Molecular Mechanisms of Mental Illness: Quantum Dot-based Single Molecule Investigation of the Dopamine Transporter and Its Protein Partners
Abstract
The human dopamine transporter (DAT) is a presynaptic transmembrane protein that facilitates the reuptake of synaptically released dopamine. Several lines of evidence indicate that DAT dysfunction is linked to neuropsychiatric disorders, such as autism spectrum disorder (ASD), bipolar disorder (BD), and attention deficit hyperactivity disorder. The lateral membrane diffusion and clustering propensity of DAT are emergent properties of functional dopamine signaling. This thesis work will be focused on interrogating DAT membrane diffusion dynamics utilizing a suite of independent labeling strategies. Using fluorescence microscopy, single particle tracking (SPT) of ASD/BD-associated DAT missense mutant A559V will be carried out by labeling them with antagonist-conjugated quantum dots (Qdots). The effects of the D2 dopamine receptor (D2R) modulation on aberrant DAT A559V diffusion will be investigated, including functional D2R antagonism and simultaneous tracking of both DAT and D2R. In addition, the propensity and extent of DAT and DAT A559V colocalization with D2R into membrane microdomains will be evaluated using a multiplexed Qdot-SPT strategy. While heterologous expression systems prove to be valid platforms in neuroscience, translating SPT studies to a native 3D neuronal architecture will be necessary for application in diagnosis and therapy for mental disease. To assess the degree to which DAT and its variants diffuse and cluster in vivo, our group has developed a novel antagonist-conjugated Qdot-SPT strategy that capitalizes on a Qdot design that yields exceptional photophysical properties and access to sterically hindered spaces in deep tissue to track native DAT in physiologically relevant environments such as brain tissue slices. This thesis work will be focused on probe optimization to reduce non-specific binding to maximize DAT membrane tracking confidence.
Description
Keywords
Dopamine transporter, Dopamine receptor, Quantum dot, Single-particle tracking