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The Role of the Golgi Protein GM130 in Cell Polarity and Tumorigenesis

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The Role of the Golgi Protein GM130 in Cell Polarity and Tumorigenesis The Role of the Golgi Protein GM130 in Cell Polarity and Tumorigenesis Dissertation submitted for the degree of Doctor of Natural Sciences (Dr. rer. nat.) Presented by Francesco Baschieri at the Faculty of Sciences Department of Biology University of Konstanz Date of examination: 27.03.2015 1. referent: Prof. Daniel F. Legler 2. referent: PD Dr. Hesso Farhan Konstanz, 2015 Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-287758 Table of Contents Abstract ................................................................................................................... 1 Zusammenfassung .................................................................................................. 2 Introduction ............................................................................................................ 4 Cell polarity ...................................................................................................... 5 Table 1 – Nomenclature of polarity proteins .............................................. 8 Par complex ............................................................................................... 8 Par6 ..................................................................................................... 9 Par3 ..................................................................................................... 9 aPKC .................................................................................................... 11 Table 2 – List of substrates of aPKC ................................................... 12 Par complex in cell migration and invasion ....................................... 12 Par complex in cancer ........................................................................ 13 Crumbs complex ....................................................................................... 17 CRB ..................................................................................................... 17 Pals1 ................................................................................................... 18 PATJ .................................................................................................... 19 Crumbs complex in cell migration and invasion ................................ 20 Crumbs complex in cancer ................................................................. 20 Scribble complex ...................................................................................... 21 Scribble .......................................................................................... 22 DLG ................................................................................................ 24 LGL ................................................................................................. 24 Scribble complex in cell migration and invasion ........................... 25 Scribble complex in cancer ........................................................... 26 Figure 1 – Schematic of polarity proteins localization in apico-basal and front-rear polarity ...................................................................................... 29 References ........................................................................................................ 30 Small Rho GTPases ........................................................................................... 37 Table 1 – Localization of small GTPases ................................................... 39 Actin regulation by Rho GTPases .............................................................. 39 Figure 1 – Main signaling routes downstream of Cdc42, Rac1 and RhoA 42 Rho GAPs .................................................................................................. 42 Rho GEFs ................................................................................................... 44 Rho GDIs .................................................................................................... 47 Figure 2 – Model of the mechanism of action of GDIs ............................. 48 References ........................................................................................................ 51 Table of Contents - I The secretory pathway .................................................................................... 58 Signaling at the secretory pathway .......................................................... 63 The Golgi Apparatus in cell polarity ......................................................... 66 The Golgi Apparatus in cell migration ...................................................... 69 Golgi and cancer ....................................................................................... 71 Figure 1 – Schematic of some of the signaling routes to and from the secretory pathway ................................................................................ 72 References ........................................................................................................ 73 Aim of the Thesis .................................................................................................... 80 Results...................................................................................................................... 82 Part 1 - Spatial Cdc42 signalling regulates polarity and tumorigenesis ......... 82 The Golgi pool of Cdc42 controls cell polarity .......................................... 82 Directed trafficking causes asymmetric distribution of Cdc42 ................ 83 The GM130-Cdc42 crosstalk does not involve GEFs ................................ 84 The GM130-Cdc42 crosstalk involves RasGRF ......................................... 84 Regulation of the GM130-RasGRF complex ............................................. 85 The GM130-RasGRF complex controls Ras activity .................................. 86 Expression of GM130 in colon cancer ...................................................... 86 Figures .............................................................................................................. 88 Figure 1. GM130 spatially regulates Cdc42 activity to control polarity ... 88 Figure 2. Models for the GM130-Cdc42 crosstalk ..................................... 91 Figure 3. Screening for GEFs that regulate the Golgi pool of Cdc42 ........ 92 Figure 4. The effect of GM130 on Cdc42 is dependent on RasGRF ......... 94 Figure 5. The GM130-RasGRF complex is regulated by serum ................ 96 Figure 6. GM130 depletion increases Ras activity ................................... 98 Figure 7. Dysregulation of GM130 in human colon cancer ...................... 99 Suppl. Figure 1. Effect of GM130 on Cdc42 activity and on polarity proteins ....................................................................................... 101 Suppl. Figure 2. GM130 depletion does not affect Golgi integrity ........... 104 Suppl. Figure 3. GM130 depletion does not affect trafficking, Golgi structure or microtubule regrowth ................................................. 106 Suppl. Figure 4. Effect of ARHGEF 9, 11 &12 on Cdc42 activity, cell migration and Golgi orientation ........................................................ 108 Suppl. Figure 5. TUBA is not involved in mediating the GM130 effect on Cdc42 ........................................................................................ 110 Suppl. Figure 6. Characterizing the GM130-RasGRF complex .................. 112 Suppl. Figure 7. Effect of GM130 depletion of ERK activation ................. 114 Suppl. Figure 8. Effect of GM130 on PC12 cell differentiation ................. 115 Suppl. Figure 9. Downregulation of GM130 in colorectal cancer. ............ 117 Additional results .............................................................................................. 118 Table of Contents - II Part 2 - Regulation of spatial Cdc42 signaling by GM130 and its effects on cell migration, invasion and polarity ................................................................ 119 GM130 expression in breast cancer .......................................................... 119 Golgi morphology in breast cancer cells ................................................... 119 GM130 depletion does not affect proliferation, apoptosis or adhesion . 120 Effect of GM130 depletion on cell motility .............................................. 120 Table 1 - Main characteristics of the cell lines analyzed .......................... 122 Figures ............................................................................................................... 123 Figure 1 ...................................................................................................... 123 Figure 2 ..................................................................................................... 124 Figure 3 ...................................................................................................... 126 Figure 4 ...................................................................................................... 128 Figure 5 ...................................................................................................... 130 References .............................................................................................................. 132 Materials and Methods .......................................................................................... 136 Cell Culture and transfection .......................................................................... 136 Plasmids .........................................................................................................
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