Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2014 A single molecular study of the regulation of SNARE-mediated membrane fusion Jaekyun Song Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Biophysics Commons, Molecular Biology Commons, and the Neuroscience and Neurobiology Commons Recommended Citation Song, Jaekyun, "A single molecular study of the regulation of SNARE-mediated membrane fusion" (2014). Graduate Theses and Dissertations. 14022. https://lib.dr.iastate.edu/etd/14022 This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. A single molecular study of the regulation of SNARE-mediated membrane fusion by Jae-Kyun Song A thesis submitted to graduate faculty in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Major: Biophysics Program of Study Committee: Yeon-Kyun Shin, Major Professor Reuben Peters Edward Yu Iowa State University Ames, Iowa 2014 Copyright ©Jae-Kyun Song, 2014. All rights reserved. ii TABLE OF CONTENTS ABSTRACT iii CHAPTER 1: GENERAL INTRODUCTION 1 Introduction 1 References 4 Figures and Captions 7 CHAPTER 2: SYNAPTOTAGMIN 1 MAY STABILIZE SNARE-COMPLEX CLAMPLED BY COMPLEXIN 1 12 Abstract 12 Introduction 13 Results 15 Discussion 18 Materials and Methods 20 References 24 Figures and Captions 28 Supplementary Data 32 CHAPTER 3: EXAMINING SYNTAXIN 1 CLUSTERING ON VARIOUS LIPIDIC ENVIRONMENTS 34 Abstract 34 Introduction 35 Results and Discussion 37 Materials and Methods 40 References 42 Figures and Captions 44 CHAPTER 4 GENERAL CONCLUSION 46 ACKNOWLEDGEMENT 48 iii ABSTRACT The presynaptic membrane fusion is mediated by a protein set called SNARE (Soluble NSF Attachment protein REceptor) proteins. SNARE proteins form a ternary SNARE-complex that comprises minimal machinery for membrane fusion; the complex consists of three SNARE proteins: Syntaxin 1, SNAP-25 and Vamp 2, also called Synaptobrevin 2. The SNARE complex is a four-helix coiled coil with four SNARE motifs; two come from SNARE-25 and one each from Syntaxin 1 and Vamp 2. It is believed that a regulatory protein Complexin binds tightly to the SNARE complex and stabilizes the complex, preventing it from driving toward fusion. However, the detailed mechanism of fusion clamping is still unclear. In our work, we constructed a single- molecule lipid-mixing assay on a supported lipid bilayer to investigate the role of Complexin 1, one of the important regulatory proteins. Moreover, we found that Synaptotagmin 1, a calcium sensor for Ca2+-triggered fusion, plays a role along with Complexin in fusion clamping. Furthermore, the supported lipid bilayer was also incorporated into a photo-bleaching assay to investigate the role of various lipids on Syntaxin 1 clustering. 1 CHAPTER 1: GENERAL INTRODUCTION Introduction Membrane fusion and SNARE proteins For eukaryotic cells, an intracellular membrane fusion plays an important role in communication between two cells because eukaryotic cytosol is separated by such a membrane. During communication, molecules must be transported from a particular cytosol to a destination cytosol without compromising membrane integrity; this can be achieved by transporting cargo-containing vesicle fusion [1]. In the brain, for normal brain function, neuronal signals should be transferred from a given neuron to others and the signal transduction is mediated by synaptic vesicle exocytosis. This vesicle exocytosis, simply speaking, includes docking/priming, hemifusion, fusion-pore opening and pore expansion (Figure 1). SNARE (soluble N-ethyl-maleimide-sensitive factor attachment protein receptors) proteins have so far been studied for nearly all intracellular membrane fusion events. In the initial studies, three SNARE proteins were discovered: Syntaxin 1 [2], SNAP-25 [3] and Vamp 2 [4] (also called Synaptobrevin [5]). Syntaxin 1 and Vamp 2 are anchored to the target membrane and vesicle membrane, respectively, by a transmembrane domain, while SNAP-25 resides on the target membrane through palmitoylation of four cysteine residues (Figure 2A). A knockout study has shown the importance of SNARE proteins in 2 synchronous neurotransmitter release and lowered vesicle fusion rate in the absence of SNAP-25 and Vamp 2 genes, respectively [6, 7]. The SNARE proteins form a coiled coil four-bundle helix designated as a SNARE complex (Figure 2B). Each SNARE protein has one (Syntaxin 1 and Vamp 2) or two (SNAP-25) SNARE motifs consisting of 60~70 amino acids (Figure 2A). Four SNARE motifs are arranged by heptad repeat so that they form a SNARE complex with 16 hydrophobic ionic layers (Figure 3A). An ionic zero layer is located in the middle of the SNARE complex and the SNARE can be classified according to the residue of the ionic zero layer; the Q-SNARE protein (Syntaxin 1 and SNAP-25) contains glutamine and the R-SNARE protein (Vamp 2) contains arginine (Figure 3B). Synaptotagmin 1 In presynaptic membranes, neurotransmitter release is triggered by a Ca2+ influx propagated by action potential, and a protein called Synaptotagmin 1 is in charge of sensing Ca2+ [8, 9]. SNARE proteins are deficient in temporal regulation with respect to neurotransmitter release. Synaptotagmin 1, a neuronal Syt isoform out of 17 superfamilies [10], is believed to regulate SNARE-mediated membrane fusion synchronously with a Ca2+ influx [11, 12]. Knockout of the Synaptotagmin 1 gene abolishes fast synchronous neurotransmitter release [9]. Synaptotagmin 1 together with Vamp 2 is anchored to a presynaptic vesicle. Synaptotagmin 1 consists of an N-terminal transmembrane domain, a variable linker, and 3 two C-terminal C2 domains – C2A and C2B (Figure 4A). The C2AB domain can bind neuronal t-SNARE proteins in a Ca2+-independent manner contributing to the docking of secretory vesicles at the plasma membrane [13-15]. Two and three Ca2+ ions bind to the Ca2+ binding pocket lined by loops of C2A and C2B domains, respectively (Figure 4B). After Ca2+ binds to a C2AB domain, it has been reported that Synaptotagmin 1 induces a positive curvature in the target membrane by inserting its loop region to facilitate fusion (Figure 4C) [16]. Complexin 1 Complexin 1 (also called synaphin 2) is another core regulatory protein like Synaptotagmin 1. Synchronous neurotransmitter release in mice was abolished after double knockout of Complexin 1 and Complexin 2 [17] and, moreover, triple knockout of Synaptotagmin 1, Complexin 1, and Complexin 2 caused newborn rats to die immediately after birth. Furthermore, many neuronal diseases such as schizophrenia, Huntington’s disease, Parkinson’s disease, Alzheimer’s disease, etc., are related to the depletion of Cpx [18]. Complexin 1 is a small soluble protein (~15 kDa) consisting of 134 amino acids partitioned by distinct functions into four distinct domains: an unstructured N-terminal domain (amino acids 1-26), an accessory alpha-helix (amino acids 27-47), a central helix (amino acids 48-70), and an unstructured C-terminal domain (amino acids 71-134) 7 -1 -1 (Figure 5A). Complexin 1 binds rapidly (5×10 M S ) and with high affinity (Km = 10 4 nM) to the groove of a SNARE complex between Syntaxin 1 and Vamp 2 in an antiparallel manner via its central helix (Figure 5B) [19, 20]. Recent studies have produced controversial results with respect to Complexin 1 that can be described by the term ‘dual function’ that includes both stimulatory and inhibitory effects. The N-terminal domain of Complexin 1 stimulates synaptic vesicle fusion [21, 22] while an accessory alpha helix inhibits such fusion by replacing the C- terminal part of Vamp 2 from the SNARE complex [21, 23]. A central alpha helix is crucial, mainly for to its role in binding to the SNARE complex, for both stimulating and clamping fusion [24, 25]. The C-terminal region also has dual functions of stimulating and clamping fusion [26]. Reference 1. Chen, Y.A. and R.H. Scheller, SNARE-mediated membrane fusion. Nat Rev Mol Cell Biol, 2001. 2(2): p. 98-106. 2. Bennett, M.K., N. Calakos, and R.H. Scheller, Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones. Science, 1992. 257(5067): p. 255-9. 3. Oyler, G.A., et al., The identification of a novel synaptosomal-associated protein, SNAP- 25, differentially expressed by neuronal subpopulations. J Cell Biol, 1989. 109(6 Pt 1): p. 3039-52. 4. Trimble, W.S., D.M. Cowan, and R.H. Scheller, VAMP-1: a synaptic vesicle-associated integral membrane protein. Proc Natl Acad Sci U S A, 1988. 85(12): p. 4538-42. 5. Baumert, M., et al., Synaptobrevin: an integral membrane protein of 18,000 daltons present in small synaptic vesicles of rat brain. EMBO J, 1989. 8(2): p. 379-84. 6. Washbourne, P., et al., Genetic ablation of the t-SNARE SNAP-25 distinguishes mechanisms of neuroexocytosis. Nat Neurosci, 2002. 5(1): p. 19-26. 5 7. Schoch, S., et al., SNARE function analyzed in synaptobrevin/VAMP knockout mice. Science, 2001. 294(5544): p. 1117-22. 8. Brose, N., et al., Synaptotagmin: a calcium sensor on the synaptic vesicle surface. Science, 1992. 256(5059): p. 1021-5. 9. Geppert, M., et al., Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse. Cell, 1994. 79(4): p. 717-27. 10. Sudhof, T.C. and J. Rizo, Synaptotagmins: C2-domain proteins that regulate membrane traffic. Neuron, 1996. 17(3): p. 379-88. 11. Chapman, E.R., Synaptotagmin: a Ca(2+) sensor that triggers exocytosis? Nat Rev Mol Cell Biol, 2002. 3(7): p. 498-508. 12. Yoshihara, M. and J.T. Littleton, Synaptotagmin I functions as a calcium sensor to synchronize neurotransmitter release.