Bowers, Matthew Robert, authorReist, Noreen, advisorTsunoda, Susan, committee memberDi Pietro, Santiago, committee memberTamkun, Michael, committee member2020-08-312022-08-242020https://hdl.handle.net/10217/211818Efficient cell-to-cell communication is critical for nervous system function. Fast, synchronous neurotransmission underlies this communication. Following depolarization of the nerve terminal, Ca2+ enters the presynaptic cell and drives fusion of vesicles with the membrane, releasing neurotransmitter. The synaptic vesicle protein, synaptotagmin, was identified as the Ca2+ sensor for this fast, synchronous neurotransmitter release. It is hypothesized that Ca2+ binding by synaptotagmin acts as an electrostatic switch. At rest, vesicles are in a state of variable priming with repulsion between negatively charged residues in synaptotagmin and the negatively charged presynaptic membrane acting as a brake to prevent fusion of the vesicles. Following binding of positively charged Ca2+ ions, this electrostatic repulsion is switched to attraction, allowing hydrophobic residues in synaptotagmin to insert into the presynaptic membrane. This insertion is thought to lower the energy barrier for fusion, resulting in the synchronous fusion of many vesicles and the chemical propagation of a signal to the postsynaptic cell. Synaptotagmin is composed primarily of two C2 domains that have negatively charged Ca2+ binding pockets, C2A and C2B. The C2B domain is thought to be the primary functional domain, with C2A playing a supporting role. While using point mutations to the C2A domain to investigate the functional roles of specific residues of the protein, I discovered that the C2A domain, may, in fact, be much more important than anticipated. In chapter 2, I created mutations disrupting the membrane penetrating hydrophobic residues of the C2A domain. Mutation of these residues was hypothesized to only partially disrupt evoked release. Surprisingly, mutation of both residues in tandem resulted in the most dramatic phenotype of a C2A domain mutation to date. This dramatic decrease in synaptic transmission is the first instance of a C2A domain mutation resulting in a phenotype worse than the synaptotagmin null mutant. In chapter 3, I generated mutations to various combinations of Ca2+-binding aspartate residues in the Ca2+ binding pocket of C2A. These mutations are hypothesized to prevent Ca2+ binding, while simultaneously neutralizing the charge of the pocket, essentially mimicking constitutive Ca2+ binding. Again surprisingly, evoked release was dramatically decreased in some of the mutants, suggesting C2A Ca2+ binding mutants disrupt Ca2+ dependent synchronous release, a finding that increases our understanding of Ca2+ binding by the domain and contradicts some interpretations of previous reports. My findings provide key mechanistic insights into the function of this critical protein. For one, investigation of the role of the C2A hydrophobic residues revealed that the downstream effector interactions mediated by these hydrophobic residues are critical to drive synchronous vesicle fusion. Also, investigation of the role of the critical Ca2+-binding residues in the C2A domain revealed that these residues each play a distinct role in driving vesicle fusion, while further suggesting Ca2+ binding by C2A is more important than originally posited. Most interestingly though, I believe the sum of my findings disproves the long-held belief that C2A is purely a facilitatory domain, prompting many questions about how these two C2 domains may work together to promote neurotransmitter release in tandem.born digitaldoctoral dissertationsengCopyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright.Text