Illuminating Molecular Mechanisms of Serotonin Transporter Regulation with Quantum Dot Single Particle Tracking

Abstract

The serotonin transporter protein (SERT) terminates serotonin signaling in the brain by enabling rapid clearance of the neurotransmitter. SERT dysfunction has been associated with a variety of psychiatric disorders, including depression, anxiety, and autism. Visualizing SERT behavior at the single molecule level in endogenous systems remains a challenge. In this dissertation, quantum dot (QD) single particle tracking (SPT) is utilized to capture SERT dynamics in both a hyperphosphorylated disease-associated mutant model, Gly56Ala SERT, and primary rat midbrain neurons. Membrane microenvironment, specifically membrane cholesterol, plays a key role in SERT regulation and has been found to affect SERT conformational state. A key focus was to determine how reduced cholesterol content affects both lateral mobility and phosphorylation of conformationally-sensitive threonine 276 (Thr276) in endogenous SERT using two different methods of cholesterol manipulation, statins and methyl-beta-cyclodextrin. Both chronic and acute cholesterol depletion increased SERT lateral diffusion, radial displacement along the membrane, mobile fraction, and Thr276 phosphorylation levels. The hyperphosphorylated Gly56Ala SERT also displayed increased lateral diffusion and radial displacement compared to the wild type SERT. In addition to the SERT studies, progress towards monovalent aptamer-QD probes is detailed. The aptamer-QDs displayed 1:1 binding with extracellular GFP and were generalizable for a variety of neuronal proteins. Overall, this work has provided new insights about endogenous neuronal SERT mobility and its associations with membrane cholesterol and SERT phosphorylation status, as well as a new method for generating monovalent aptamer-QDs.