Degree

Doctor of Philosophy (PhD)

Department

Biological Sciences

Document Type

Dissertation

Abstract

Protein-protein interactions vary among cell types and the localization of these interactions help to define protein function. The function of cystic fibrosis transmembrane conductance regulator (CFTR) is well established in the context of epithelial tissue due to the debilitating effects of its mutations that cause the disease cystic fibrosis. While in neurons a role has been established in our lab for CFTR in the regulation of cytosolic Cl- in retinal amacrine cells (ACs). This research sets out to learn if CFTR has a synaptic role. Exploring this potential role, localization of CFTR was examined using fluorescent and immunogold labeled transmission electron microscopy to achieve subcellular resolution of CFTR throughout the adult chicken. CFTR was found in areas of the retina that include the ellipsoidal mitochondria, and multiple locations of the inner retina including conventional and ribbon synapses. A role in synaptic transmission was tested by recording spontaneous postsynaptic quantal currents that were found to increase in frequency upon pharmacological inhibition of CFTR. This elevation in frequency points to a presynaptic role. Interestingly, in epithelia there is an established functional interaction between CFTR and the SNARE protein Syntaxin 1A. This interaction was verified in the retina and brain by a CFTR baited co-immunoprecipitation of Syntaxin 1A and fellow SNARE SNAP25. Further verification by an in vitro binding assay showed a more than 2-fold increase of STX1A and SNAP25 when the co-immunoprecipitation was supplemented with purified CFTR protein. The localization of this interaction in ACs was explored using fluorescent microscopy and proximity ligation assay showing a strong co-localization between CFTR and STX1A in the processes of retinal ACs where synapses occur. To broaden our understanding of observed protein interactions with CFTR, eluate from a bait CFTR co-immunoprecipitation was analyzed via mass spectrometry. Our analysis found 621 CFTR interactors in brain tissue. Following data filtering, 89 proteins were found to be a synaptic protein using the databases Gene Ontology, UniProtKB, and with literature searches. Overall, the broad scope of proteins identified in our analysis suggest that CFTR has multiple functions in the nervous system.

Date

4-20-2025

Committee Chair

Evanna Gleason

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