Shigeki Watanabe, Ph.D. - US grants

Physical forces determine how the membrane of a living cell changes shape. Endocytosis is a membrane process that is essential for bringing molecules into the cell. Viruses often gain entry into cells by hijacking the endocytic process, and thus it is critical to understand how physical forces contribute to membrane remodeling and, specifically, to endocytosis. One place where endocytosis is important is in the transfer of packets of material from one nerve cell to another at a nerve synapse. How this type of endocytosis occurs remains a mystery. Recent experiments have discovered a novel, ultrafast, endocytic mechanism at synapses that occurs within 50 milliseconds. The mechanisms underlying the ultrafast endocytosis, however, remain poorly understood. The goal of this project is to gain mechanistic insights into this novel endocytic pathway. Since defects in endocytosis at nerve synapses may cause neurodegenerative diseases, the work will lay the foundation to understand synapses in different diseases. To educate citizens and attract young people into science research, the investigators will create opportunities for high-school students to participate in research-based scientific activities, deliver guest lectures about the research at high schools and host high school teachers in the lab.

In this research, we will take synergistic experimental and computer modeling approaches to test these models. The Watanabe lab will perform localization and perturbation experiments to reveal the molecular organization and requirements. The Agrawal lab will use the molecular information to build computational modeling to test the passive and active mechanisms and predict the time-scales. The work will be the first unified effort to investigate vesicle dynamics regulated by elastic and dissipative forces. In addition, the research will provide a validated modeling framework for future exploration of membrane-protein dynamics and cellular interface remodeling.

At chemical synapses, neurons communicate each other through release of neurotransmitters. Synaptic transmission occurs in two kinetically distinct phases. Within milliseconds of an action potential, synaptic vesicles fuse with the plasma membrane synchronously across multiple synapses. Following synchronous fusion, synaptic vesicles continue to fuse with the plasma membrane over tens to hundreds of milliseconds. This phase of neurotransmission is called asynchronous release, and it becomes more apparent in pathophysiological conditions. Despite extensive research over the last 50 years, the nano-scale organization of synaptic vesicle fusion sites is poorly understood. Where do synchronous and asynchronous fusions take place within an active zone? Are fusion sites determined by the locations of calcium channels? Which calcium sensors are responsible for asynchronous fusion? Is there a separate pool of vesicles for asynchronous fusion? Or are the same pool of vesicles consumed for both phases? To address these questions, we have developed zap-and-freeze electron microscopy to follow membrane dynamics millisecond- by-millisecond. This technique couples electrical stimulation of neurons with high-pressure freezing to capture rapid membrane trafficking events at mammalian central synapses with unprecedented spatial (1 nm) and temporal (1 ms) resolution. Using this approach in combinations with advanced genetics, molecular biology, and biochemical techniques, we will determine how fusion sites are organized at mammalian central synapses. Defects in synaptic transmission play a causal role in neurological disorders. The proposed research aims to understand the molecular mechanisms underlying synaptic transmission with the ultimate goal of understanding the pathogenesis of these diseases.