Molecular function of RIM1α: role of phosphorylation sites Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Johannes Alexander Müller aus Bonn Bonn, Juli 2019 Angefertigt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn. 1. Gutachter: Prof. Dr. Susanne Schoch McGovern 2. Gutachter: Prof. Dr. Thorsten Lang Tag der Promotion: 09.10.2019 Erscheinungsjahr: 2020 Declaration: Parts of this thesis are already published in: Engholm-Keller K, Waardenberg AJ, Müller JA, Wark JR, Fernando RN, Arthur JW, Robinson PJ, Diet- rich D, Schoch S, Graham ME. The temporal profile of activity-dependent presynaptic phospho-signalling reveals long-lasting patterns of poststimulus regulation. PLoS Biology 2019; 17(3): e3000170 Marvin JS, Scholl B, Wilson DE, Podgorski K, Kazemipour A, Müller JA, Schoch S, Quiroz FJU, Rebola N, Bao H, Little JP, Tkachuuk AN, Cai E, Hantman AW, Wang SS, DePiero VJ, Borghuis BG, Chapman ER, Dietrich D, DiGregorio DA, Fitzpatrick D, Looger LL. Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR. Nature Methods 2018; 15(11): 936-939 For a full list of publications, book chapters and conference presentations (also including works, that are not part of this thesis) please refer to section 11 (Publications). Finally. Index i Index List of Abbreviations vi List of Figures viii List of Tables xi 1 Summary 1 2 Introduction 3 2.1 Synapses . 3 2.2 The presynaptic terminal - a highly specialized subcellular compartment . 4 2.3 Generation, fusion and recycling of synaptic vesicles . 5 2.4 Heterogeneity of presynaptic vesicle pools . 9 2.5 The active zone . 10 2.5.1 The molecular architecture of the cytomatrix at the active zone . 10 2.5.2 RIM protein family . 11 2.6 Ca2+ - channels and - influx are determinants of vesicle release . 15 2.7 Presynaptic plasticity . 16 2.7.1 Short-Term Synaptic Plasticity . 17 2.7.2 Presynaptic Long-Term Plasticity - LTP and LTD . 17 2.7.3 Homeostatic Plasticity . 19 2.8 Phosphorylation: a molecular switch to control synaptic function . 22 2.9 SRPK2 a novel player among presynaptic kinases . 23 2.10 Optical tools to measure neurotransmitter release . 24 2.10.1 Membrane staining based methods . 24 2.10.2 pH - sensitive fluorescence reporters . 25 2.10.3 Neurotransmitter binding sensors . 25 3 Aims of the Project 27 Index ii 4 Material and Methods 28 4.1 Molecular Biology . 28 4.1.1 Polymerase Chain Reaction (PCR) and site-directed mutagenesis . 28 4.1.2 Restriction cloning of DNA fragments and fragmented cloning of RIM1 . 29 4.1.3 Transformation of and DNA preparation from competent bacteria . 29 4.2 Cell culture . 30 4.2.1 Human Kidney Embryo (HEK) 293T cell culture . 30 4.2.2 Primary neuron culture . 31 4.3 Virus preparation . 32 4.3.1 Lenti-viral particles . 32 4.3.2 Recombinant adeno-associated viral (rAAV) particles . 33 4.4 Biochemistry . 33 4.4.1 Lysis of cells . 33 4.4.2 Protein synthesis induction and purification . 33 4.4.3 GST-Pull down assay . 34 4.4.4 Gel-electrophoresis and western blotting . 34 4.5 Mass - spectrometry . 34 4.6 Bioinformatics . 35 4.7 Microscopy and image analysis . 35 4.7.1 Confocal imaging . 35 4.7.2 FM imaging . 35 4.7.3 iGluSnFR Imaging . 38 4.7.4 Fluorescence recovery after photo-bleaching (FRAP) . 40 4.8 Statistics and data presentation . 41 5 Results 43 5.1 FM imaging to investigate vesicle fusion and neurotransmitter release . 43 5.1.1 FM imaging approach to resolve reduced release probability in cultured neurons . 43 5.1.2 Potassium induced vesicle fusion is Ca2+ dependent . 45 5.2 iGluSnFR: a novel tool for the investigation of synaptic release parameters . 46 5.2.1 Low affinity variant of iGluSnFR to resolve high stimulation frequencies . 47 5.2.2 iGluSnFR sensors allow sub-µm localization of release sites . 48 5.2.3 Estimation of glutamate diffusion speed in a given biological system . 50 5.2.4 Estimation of vesicular release probability with binomial release model . 51 Index iii 5.3 Molecular rescue of RIM1α KO and RIM1/2 cDKO with GFP-RIM1α ............ 53 5.3.1 Stimulation strength is crucial to resolve reduced release probability . 53 5.3.2 Release probability in RIM1α KO neurons is strongly reduced . 54 5.3.3 Reduced release probability of RIM1α KO neurons is rescued by expression of GFP-RIM1α ........................................ 56 5.3.4 RIM1/2 cDKO neurons phenocopy reduced synaptic release probability of RIM1α KO neurons . 58 5.3.5 The vesicular release probability in RIM1/2 cDKO synapses is reduced compared to control neurons . 59 5.3.6 GFP-RIM1α rescues synaptic release probability of RIM1/2 cDKO . 61 5.4 Identification of phosphorylation sites in RIM1α ........................ 62 5.4.1 Bioinformatic identification of RIM1α phosphorylation sites . 62 5.4.2 Activity regulated phosphorylation helps to identify phosphorylation sites of RIMα 63 5.4.3 Depolarization of neurons activates different kinases and phosphatases . 64 5.5 Phosphorylation sites in RIM1α that are relevant for basal release . 66 5.5.1 Mutations of T812/814 and S1600 to alanine are not able rescue the reduced re- lease probability of RIM1α KO neurons . 67 5.5.2 S991A, T812/814A and S1600A fail to rescue reduced release probability when physiologically stimulated . 69 5.5.3 Phospho-mimicry rescues release deficiency . 71 5.6 Localization and mobility of GFP-RIM1α variants in synaptic structures . 73 5.6.1 GFP-RIM1α with release relevant mutations are present in synaptic terminals . 73 5.6.2 GFP-RIM1α(S1600A) shows altered persistence in CAZ . 74 5.7 The importance of the C-Terminus for synaptic release and CAZ integration . 78 5.8 Phospho-dependent protein interactions of RIM1α ...................... 79 5.9 Synaptic vesicle release correlates with the protein levels of SRPK2 . 80 5.9.1 SRPK2 over-expression increases and knock-down decreases synaptic release probability . 80 5.9.2 SRPK2 OE fails to increase glutamate release when RIM1 and RIM2 are ablated . 82 5.9.3 SRPK2 overexpression mediated increase of neurotransmitter release depends on phosphorylation sites in RIM1α ............................. 84 5.9.4 When SRPK2 is overexpressed, neurons fail to induce presynaptic homeostatic scaling . 86 5.9.5 Homeostatic scaling is dependent on RIM and its phosphorylation state . 87 Index iv 6 Discussion 89 6.1 GFP-RIM1α fully rescues reduced synaptic release probability in RIM1α KO and RIM1/2 cDKO neurons . 89 6.2 The importance of RIM1α phosphorylation in synaptic function . 92 6.3 SRPK2, a novel kinase in the presynaptic terminal, regulates neurotransmitter release and influences presynaptic homeostatic scaling . 97 6.4 General implications of the results for presynaptic plasticity . 100 6.5 Experimental and technical considerations . 102 7 Outlook 107 8 Contributions 108 9 Appendix 109 9.1 Experimental data support two-pool model . 109 9.2 FM analysis . 110 9.3 Fitting procedure for binomial analysis of vesicular release probability . 111 9.4 Pooling of WT/dCre and Cre/GFP-Cre experiments . 112 9.5 Amount of not fittable structures in different conditions . 113 9.6 Involvement of CamKII in SRPK2-RIM signaling cascade . 114 9.7 Elevated Caclium conentration further increased release probability of neurons that over- expressed SRPK2 . 115 9.8 Websites and Tools for Bioinformatics . 115 9.9 List of Antibodies and Primers . 116 10 References 119 11 Publications 141 11.1 Journal Articles . 141 11.2 Book Chapters . 142 11.3 Poster Presentations . 142 12 Acknowledgements 143 Index v vi List of Abbreviations A A alanine (mutation) AMPA α-amino-3-hydroxy-5-methyl-4- GST glutathion-s-transferase isoxazolepropionic H acid h hour AP action potential HEK293T Human embyonic kindey 293 T approx. approximately cells a.u. arbitrary unit Hz hertz AZ active zone I B i.e. id est BP band pass IRES internal ribosomal entry site C K CaCl2 calcium chloride K+ potassium CamK calmodulin kinase kDa kilo Dalton cAMP cyclic adenosine kDead kinase dead monophosphate KCl potassium chloride CAZ cytomatrix at the active zone KO knock-out cDKO conditional double knock-out L cm2 square centimeters LB Luria Bertani Co2+ cobalt LED Light emitting diode CO2 carbon dioxide LP long pass ctrl. control LTD long-term depression °C degree Celsius LTP long-term potentiation D M dCre delta Cre-recombinase mA milli ampere DF/F relative change in fluorescence mf mossy fiber DG dentate gyrus µg microgram DIV days in vitro MgCl22 magnesium chloride DNA deoxyribonucleic acid min minute E minusC deleted C-terminus E glutamate (mutation) µl microlitre e.g. example given ml millilitre EM electron microscopy µM mircomolar EM-CCD Electron multiplying µm micrometre charge-coupled device mm millimetre EPSP excitatory postsynaptic potential mM millimolar et al. et alia ms milliseconds etc. et cetera MS mass spectrometry F mut. mutation fEPSP field EPSP N FRAP fluorescence recovery after Na2HPO4 di-sodium hydrogen phosphate photobleaching NaCl sodium chloride FRET Förster resonance energy nm nano metre transfer NMDA N-Methyl-D-Aspartate G NMJ neuromuscular junction GFP green fluorescent protein ns not significant vii O OE over-expression P PBS phosphate buffered saline PCR polymerase chain reation PhD philosophiae doctor PKA protein kinase A PKC protein kinase C PP1 protein phosphatase 1 PSD postsynaptic density PTM post-translational modification pves vesicular release probability px pixel R RIM Rab3-interacting molecule RIM-BP RIM binding protein ROI region of interest RRP readily releasable pool RtP Resting pool S s second SDS sodium dodecyl sulfate shRNA short hairpin RNA SRPK serine/arginine-rich protein kianse SSA (SSB, splicing site A (B, C) SSC) struct.