Abstract
PURPOSE: The actin cytoskeleton of trabecular meshwork (TM) cells plays a role in regulating aqueous humor outflow. Many studies have investigated stress fibers, but F-actin also assembles into other supramolecular structures including filopodia. Recently, specialized filopodia called tunneling nanotubes (TNTs) have been described, which communicate molecular signals and organelles directly between cells. Here, we investigate TNT formation by TM cells. METHODS: Human TM cells were labeled separately with the fluorescent dyes, DiO and DiD, or with mitochondrial dye. Fixed or live TM cells were imaged using confocal microscopy. Image analysis software was used to track fluorescent vesicles and count the number and length of filopodia. The number of fluorescently labeled vesicles transferred between cells was counted in response to specific inhibitors of the actin cytoskeleton. Human TM tissue was stained with phalloidin. RESULTS: Live-cell confocal imaging of cultured TM cells showed transfer of fluorescently labeled vesicles and mitochondria via TNTs. In TM tissue, a long (160 μm) actin-rich cell process bridged an intertrabecular space and did not adhere to the substratum. Treatment of TM cells with CK-666, an Arp2/3 inhibitor, significantly decreased the number and length of filopodia, decreased transfer of fluorescently labeled vesicles and induced thick stress fibers compared to vehicle control. Conversely, inhibiting stress fibers using Y27632 increased transfer of vesicles and induced long cell processes. CONCLUSIONS: Identification of TNTs provides a means by which TM cells can directly communicate with each other over long distances. This may be particularly important to overcome limitations of diffusion-based signaling in the aqueous humor fluid environment.