Representative radial sections of TM tissue postperfusion stained with hematoxylin and eosin (C, E, G), or immunostained with anti-Myo10 (red; D, F, H). DAPI stained nuclei blue. TM, trabecular meshwork; SC, Schlemm's canal. Scale bar: 20 μm.

Figure 3

Effects of filopodia/TNT inhibition on MMP activity by TM cells. (A) Myo10 localizes to podosome-like structures in human TM cells, where is partially colocalizes with (B) cortactin, a podosome biomarker, (C) MMP2, and (D) MMP14. Scale bars = 10 μm. (E) ADAMTS4 and MMP activity assays of human TM cells treated with CK-666 or shMyo10-silenced TM cells. MMP activity in the medium was significantly reduced by CK-666 (compared to DMSO vehicle) and shMyo10 (compared to shCtrl). *P = 0.048, **P = 0.00001; n = 3 from 3 biological replicates. (F) Representative Western immunoblots of MMP14 and MMP2 in CK-666 treated and shMyo10-silenced human TM cells compared to their respective controls. Densitometry of n = 4 immunoblots from three biological replicates for each treatment. *P = 0.041, **P = 0.001.

Figure 4

ECM protein levels after CK-666 and shMyo10 treatment in TM cells. (A) Representative Western immunoblots of FN, TNC collagen type I (COLI), SPARC, TSP-1, and collagen type IV (COLIV) in human TM cell medium after 24 hours of CK-666 or DMSO (vehicle) treatment. (B) Densitometry of Westerns (n = 4 from three biological replicates) for each protein. Error bars are the SEM. *P < 0.05 by ANOVA. (C) Densitometry of Western immunoblots for SPARC and TSP-1 following shMyo10 silencing. Error bars are the SEM; n = 3 from three biological replicates. *P = 0.0005 by ANOVA.

Figure 5

Phagocytosis assay in shMyo-silenced and CK-666-treated TM cells. TM cells were imaged for 16 hours by the Incucyte ZOOM in the presence of opsonized pHrodo Red S. aureus bBioparticles. Fluorescence intensity was measured in (A) shMyo10-silenced and (B) CK-666-treated TM cells compared to their relevant controls (n = 27 from n = 3 biological replicates). Error bars are the SEM. (C) Representative images from the phagocytosis assay at 0, 4, 12, and 16 hours after application of the pHrodo bioparticles.

Figure 6

Schematic of the actin cytoskeleton of a TM cell summarizing data found in this study as well as that found in the literature. Inhibiting filopodia/TNTs by shMyo10 silencing lentivirus or CK-666 inhibition of the Arp2/3 complex decreases outflow and reduces vesicle transfer via TNTs. Conversely, relaxing actin stress fiber formation using Rho kinases inhibitors increases outflow and vesicle transfer via TNTs.