As a biophysicist and a joint faculty member in both the division of Life Sciences and the department of Physics, I have worked on interdisciplinary fields which involve physics and biology. In particular, I have worked on neuroscience and biophysics using advanced fluorescence imaging techniques.
In neuroscience, my lab has studied the exocytosis and motion of vesicles at single presynaptic levels in living neurons to understand synaptic transmission and their roles in neurodegeneration in Huntington’s disease (a neurodegenerative disease). First, we found the significant decrease in the release and transport of brain-derived neurotrophic factor (BDNF), which is essential for neuronal survival and synaptic activity, in neurons from zQ175 mice (HD mice). The decrease in exocytosis and transport in HD suggest that decreased delivery of BDNF from cortical neurons to the striatum may play a key role in the vulnerability of striatal neurons in HD. We also found an alteration in in synaptic vesicle release in neurons at presynaptic terminals in HD during electrical stimulation using real-time imaging of FM 1-43‒labeled synaptic vesicles. Furthermore, we investigated the exocytosis and motion of single synaptic vesicles in living neurons using our novel real-time three-dimensional (3D) nanometer-accuracy tracking of single synaptic vesicles with an accuracy of tens of nanometers up to the moment of exocytosis. We found that the displacement and motion of single exocytosed synaptic vesicles in HD neurons was significantly altered and that synaptic vesicles with high release probability tend to reside at the similar location as vesicles with low release probability in HD neurons (but not in WT neurons). Interestingly, we also found that non-releasing HD vesicles have a higher frequency of irregular oscillatory motion compared with non-releasing WT neurons, resulting in a significantly larger radius of gyration. These results provide the first direct evidence that the exocytosis, dynamics, and pool of single synaptic vesicles are altered of the early stages of this devastating neurodegenerative disease and suggest that the defect in the function of synaptic vesicles play an important role in neurodegeneration.
Our real-time 3D nanometer-accuracy tracking of single synaptic vesicles has provided detailed mechanisms about exocytosis of single synaptic vesicles. Single vesicles are known to undergo multiple rounds of kiss-and-run fusion. Using real-time 3D nanometer-accuracy tracking of quantum dot (Qdot)‒conjugated antibodies against the luminal domain of synaptotagmin 1 (Syt1) to label single synaptic vesicles, we found that the majority of these synaptic vesicles after the first kiss-and-run fusion undergo a second exocytosis within 10 sec of the first event and within 120 nm of the first fusion site, indicating that after undergoing kiss-and-run fusion, the vesicle remains relatively close to its original fusion site and release repeatedly at brief intervals, which enables the neuron to maintain bursting activity. The real-time 3D tracking also provided the detailed motion of single synaptic vesicles before exocytosis. We found that single synaptic vesicles have three distinct patterns of motion dynamics in terms of fusion time and net displacement during the stimulation before the first fusion. These distinct motion dynamics based on their location in presynaptic terminals may support synaptic transmission at different types of neuronal stimulations. Using Qdot-conjugated antibodies against the luminal domain of the vesicular GABA transporter (VGAT), we selectively labeled single GABAergic vesicles and found that net displacement during fusion differs significantly between inhibitory synaptic vesicles and Syt1-labeled synaptic vesicles (mainly excitatory synaptic vesicles). Interestingly, early releasing inhibitory vesicles are closer to their fusion site compared to late releasing vesicles, similar to Syt1-labeled vesicles. Thus, we concluded that inhibitory synaptic vesicles have unique dynamics distinct from excitatory vesicles though they share the similar properties of synaptic vesicle pool.
In biophysics, we developed several new imaging techniques which will contribute to addressing biological questions. We developed an automatic method for real-time subpixel-accuracy tracking of single mitochondria in living cells, a method for simultaneous nanometer-accuracy localization and FRET measurements, and a method for measuring force and FRET in living cells. Using these methods and other advanced imaging methods, we have addressed important biological questions. We investigated the detailed mechanism of store-operated Ca2+ entry using the single particles tracking and live-cell FRET measurements. We observed the significant decrease in the motion and significant increase in the non-Gaussianity of single STIM1 (a Ca2+ sensor in the endoplasmic reticulum (ER) membrane) and Orai1 (a pore-forming subunit of the Ca2+ release-activated calcium (CRAC) channel in the plasma membrane) in the ER and plasma membrane after store depletion. Detailed analyses and simulations revealed that that single STIM1 and Orai1 particles are confined in the compartmentalized membrane both before and after store depletion, and the changes in the motion after store depletion are explained by increased confinement and polydispersity of STIM1-Orai1 complexes formed at the ER-plasma membrane junctions. Live-cell FRET revealed that IDSTIM (inactivation domain of STIM1) binds to and inhibits CC1-CAD (coiled-coil domain 1-CRAC activating domain) and that this inhibitory effect of IDSTIM on CC1-CAD is abolished by either CC1α1 deletion or leucine substitution. We also found that the conserved short linker between CC1-CAD and IDSTIM facilitates the IDSTIM-mediated inactivation of STIM1. Lastly, we studied the stepping mechanism of single myosin X (the first anti-parallel coiled-coil myosin dimer) molecules. In vitro motility of single myosin X revealed that myosin X take variable-size step with multiple peaks, which allows myosin X to preferentially move along actin bundles. We also found that myosin X undergo frequent near simultaneous steps, which is a strikingly different mechanism that myosin V and VI use. We also found that one head takes 2-3 steps less frequently before the other head takes a step. In addition, we studied the motion and function of myosin X in living cells using our real-time nanometer-accuracy tracking system and labeling myosin X with tetramethylrhodamine. Our real-time nanometer-accuracy tracking showed that myosin X take highly variable step sizes with multiple peaks in living cells, which is consistent with our in vitro results. In addition, introducing a mutation in myosin X prevented the induction and elongation of filopodia, confirming that myosin X are required for the formation of filopodia living cells.