Deciphering The Role Of Gtpases And The Cytoskeleton In Aquaporin-4 Vesicular Trafficking

Abstract

The AQP4 water channel is abundantly expressed in brain glial cells and contributes to swelling following brain trauma. Cell surface localization of AQP4 in mammalian cells fluctuates rapidly in response to changes in oxygen and tonicity, suggesting a role for vesicular trafficking in its translocation, but the molecular mechanism of this is not fully understood. The aim of this thesis was to decipher the role of key elements of vesicular trafficking in the movement of AQP4 to the plasma membrane. Early and recycling endosomes were selected as likely candidates of rapid AQP4 translocation. HEK293 cells were transiently transfected with AQP4-EGFP and either mCherry-Rab5 or mCherry-Rab11. It was found that AQP-EGFP colocalized with mCherryRab5 positive early endosomes and mCherry-Rab11 positive recycling endosomes. Under hypotonic conditions, AQP4-EGFP translocated from intracellular vesicles to the plasma membrane within 90 seconds. Co-expression of dominant negative forms of the mCherrytagged Rab5 and Rab11 with AQP4-EGFP resulted in no changes in the relative membrane expression of AQP4 following changes in tonicity. Inhibition of dynamin impaired internalisation of AQP4. Cytoskeleton modifying compounds also affected AQP4 translocation to and from the plasma membrane. Findings in HEK293 cells were confirmed and validated in human primary astrocytes using cell surface biotinylation. Through the work completed in this thesis, dynamin has been shown to be necessary for the internalisation of AQP4. Whether the mechanism is either clathrin-dependent or clathrin independent would need to be determined with further studies. The role of actin and microtubules is less clear; the results showed an involvement of actin and microtubules, but future work will determine whether F-actin or G-actin is involved, as well as whether microtubules are required for AQP4 vesicular targeting, or whether they are required for untargeted movement within the cell.