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Kate E Keller, John M Bradley, Ted S Acott; Unidirectional transfer of cellular cargo between trabecular meshwork cells via novel actin-based filopodia. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3265. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Various glaucoma drugs targeting the actin cytoskeleton of trabecular meshwork (TM) cells increase outflow facility. However, the underlying mechanisms by which these drugs act are only partially understood. A novel class of specialized filopodia including cytonemes and tunneling nanotubes (TNTs) has been described. These actin-based structures allow the direct transfer of molecular cargo between cells over long distances. Here, we investigated the existence of TNTs and/or cytonemes in TM cells and tissue.
Half of a flask of TM cells was labeled with the fluorescent membrane dye DiO (488 nm; Invitrogen) and the other half with DiD or Mitotracker Red (647 nm). The cells were mixed 1:1 at 1 x 105 cells/well, cultured overnight and then fixed and immunostained with CD44. Additionally, live-cell microscopy using a Deltavision Core DV microscope was performed. To measure the amount of DiO transferred between cells, unlabeled and DiO-labeled cells were cultured in separate chambers and then co-cultured for 48 hours. Transfer of DiO fluorescence to unlabeled cells was quantitated using a plate reader. Alexa-fluor phalloidin was used to stain F-actin in human TM cadaver tissue.
Confocal images of CD44-immunostained cells show the formation of long filopodia (~ 240 µm) extending between DiO-labeled TM cells and DiD-stained cells. DiO-stained vesicles were clearly visible in the filopodia and in the cytoplasm of a DiD-stained cell. Conversely, no DiD was present in the DiO cell. In other experiments, Mitotracker-labeled mitochondria were found in a DiO-labeled cell. Live-cell microscopy showed the direct transfer of DiO-stained vesicles via tubular conduits formed between adjacent TM cells. In the co-culture assay, approximately 23% of DiO was transferred to unlabeled cells after 48 hours. Low-dose Latrunculin B, an actin depolymerizer, significantly reduced DiO transfer; p<0.05. In human TM tissue, Phalloidin staining detected a long cell process (~160 µm) extending across an intertrabecular space.
Our results support the formation of cytonemes in TM cells and tissue and show the unidirectional transfer of cellular cargo via tubular conduits. Cytonemes and TNTs may provide a means by which cells in different anatomical regions of the TM can directly and rapidly communicate with each other to modify outflow resistance.
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