Phosphorylation of Synaptotagmin 4 captures transiting dense core vesicles at active synapses Dissertation for the award of the degree “Doctor rerum naturalium” (Dr.rer.nat.) of the Georg-August-Universität Göttingen within the IMPRS Neuroscience program of the Georg-August University School of Science (GAUSS) submitted by Vinita Bharat from Delhi, India Göttingen, 2016 Thesis Committee Camin Dean,PhD Department of Trans-Synaptic Signaling (Reviewer) European Neuroscience Institute, Göttingen Prof. Dr.rer.nat.Nils Brose Department of Molecular Neurobiology (Reviewer) Max Planck Institute of Experimental Medicine Göttingen Prof. Dr. Reinhard Jahn Department of Neurobiology Max Planck Institute for Biophysical Chemistry Göttingen Members of the Examination Board Camin Dean,PhD Department of Trans-Synaptic Signaling (Reviewer) European Neuroscience Institute, Göttingen Prof. Dr.rer.nat.Nils Brose Department of Molecular Neurobiology (Reviewer) Max Planck Institute of Experimental Medicine Göttingen Further members of the Examination Board Prof. Dr. Reinhard Jahn Department of Neurobiology Max Planck Institute for Biophysical Chemistry Göttingen Prof. Dr.Thomas Dresbach Department of Anatomy and Embryology University Medical Centre Göttingen Prof. Dr. Michael Hörner Department of Cellular Neurobiology European Neuroscience Institute Göttingen Prof. Dr. Ralf Heinrich Department of Neurobiology Schwann-Schleiden Research Centre Göttingen Date of the oral examination: 26th April 2016 Affidavit I hereby declare that the presented thesis entitled “Phosphorylation of Synaptotagmin 4 captures transiting dense core vesicle at active synapses” has been written independently and with no other sources and aids than quoted. Göttingen, 21st March 2016 Vinita Bharat Abstract Synaptic modulation requires fast recruitment of neuronal dense core vesicles (DCVs) containing various neuropeptides and neurotrophins at nerve terminals. DCVs undergo long-range trafficking in axons to deliver cargoes at release sites. However, the question of whether and how specific sites capture these transiting vesicles upon neuronal activity is open. In this study, we have used a Synaptotagmin (Syt) isoform, Syt4, as a DCV marker to investigate trafficking and activity-dependent capture of DCVs in hippocampal neurons. We found that Syt4-harboring vesicles are highly mobile on microtubules and switch directions only at the distal end of axons in hippocampal neurons. We examined the effects of phosphorylation of Syt4 at S135 on trafficking, capture and fusion of DCVs in mature neurons. We found that phosphomimetic Syt4 vesicles traffic less and are more concentrated at synapses. Conversely, phosphodeficient Syt4 vesicles had the most processivity and were least localized at synapses. We also found that disrupting actin, which is enriched at pre-synaptic sites, enhances the mobility of phosphomimetic vesicles. We found that the motor protein Kif1A is associated with Syt4 vesicles but phosphomimetic vesicles had less interaction with Kif1A. Over-expression of Kif1A rescued the trafficking of phosphomimetic Syt4 vesicles. In addition, we found that c-Jun N-terminal kinase (JNK) phosphorylates Syt4 at S135 specifically causing decreased motility of transiting DCVs. Furthermore, increased neuronal activity promoted capture of transiting vesicles at synapses via a JNK phosphorylation dependent mechanism. Phosphorylation of Syt4 did not affect the fusion of vesicles at synaptic and non-synaptic sites in hippocampal neurons. Together, this study reveals a JNK-dependent phosphorylation mechanism involved in trafficking and capture of Syt4 harboring DCVs in hippocampal neurons. We propose a mechanism whereby JNK at active synapses phosphorylates Syt4 at S135 on transiting DCVs, promoting destabilization of Syt4-Kif1A binding and allowing capture of DCVs at synapses by actin. This mechanism would potentially allow fast recruitment of dense core vesicles to active synapses, ensuring the efficient delivery of neuropeptides and neurotrophins to specific sites in hippocampal neurons whenever needed. Table of Contents 1. Introduction ............................................................................................................. 1 1. 1 NEURONS – “BUILDING BLOCKS” OF THE NERVOUS SYSTEM ................................................................. 1 1. 2 SYNAPSE- THE “GAP” THAT CONNECTS TWO NEURONS ....................................................................... 1 1. 3 NEUROSECRETORY VESICLES – “VEHICLES” FOR NEUROTRANSMISSION .................................................. 2 1.3.1 Synaptic vesicles (SVs) ....................................................................................................... 2 1.3.2 Dense core vesicles (DCVs) ................................................................................................ 4 1. 4 BIOGENESIS OF DCVS ................................................................................................................... 6 1. 5 INTRACELLULAR TRANSPORT OF DCVS ............................................................................................. 6 1.5.1 Cytoskeletal elements ....................................................................................................... 6 1.5.2 Motor proteins for vesicle trafficking................................................................................ 8 1. 6 KINESIN-3 FAMILY, UNC-104/KIF1A: A MOTOR PROTEIN FOR DCVS .................................................... 9 1. 7 “SUSHI-MODEL” FOR DENSE CORE VESICLE TRANSPORT .................................................................... 11 1. 8 SYNAPTOTAGMIN 4: A MEMBRANE PROTEIN OF DCVS...................................................................... 13 1. 9 JNK AND ITS ROLE IN AXONAL TRANSPORT ...................................................................................... 14 1. 10 CAPTURE OF TRANSITING DCVS .................................................................................................. 16 1. 11 FUSION AND RELEASE OF CARGO FROM DCVS ............................................................................... 17 1. 12 AIM AND SCOPE OF THESIS ......................................................................................................... 19 2. Materials and Methods ............................................................................................ 21 2.1 MATERIALS ................................................................................................................................ 21 2.1.1 Antibodies used ............................................................................................................... 21 2.1.2 Mammalian Expression constructs ................................................................................. 22 2.1.3 Buffers and Solutions ...................................................................................................... 23 2.1.4 Chemicals used ................................................................................................................ 24 2.1.5 Mouse lines ..................................................................................................................... 25 2.1.5.1 Genotyping ................................................................................................................... 25 2.2 METHODS ................................................................................................................................. 27 2.2.1 CELL CULTURE ......................................................................................................................... 27 2.2.1.1 Dissociated rat hippocampal neuron culture preparation ........................................... 27 2.2.1.2 Dissociated mouse hippocampal neuron culture preparation ..................................... 28 2.2.1.3 HEK 293T cell culture ................................................................................................... 28 2.2.2 TRANSFECTION PROTOCOLS ....................................................................................................... 28 2.2.2.1 Plasmid DNA amplification .......................................................................................... 29 2.2.2.2 Lipofectamine 2000 Transfection ................................................................................ 29 2.2.2.3 Calcium phosphate transfection .................................................................................. 30 2.2.3 IMAGING PROTOCOLS ............................................................................................................... 30 2.2.3.1 Immunocytochemistry (ICC) and fixed sample imaging .............................................. 30 2.2.3.2 Live cell imaging ........................................................................................................... 31 2.2.4 BIOCHEMISTRY EXPERIMENTS .................................................................................................... 32 2.2.4.1 SDS-PAGE ..................................................................................................................... 33 2.2.4.2 Western blotting .......................................................................................................... 33 2.2.4.3 Co-immunoprecipitation .............................................................................................. 34 2.2.4.4 Immuno-isolation of synaptic vesicles ......................................................................... 34