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Browsing by Author "Reist, Noreen, advisor"

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    An investigation of synaptic vesicle docking and priming and a proposed method for quantitatively measuring both in Drosophila using electron tomography
    (Colorado State University. Libraries, 2023) Twiggs, Jasmin A., author; Reist, Noreen, advisor; Hoerndli, Frederic, committee member; Hoke, Kim, committee member; Tamkun, Michael, committee member
    The nervous system, as the body's command center, plays a crucial role in cellular communication within the brain and between the brain and other body systems. Neurons, the individual cellular units, transmit electrical information and communicate with other cells through neurotransmitter release in response to electrical stimuli. Chapter 1 introduces the foundational concepts of neuronal structure and function and delves into the mechanisms underlying neurotransmitter release. Special attention is given to the neuromuscular junction (NMJ), a well-studied chemical synapse crucial for muscle movement. The synaptic vesicle cycle is introduced, with particular emphasis on docking and priming. The significance of active zones, specialized sites for efficient signal transmission, and their associated structural components are underscored. Synaptotagmin, a pivotal protein in calcium-triggered vesicle fusion, is discussed with emphasis on its C2B polylysine motif. Throughout the chapter, the utility of Drosophila as a model system for studying synaptic processes, particularly at the NMJ, is emphasized. In sum, Chapter 1 provides the foundational knowledge essential for comprehending the intricate cellular and molecular facets of synaptic communication within the nervous system, serving as a precursor to subsequent chapters' investigations. Chapter 2 examines synaptotagmin's C2B polylysine motif and its role in synaptic vesicle docking at the Drosophila NMJ. It explores the polylysine motif's potential involvement in endocytosis, demonstrates an unaffected interaction with AP-2, and uses electron microscopy to find no significant changes in vesicle distribution. The findings suggest that the reduced neurotransmitter release in the polylysine mutant is likely due to an impairment in vesicle priming. Chapter 3 introduces a method for studying synaptic vesicle docking and priming in Drosophila, using electron tomography. I address the limitations of conventional electron microscopy and underscore the need for higher-resolution techniques to assess molecular structures that mediate physiological processes. Chapter 3 also emphasizes the significance of the contact area between docked vesicles and the presynaptic membrane as a correlate of vesicle priming. The protocol, expected results, and key considerations are discussed. The methods presented in Chapter 3 offer a promising approach for understanding synaptic processes. In Chapter 4, I discuss key considerations for when standard electron microscopy can be used for assessing vesicle docking. Then, I discuss how the electron tomography method presented in Chapter 3 could not only confirm the results found in Chapter 2, that the synaptotagmin C2B polylysine motif is not implicated in vesicle docking but could also be used to directly test the mutant's role in priming. Specific aims for future studies on the synaptotagmin polylysine mutation in Drosophila are presented, potential results and interpretations are discussed. Finally, I showcase interesting, unpublished findings from electron tomograms I have taken at the Drosophila NMJ and discuss their potential significance.
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    Reevaluating the functional role of the C₂A domain of synaptotagmin in neurotransmitter release
    (Colorado State University. Libraries, 2020) Bowers, Matthew Robert, author; Reist, Noreen, advisor; Tsunoda, Susan, committee member; Di Pietro, Santiago, committee member; Tamkun, Michael, committee member
    Efficient 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.
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    Synaptotagmin in asynchronous neurotransmitter release and synaptic disease
    (Colorado State University. Libraries, 2018) Shields, Mallory Catherine, author; Reist, Noreen, advisor; Garrity, Deborah, committee member; Tamkun, Michael, committee member; Tsunoda, Susan, committee member
    The majority of cell-to-cell communication relies on the stimulated release of neurotransmitter. Two forms of Ca2+-dependent stimulated release, synchronous and asynchronous, have been identified. Synchronous release is the initial release that occurs within milliseconds of stimulation. Critical for efficient synaptic communication, synchronous release is the dominant form of release at most synapses. Alternatively, asynchronous release occurs over longer time periods, with implications in synaptic plasticity and development. However, its mechanisms are poorly understood. Both synchronous and asynchronous release rely on Ca2+ sensors to confer their distinct characteristics. Synaptotagmin 1 is widely accepted as the Ca2+ sensor for fast, synchronous release, but its role in asynchronous release is unclear. Previous studies have led to the hypothesis that synaptotagmin 1, particularly Ca2+ binding by its C2A domain, is needed to inhibit aberrant asynchronous fusion events. However, recent studies have raised questions regarding the interpretation of the results that led to this conclusion. In chapter 2, I have directly tested the effect of Ca2+ binding by synaptotagmin 1's C2A domain on asynchronous release utilizing an alternant Ca2+-binding mutant. This novel mutation was designed to block Ca2+ binding without introducing the artifacts of the original Ca2+-binding mutation. By investigating asynchronous events in vivo at the Drosophila neuromuscular junction, I found no significant effect on asynchronous release when C2A Ca2+ binding was blocked. Thus, I conclude that Ca2+ binding by synaptotagmin's C2A domain is not needed for regulation of asynchronous release, in contrast to the previous study that inadvertently introduced an artifact described below. To prevent Ca2+ binding, the original aspartate to asparagine mutations (sytD-N) removed some of the negatively-charged residues that coordinate Ca2+. This simultaneously introduced aberrant fusion events, because it also interrupted the electrostatic repulsion between synaptotagmin's negatively-charged C2A Ca2+-binding pocket and the negatively-charged presynaptic membrane which is required to clamp constitutive SNARE-mediated fusion. Previous Reist lab results demonstrate that the sytD-N mutations in the C2A domain are likely behaving as ostensibly constitutively bound Ca2+. Indeed, I report that the sytD-N mutation displays slower release kinetics. To directly test if this mutation is the cause of the increase in asynchronous events, I generated additional mutations that prevent interactions with the presynaptic membrane coupled to the originally published sytD-N mutations. In chapter 3 of this dissertation, I investigated these novel mutations at the Drosophila neuromuscular junction. I reported no increase in asynchronous release relative to control, providing evidence that the increased asynchronous events in sytD-N mutants are a result of the original mutation acting as an asynchronous sensor. Together, my results contradict the current hypothesis in the field and provide the likely mechanism for the increased asynchronous release observed in the original study. This dissertation also investigated the relatively new role for synaptotagmin mutations in the etiology of neuromuscular disease. With increased availability of high-throughput sequencing, over 20 candidate genes have been implicated in different forms of congential myasthenic syndromes. These inherited disorders are caused by mutations in genes needed for effective neuromuscular signaling. Two families, presenting with similar myasthenic syndromes, carry point mutations in the C2B Ca2+ binding pocket of synaptotagmin, expressed as an autosomal dominant disorder. One of theses families contains a proline to leucine substitution (sytP-L) a residue that had not been previously investigated for synaptotagmin function. In chapter 4, I investigated the functional importance of this mutation and created a disease model for this familial condition by driving the expression of a homolous proline-leucine synaptotagmin substitution in the central nervous system of Drosophila. I demonstrated that the proline residue plays a functional role in efficient transmitter release by testing its function in an otherwise synaptotagmin null genetic background. Additionally, this mutation displayed characteristics similar to the human disorder when expressed in a heterozygous synaptotagmin background, similar to the familial expression. Namely, the sytP-L mutants exhibited a decreased release probability, which resulted in decreased evoked responses that facilitate upon high frequency stimulation, a rightward shift in Ca2+ sensitivity, and behavioral deficits, including decreased motor output and increased fatigability. Thus, these studies establish the causative nature of the sytP-L mutation in this rare form of congenital myasthenic syndrome and highlight the utility of the Drosophila system for disease modeling.