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In Vitro Single Vesicle Fusion Study of Ca2+-Triggered SNARE-Mediated Membrane Fusion" (2017)

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In Vitro Single Vesicle Fusion Study of Ca2+-Triggered SNARE-Mediated Membrane Fusion Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2017 In vitro single vesicle fusion study of Ca2+- triggered SNARE-mediated membrane fusion Jaewook Kim Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Biophysics Commons Recommended Citation Kim, Jaewook, "In vitro single vesicle fusion study of Ca2+-triggered SNARE-mediated membrane fusion" (2017). Graduate Theses and Dissertations. 15551. https://lib.dr.iastate.edu/etd/15551 This Dissertation 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]. In vitro single vesicle fusion study of Ca2+-triggered SNARE-mediated membrane fusion by Jaewook Kim A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Biophysics Program of Study Committee: Yeon-Kyun Shin, Major Professor Adam Barb Eric Underbakke Scott Neslon Nigel Reuel Iowa State University Ames, Iowa 2017 Copyright © Jaewook Kim, 2017. All rights reserved. ii TABLE OF CONTENTS Page ABSTRACT………………………………. ............................................................. vi CHAPTER 1 GENERAL INTRODUCTION ....................................................... 1 1.1 Ca2+ triggered synaptic membrane fusion ................................................................ 1 1.2 SNAREs………………………………. ................................................................... 2 1.3 SNARE complex ...................................................................................................... 3 1.4 Accessory proteins .................................................................................................... 4 1.4.1 Synaptotagmin 1 ............................................................................................. 4 1.4.2 Complexin ....................................................................................................... 6 1.5 In vitro assays .......................................................................................................... 7 1.5.1 Single vesicle-to-vesicle fusion assay ............................................................. 7 1.5.2 Single vesicle-to-SBL fusion assay content mixing......................................... 9 1.6 Perspectives .............................................................................................................. 10 1.7 References ................................................................................................................. 12 1.8 Figures and legends................................................................................................... 18 CHAPTER 2 PRE-INCUBATION OF T-SNARES WITH COMPLEXIN I INCREASES CONTENT-MIXING EFFICIENCY ................ 23 2.1 Abstract ......................................................................................................... 23 2.2 Introduction .................................................................................................... 24 2.3 Results ......................................................................................................... 26 2.3.1 Single-vesicle content-mixing assay ..................................................... 26 2.3.2 Bell-shaped response of Ca2+-triggered vesicle fusion to Cpx ............. 27 iii 2.3.3 Cpx contributes little to the synchronization of Ca2+-triggered vesicle fusion ......................................................................... 29 2.3.4 The N-terminal of Cpx is essential for the enhancement of the fusion probability ................................................................................. 29 2.4 Discussion ...................................................................................................... 30 2.5 Materials and methods ................................................................................... 32 2.6 References ...................................................................................................... 37 2.7 Figures and legends........................................................................................ 41 CHAPTER 3 BOTULINUM TOXINS A AND E INFLICT DYNAMIC DESTABILIZATION ON T-SNARE TO IMPARE SNARE ASSEMBLY AND MEMBRANE FUSION .................... 45 3.1 Abstract ......................................................................................................... 45 3.2 Introduction .................................................................................................... 46 3.3 Results ......................................................................................................... 48 3.3.1 Single-vesicle content-mixing assay ..................................................... 48 3.3.2 Cleavage of SC by botulinum toxins increases the dynamics of SC .... 51 3.3.3 Cleavage of SC by BoNT E impairs ternary SNARE complex formation ........................................................................................ 52 3.3.4 Cleavage of SC by botulinum toxins decreases/abolishes Ca2+-triggered vesicle fusion ......................................................................... 54 3.4 Discussion ...................................................................................................... 57 3.5 Materials and methods ................................................................................... 59 3.6 References ...................................................................................................... 62 iv 3.7 Figures and legends........................................................................................ 66 CHAPTER 4 PRODUCTIVE AND NON-PRODUCTIVE PATHWAYS FOR SYNAPTOTAGMIN 1 TO SUPPORT CA2+ TRIGGERED FAST EXOCYTOSIS ........................................................... 71 4.1 Abstract ......................................................................................................... 71 4.2 Introduction .................................................................................................... 72 4.3 Results ......................................................................................................... 74 4.3.1 SNAREs are capable of driving fast membrane fusion ........................ 74 4.3.2 Syt1 increases the probability of fast membrane fusion in the presence of Ca2+ ........................................................................ 76 4.3.3 Dissection of membrane fusion steps ................................................... 78 4.4 Discussion ...................................................................................................... 79 4.5 Materials and methods ................................................................................... 82 4.6 References ...................................................................................................... 86 4.7 Figures and legends........................................................................................ 90 CHAPTER 5 SUMMARY AND CONCLUSIONS ........................................... 96 5.1 Conclusions .................................................................................................... 96 ACKNOWLEDGMENT ......................................................................................... 99 v ABSTRACT SNARE-mediated Ca2+ triggered membrane fusion is essential for neuronal communication. The speed at which this process is orchestrated is further emphasized because it serves as a temporal limit for cognitive and physical activities. However, attempts to recapitulate SNARE-mediated membrane fusion with SNAREs and synaptotagmin 1 (Syt1) between two single proteoliposomes came short in respect to Ca2+ sensitivity, speed and fusion efficiency compared to in vivo observations. This discrepancy raises concerns if there are some critical protein machinery that are missing or if the topology of the proteoliposome fusion assay does not faithfully represent synaptic vesicle and plasma membrane. Some suspect that the discrepancy might be due to the tight membrane curvature of proteoliposomes which may not mimic the relaxed curvature of the plasma membrane well. Others wonder if our long-standing dogma that SNAREs are the core membrane fusion machinery is valid. In this study we investigate the role of complexin (Cpx) in a well-defined in vitro environment. Specifically, we observed Ca2+-triggered SNARE-mediated content-mixing between two proteoliposome pairs with total internal reflection (TIR) microscopy. We find that Cpx enhances Ca2+-triggered vesicle fusion with the yield changing from approximately 10% to 70% upon increasing Cpx from 0 to 100 nM. Unexpectedly, however, the fusion efficiency becomes reduced when Cpx is increased further, dropping to 20% in the µM range, revealing a bell-shaped dose-response curve. With our Cpx assisted in vitro single vesicle-to-vesicle fusion assay which has high efficiency and physiologically relevant Ca2+ sensitivity, we investigated the inhibitory of vi effects botulinum toxins (BoNT) A and E. BoNT A and E both block neurotransmitter release by specifically cleaving the C-terminal ends of SNAP-25, a plasma membrane SNARE protein. Here, we find that SNAP-25A and E, the cleavage products of BoNT A and E respectively, terminate membrane
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