June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Unidirectional transfer of cellular cargo between trabecular meshwork cells via novel actin-based filopodia
Author Affiliations & Notes
  • Kate E Keller
    Ophthalmology, Casey Eye Insitute - OHSU, Portland, OR
  • John M Bradley
    Ophthalmology, Casey Eye Insitute - OHSU, Portland, OR
  • Ted S Acott
    Ophthalmology, Casey Eye Insitute - OHSU, Portland, OR
  • Footnotes
    Commercial Relationships Kate Keller, None; John Bradley, None; Ted Acott, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 3265. doi:https://doi.org/
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    • Get Citation

      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)

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Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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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