Engineering Genetically Encoded Sensors to Study Purinergic Signaling
Abstract
Extracellular adenosine triphosphate (ATP) is a key purinergic signal that mediates cell-to cell communication both within and between organ systems. We address the need for a robust and minimally invasive approach to measuring extracellular ATP by re-engineering the ATeam ATP sensor to be expressed on the cell surface. Using this approach, we image real-time changes in extracellular ATP levels with a sensor that is fully genetically-encoded and does not require an exogenous substrate. Using live-cell microscopy, we characterize sensor performance when expressed on cultured Neuro2A cells, and we measure both stimulated release of ATP and its clearance by ectonucleotidases. In addition, we increase the affinity of ATeam threefold by mutating the hinge region (R103A/R115A) of the epsilon subunit. Thus, this proof-of-principle demonstrates a sensor to report extracellular ATP dynamics that may be useful for studying purinergic signaling in living specimens.
Degree
M.S.
Advisors
Tantama, Purdue University.
Subject Area
Biochemistry
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.