Functional analysis of the latrophilin homolog dCirl in Drosophila melanogaster Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Julius-Maximilians-Universität Würzburg vorgelegt von Jennifer Gehring aus Würzburg Physiologisches Institut – Lehrstuhl für Physiologie Schwerpunkt Neurophysiologie Würzburg, 2014 Eingereicht am: ………………………… Mitglieder der Promotionskommission: Vorsitzender: ………………………… Gutachter: Prof. Dr. Manfred Heckmann Gutachter: Prof. Dr. Christian Stigloher Tag des Promotionskolloquiums: ………………………... Doktorurkunde ausgehändigt am: .................................. I Erklärung Ich versichere hiermit, dass ich die vorliegende Dissertation mit dem Titel “Functional analysis of the latrophilin homolog dCirl in Drosophila melanogaster” eigenständig angefertigt und mich dabei keiner anderen als der von mir angegebenen Quellen und Hilfsmittel bedient habe. Die Dissertation wurde in der jetzigen oder einer ähnlichen Form noch bei keiner anderen Hochschule eingereicht. Würzburg, 2014 ____________________________ Jennifer Gehring II Contents Contents 1. Summary............... ................................................................................................. 1 2. Introduction........... .................................................................................................. 4 2.1 Adhesion class G-protein coupled receptors ..................................................... 4 2.1.1 Latrophilin – a prototype of adhesion-GPCRs ............................................ 6 2.1.2 Structural characteristics of latrophilin ........................................................ 6 2.1.3 Functional characteristics of latrophilin ....................................................... 7 2.1.4 Mammalian latrophilin homologs ................................................................ 8 2.1.5 Latrophilin in C. elegans ............................................................................. 9 2.2 Drosophila melanogaster ................................................................................ 10 2.2.1 Strength of the fruit fly as a model organism ............................................ 10 2.2.2 Mechanosensation in Drosophila .............................................................. 11 2.2.3 The Drosophila neuromuscular junction ................................................... 13 2.2.3.1 Structural organisation of the Drosophila NMJ ................................... 13 2.2.3.2 Molecular organisation of the NMJ ..................................................... 15 2.2.3.3 Mechanism of synaptic vesicle exocytosis ......................................... 16 2.3 Reconstitution of split GFP fragments ............................................................. 17 2.3.1 GRASP – GFP reconstitution across synaptic partners ............................ 17 2.3.2 Analysing intracellular membrane protein interactions with split GFP fragments .......................................................................................................... 18 2.4 Study objectives .............................................................................................. 19 3. Materials and Methods ......................................................................................... 21 3.1 Molecular biology ............................................................................................ 21 3.1.1 Material ..................................................................................................... 21 3.1.2 Transgene construction ............................................................................ 21 3.1.3 Ends-out targeting of dCirl ........................................................................ 23 3.1.4 Reverse Transcription PCR ...................................................................... 23 3.1.5 dCIRL antibody production ....................................................................... 23 3.1.6 Western Blot ............................................................................................. 23 3.2 Drosophila melanogaster ................................................................................ 24 3.2.1 Fly rearing ................................................................................................ 24 3.2.2 Transgenesis ............................................................................................ 24 3.2.3 Transgenic lines used in thesis................................................................. 24 3.3 Immunohistochemistry .................................................................................... 25 3.3.1 Materials ................................................................................................... 25 3.3.2 Larval body wall preparation ..................................................................... 26 3.3.3 Fixation and staining procedures .............................................................. 26 3.4 Image acquisition ............................................................................................ 27 3.4.1 Confocal microscopy ................................................................................ 27 3.4.2 Image processing ..................................................................................... 28 3.5 Larval behavioural studies .............................................................................. 28 3.5.1 Survival analysis ....................................................................................... 28 3.5.2 Wandering behaviour ............................................................................... 28 III Contents 3.5.3 Touch sensitivity assay ............................................................................. 28 4. Results.................. ................................................................................................ 30 4.1 Functional characterisation of dCirl ................................................................. 30 4.1.1 Confirmation of dCirlKO as a null mutant ................................................... 30 4.1.2 Loss of dCirl does not influence development in Drosophila .................... 32 4.1.3 Genomic engineering of dCirl fusion proteins ........................................... 32 4.1.4 dCIRL is expressed in the larval and adult nervous system ..................... 34 4.1.5 Diminished wandering behaviour of dCirlKO larvae ................................... 36 4.1.6 Expression pattern of dCirl-promoter-driven GFP ..................................... 37 4.1.7 Loss of dCIRL does not affect synaptic transmission of glutamatergic motoneurons ..................................................................................................... 39 4.1.8 Loss of dCIRL increases content of the scaffold protein Discs-large ........ 41 4.1.9 Transcriptional expression of dCirl in the peripheral nervous system ....... 42 4.1.10 Loss of dCirl reduces gentle touch sensitivity ......................................... 44 4.1.11 Expression of dCirl in chos is required for normal response to gentle touch .................................................................................................................. 46 4.1.12 Cho morphology is largely unaffected in larvae lacking dCirl ................. 48 4.2 Characterisation of intracellular protein interactions using the split-GFP system .............................................................................................................................. 50 4.2.1 Expression pattern of spGFP docking and fusion sensors ....................... 51 4.2.2 Analysing the localisation of dCIRL using spGFP fusion proteins ............ 55 5. Discussion……….................................................................................................. 58 5.1 Functional analysis of dCirl ............................................................................. 58 5.1.1 Verification of the dCirlKO null allele .......................................................... 58 5.1.2 Expression of dCIRL in the larval and adult nervous system .................... 59 5.1.3 Loss of dCirl results in locomotion defects ............................................... 62 5.1.4 Diminished touch sensation of dCirlKO larvae ........................................... 64 5.1.5 Molecular mechanism of dCIRL signalling ................................................ 68 5.2 Optical sensors for protein-protein interactions ............................................... 70 6. Supplementary….. ................................................................................................ 74 6.1 Appendices ..................................................................................................... 74 6.1.1 Supplement figures ................................................................................... 74 6.1.2 Table of figures ......................................................................................... 76 6.1.3 Abbreviations ............................................................................................ 77 6.1.4 Strain list ................................................................................................... 79 6.1.5 Plasmid list ............................................................................................... 83 6.2 Acknowledgements ......................................................................................... 86 6.3 Curriculum vitae .............................................................................................