June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Influence of hyaluronan depletion on CD44 and tunneling nanotubes in trabecular meshwork cells
Author Affiliations & Notes
  • Kate E Keller
    Casey Eye Institute, Oregon Health & Science University, Portland, Oregon, United States
  • Yong-feng Yang
    Casey Eye Institute, Oregon Health & Science University, Portland, Oregon, United States
  • Ying YIng Sun
    Casey Eye Institute, Oregon Health & Science University, Portland, Oregon, United States
  • Footnotes
    Commercial Relationships   Kate Keller None; Yong-feng Yang None; Ying YIng Sun None
  • Footnotes
    Support  NIH R01 EY019643, R01 EY032590, P30 EY010572, and unrestricted departmental funding from Research to Prevent Blindness (New York, NY)
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 3448. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Kate E Keller, Yong-feng Yang, Ying YIng Sun; Influence of hyaluronan depletion on CD44 and tunneling nanotubes in trabecular meshwork cells. Invest. Ophthalmol. Vis. Sci. 2023;64(8):3448.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose : Tunneling nanotubes (TNTs) are actin-rich membrane protrusions. TNT biogenesis involves rearrangement of the plasma membrane and intracellular actin cytoskeleton. We hypothesized that extracellular molecules tethered to cell surface receptors stabilize the membrane during actin rearrangement into TNTs. In this study, we focused on hyaluronan (HA), a glycosaminoglycan that binds CD44, which in turn, interacts with actin. Since there are reduced HA levels and hypophosphorylated CD44 in glaucoma TM, we investigated the effects of HA depletion on CD44, the actin-binding protein Myosin-X (Myo10), which induces TNT formation, and cellular communication via TNTs in trabecular meshwork (TM) cells.

Methods : HA levels were reduced by two methods: digestion with hyaluronidase, or by treatment with 1mM 4-methylumbelliferone (4MU). These treatments were added to primary human TM cells for 0-60 minutes, or for up to 2 days. In some experiments, TM membrane fractions were isolated from cytosolic proteins using MEM-Per reagent. Western immunoblots were performed. Confocal microscopy was used to evaluate the localization of phosphorylated CD44. A vesicle transfer assay determined cellular communication via TNTs. Live cell imaging monitored TM cell phenotype and cell migration.

Results : CD44 is reduced in the membrane following 15-60 minutes of 4MU treatment, while there is a concomitant increase in the cytosol. Confocal microscopy showed increased phosphorylated CD44 in the cytosol of 4MU-treated TM cell at 60 minutes. After 24 hours of 4MU treatment, Myo10 protein levels increased 30% (p=0.008). There was a slight increase (5%) in vesicles being shared between cells at 24 hours following HA depletion with 4MU, but the results were not statistically significant (p=0.228). Live cell imaging showed that 4MU treatment induced a more spindle-like TM cell morphology and reduced cell migration compared to vehicle-treated cells over 48 hours.

Conclusions : Our results suggest that depletion of HA causes rapid changes to the localization and phosphorylation of CD44, which could affect downstream signaling pathways. Increased Myo10 levels suggest TNT induction, and although vesicle transfer via TNTs was not significant at 24 hours, longer time points should be assessed. Together, our results show that the HA-CD44-actin interactions are important to maintain normal phenotype and function of TM cells.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×