Regulation of Drosophila Melanogaster Body Fat Storage by Store-Operated Calcium Entry
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Regulation of Drosophila melanogaster body fat storage by store-operated calcium entry Dissertation for the award of the degree “Doctor rerum naturalium” of the Georg-August-Universität Göttingen within the doctoral degree program (Genes and Development) of the Georg-August University School of Science (GAUSS) Submitted by Yanjun Xu From Kaifeng Göttingen, 2017 Thesis advisory committee Prof. Dr. Ronald P. Kühnlein Institute of Molecular Biosciences, University of Graz & Research group of Molecular Physiology Max Planck Institute for Biophysical Chemistry Prof. Dr. Ahmed Mansouri Research group of Molecular Cell Differentiation, Max Planck Institute for Biophysical Chemistry & Department of Clinical Neurophysiology University Medical Center Göttingen, Georg-August University, Göttingen Prof. Dr. Jörg Großhans Institute of Developmental Biochemistry University Medical Center Göttingen, Georg-August University Göttingen Members of the examination board: Referee: Prof. Dr. Ronald P. Kühnlein Institute of Molecular Biosciences, University of Graz & Research group of Molecular Physiology Max Planck Institute for Biophysical Chemistry Co-referee: Prof. Dr. Ahmed Mansouri Research group of Molecular Cell Differentiation, Max Planck Institute for Biophysical Chemistry & Department of Clinical Neurophysiology University Medical Center Göttingen Georg-August University Göttingen Other members of the examination board: Prof. Dr. Jörg Großhans Institute of Developmental Biochemistry University Medical Center Göttingen, Georg-August University Göttingen Prof. Dr. André Fiala Department of Molecular Neurobiology of Behavior Johann-Friedrich-Blumenbach-Institute for Zoology and Anthropology, Georg-August University Göttingen Prof. Dr. Ernst Wimmer Department of Developmental Biology Johann-Friedrich-Blumenbach-Institute for Zoology and Anthropology, Georg-August University Göttingen Dr. Nico Posnien Emmy-Noether Group Department of Developmental Biology Johann-Friedrich-Blumenbach-Institute for Zoology and Anthropology, Georg-August University Göttingen Date of oral examination: April 28, 2017 1 Pre-release parts of the dissertation Publication 1. Baumbach, J., Xu, Y*., Hehlert, P., and Kühnlein, R.P. (2014b). Gαq, Gγ1 and Plc21C control Drosophila body fat storage. J. Genet. Genomics 41, 283–292. doi:10.1016/j.jgg.2014.03.005. *Indicates equal contribution. This publication was collaborated with Dr. J. Baumbach, Dr. P. Hehlert, and Dr. R. P. Kühnlein. The Figure 3, and Figure 4A in the thesis were collaborated with Dr. J. Baumbach and based on the publication Fig. 1D, Fig. 2A. The Figure 4B in the thesis was kindly provided by Dr. J. Baumbach and based on the publication Fig. 2B. 2. Gáliková, M., Diesner, M., Klepsatel, P., Hehlert, P., Xu, Y., Bickmeyer, I., Predel, R., and Kühnlein, R.P. (2015). Energy homeostasis control in Drosophila adipokinetic hormone mutants. Genetics 201, 665–683. doi: 10.1534/genetics.115.178897. Startle-induced climbing assay and its material & method part were carried out and wrote by Yanjun Xu. The material & method writing of startle-induced climbing assay in the PhD thesis was based on this publication. 3. Gáliková, M., Klepsatel, P., Xu, Y., and Kühnlein, R.P. (2017). The obesity-related adipokinetic hormone controls feeding and expression of neuropeptide regulators of Drosophila metabolism. Eur. J. Lipid Sci. Technol. 119. 1600138. doi:10.1002/ejlt.201600138. qPCR primers (they target following gene respectively: CCHa2, Tk, Crz, ImpL2) were designed by Yanjun Xu, which were also used in the PhD thesis. 2 Table of contents I. Overview of figures ..................................................................................... 7 II. Overview of tables ..................................................................................... 8 III. Summary ................................................................................................... 9 IV. Acknowledgements ................................................................................ 10 V. Terms and abbreviations ........................................................................ 11 1 Introduction ............................................................................................... 18 1.1 The contributors of obesity .......................................................................................19 1.1.1 Energy balance and obesity ................................................................................... 19 1.1.2 Gene, environment and interaction ........................................................................ 22 1.2 Drosophila melanogaster as an emerging model for obesity research .....24 1.2.1 Powerful model system .......................................................................................... 25 1.2.2 Energy metabolic organ systems in Drosophila and human .................................. 29 1.2.3 Energy reserve storage in Drosophila and human ................................................. 30 1.2.3.1 Neutral lipid biosynthesis ................................................................................................................... 31 1.2.3.2 Lipid droplet (LD) biogenesis ............................................................................................................ 33 1.2.3.3 LD formation, growth and expansion ............................................................................................ 33 1.2.4 Mobilization of the energy storage in Drosophila and human ................................ 34 1.2.5 Inter-organ regulation of energy homeostasis in Drosophila and human .............. 35 1.2.5.1 Inter-organ regulation of food intake ........................................................................................... 36 1.2.5.2 Inter-organ regulation of energy storage .................................................................................... 37 1.2.5.3 Inter-organ regulation of energy mobilization.......................................................................... 38 1.2.6 Cellular regulation of energy homeostasis in Drosophila and human .................... 39 2 Materials and methods ............................................................................. 46 2.1 Fly stocks, husbandry and genetics ..............................................................................46 2.1.1 Fly stocks ................................................................................................................ 46 2.1.2 Fly husbandry ......................................................................................................... 48 2.1.3 Fly genetics for non-conditional RNAi expression and CaLexA expression .......... 49 3 2.1.4 Fly genetics for temperature-shift induced RNAi expression ................................. 49 2.1.5 Fly genetics for transient drug-feeding induced RNAi expression ......................... 50 2.1.6 Stim cDNA or RNAi resistant cDNA construct transformation ............................... 50 2.1.7 Genetic crosses for combining transgenes or mutations together ......................... 51 2.2 Molecular Biology ...............................................................................................................52 2.2.1 Molecular cloning .................................................................................................... 52 2.2.1.1 Preparation of chemical competent bacteria and their transformation ......................... 52 2.2.1.2 Colony picking, E.coli culture and plasmid isolation ............................................................... 53 2.2.1.3 DNA restriction analysis and ligations .......................................................................................... 53 2.2.1.4 Vector construction of pUASTattB-Stim RNAi1 resistant cDNA RA ................................... 53 2.2.2 Single fly genotyping by polymerase chain reaction (PCR) ................................... 55 2.2.3 RNA-seq analysis of adult fly fat body tissues ....................................................... 56 2.2.4 Reverse Transcription and quantitative PCR (RT-qPCR) ...................................... 58 2.2.4.1 SYBR® green I RT-qPCR ..................................................................................................................... 58 2.2.4.2 TaqMan RT-qPCR ................................................................................................................................... 60 2.3 Biochemical experiments ................................................................................................64 2.3.1 Bicinchoninic acid (BCA) and Bradford protein assay ............................................ 64 2.3.2 Western blotting ...................................................................................................... 65 2.3.3 Coupled colorimetric assay (CCA) for TAG equivalents quantification .................. 66 2.3.4 Glycogen assay ...................................................................................................... 67 2.3.5 Thin layer chromatography (TLC) for neutral lipids determination ......................... 67 2.3.6 14C incorporation lipogenesis assay ....................................................................... 68 2.4 Microscopy, staining, and imaging ...............................................................................69 2.4.1 Dissection, microscopy and imaging of adult flies .................................................. 69 2.4.2 Ex vivo staining of adult fat body cells