Shigeki Watanabe

My lab studies the cellular and molecular basis of synaptic transmission and plasticity. Neuronal signaling events at synapses determine circuit responses and result in specific behavioral outputs. This signaling is dynamic – modulated by synapse activity history and perceived stimuli. Three basic parameters control the synaptic output: 1) the probability of synaptic release, 2) the number of synaptic vesicles available for exocytosis, and 3) the number of postsynaptic receptors and their alignment to fusion sites. We aim to elucidate how these parameters are regulated at the ultrastructural level and how they are influenced by other cells such as astrocytes and microglia.

1. Spatial, temporal, and molecular control of synaptic release. The probability of synaptic release is controlled by many factors including the amount of ion flux (i.e. Ca2+, Na+, K+), Ca2+ buffering capacity, sensitivity of vesicles to Ca2+, distance of vesicles to Ca2+ source, and fusion-competence of vesicles. We developed a technique, zap-and-freeze, that couples electrical stimulation with high-pressure freezing to characterize vesicle fusion with millisecond temporal resolution. Using this approach along with protein localization methods, we are investigating spatial, temporal, and molecular control of vesicle fusion in different types of neurons in the mammalian central nervous system and how astrocytes contribute to these functions.

2. Cellular and molecular basis of vesicle regeneration and proteostasis. The recycling process regulates, in part, availability of synaptic vesicles and is tightly coupled to proteostasis. This process requires a series of membrane remodeling. Using a combination of genetics, biochemistry, and advanced electron microscopy approaches, we are investigating cellular, molecular, and structural basis of membrane remodeling at synapses. Currently, we are focusing on four fundamental events at synapses: endocytosis, endosomal membrane budding (protein sorting mechanism for recycling and degradation), protein degradation (multivesicular body and autophagosome formation), and protein aggregate clearance by microglia. We are developing approaches to visualize the interactions of proteins in situ using cryo-electron tomography, with the ultimate goal to visualize these processes even in human brain tissues without fluorescently labelling proteins.

3. Neurotransmitter receptor trafficking. Neurotransmitter receptors diffuse into and out of the post-synaptic receptive field and are transiently anchored in “slots” within the field: occupancy of the slots by the receptors and alignment of the receptors to presynaptic fusion sites can modulate synaptic strength. To visualize receptors in electron micrographs, we have developed small-metal affinity staining of His-tag (SMASH) approach to label His-tagged proteins with nickel-coupled gold nanoparticles. Using a combination of approaches we have developed, we are currently investigating the endocytic mechanism that internalizes glutamate receptors at synapses. We will extend our study to other neurotransmitter receptors as well as neuropeptide receptors in the future.