Single quantum dot approach for molecular dissection of serotonin transporter regulation in living cells
Chang, Chia Hua
The presynaptic serotonin (5-HT) transporter (SERT) is targeted by widely prescribed antidepressant medications. Altered SERT expression or regulation has been implicated in multiple neuropsychiatric disorders, including anxiety, depression and autism. It has been previous reported that SERT is regulated by lipid raft, a cholesterol-enriched subdomain in the plasma membrane that has been frequently reported a platform to facilitate neuronal signaling. To better understand the membrane diffusion dynamics of SERT, we developed a single quantum dot (QDot) tracking approach that exploits antagonist-conjugated single QDots to monitor, for the first time, single SERT proteins on the surface of serotonergic cells. We document two pools of SERT proteins defined by lateral mobility, one that exhibits relatively free diffusion, and a second, localized to cholesterol and GM1 ganglioside-enriched microdomains, that displays restricted mobility. Receptor-linked signaling pathways that enhance SERT activity mobilize transporters that, nonetheless, remain confined to membrane microdomains. Mobilization of transporters arises from a p38 MAPK-dependent untethering of the SERT C terminus from the juxtamembrane actin cytoskeleton. Our studies establish the utility of single QDot tracking approach for analysis of the behavior of single membrane proteins and reveal a physical basis for signaling-mediated SERT regulation. In line with our single SERT analysis, single QDot-labeled ganglioside GM1 was incorporated in this thesis that aimed to quantitatively measure the diffusion dynamics and membrane compartmentalization of lipid raft in living RN46A cells. Diffusion measurements revealed that single QDot-labeled GM1 ganglioside complexes undergo slow, confined lateral diffusion with a diffusion coefficient of 7.87 × 10-2 μm2/s and a confinement domain about 200 nm in size. Further analysis of their trajectories showed lateral confinement persisting on the order of tens of seconds, comparable to the time scales of the majority of cellular signaling and biological reactions. Hence, our results provide further evidence in support of the putative function of lipid rafts as signaling platforms. Finally, we discussed the recent progress of single-QDot techniques, with emphasis on their applications in exploring membrane dynamics and intracellular trafficking. In recent years, single QDot imaging approach has been introduced as a sub-category of single molecule fluorescent techniques for revealing the single protein/vehicle dynamics in real-time. One of the major advantages of using single QDots is the high signal-to-noise ratio, which is beneficial due to the unique photophysical properties of QDot such as extraordinarily high molar extinction coefficients and large Stokes shifts. Although there are some limitations due to the physical nature of the QDots, advances in QDot synthesis and surface chemistry show significant potential to eliminate these pitfalls. Considering the applications of a single QDot approach in the past few years, we are optimistic that the use of single QDots will largely advance our understanding in the biological research field.