From the Max-Delbrück-Center for Molecular Medicine Berlin Head of the research group: Prof. Dr. M. Bader “Individual functions and substrate specificities of importin α subtypes” Dissertation for Fulfillment of Requirements for the Doctoral Degree of the University of Lübeck from the Department of Natural Sciences Submitted by Stefanie Hügel from Hennigsdorf Lübeck 2013 First referee: Prof. Dr. M. Bader Second referee: Prof. Dr. N. Tautz Date of oral examination: 21.02.13 Approved for printing. Lübeck, 22.02.2013 2 1 TABLE OF CONTENTS 1 TABLE OF CONTENTS _______________________________________________________ 3 2 ABSTRACT _______________________________________________________________ 6 3 ZUSAMMENFASSUNG ______________________________________________________ 7 4 INTRODUCTION ___________________________________________________________ 9 4.1 Nucleocytoplasmic protein transport ____________________________________________ 9 4.1.1 The nuclear pore complex – gateway to the nucleus ______________________________________ 10 4.1.1 Transport factors __________________________________________________________________ 11 4.1.2 Nothing happens without RanGTP _____________________________________________________ 12 4.2 The classical nuclear protein import pathway ____________________________________ 13 4.2.1 Structure and function of importin α ___________________________________________________ 13 4.2.2 Importin α:β mediated nuclear protein import___________________________________________ 15 4.2.3 The importin α protein family ________________________________________________________ 16 4.2.4 Alternative roles of importin α proteins ________________________________________________ 18 4.2.5 Importin α7 is essential for zygotic genome activation and early mouse development ___________ 20 4.2.6 Importin α3 _______________________________________________________________________ 21 4.2.7 Substrate specificities of α importins __________________________________________________ 21 4.2.7.1 Importin α5 and STAT1 _________________________________________________________ 21 4.2.7.2 Importin α3 and RCC1 __________________________________________________________ 22 4.2.7.3 Importin α7 and Keap1 _________________________________________________________ 23 5 OBJECTIVES OF THIS STUDY ________________________________________________ 24 6 RESULTS & DISCUSSION ___________________________________________________ 25 1. Binding partners of importin α7 (Part A) __________________________________________ 25 1.1. Identification of importin α7 binding partners from ovary tissue ____________________________ 25 1.2. Involvement of importin α7 binding partners from ovary tissue in RNA processing, chromosome organization and chromatin modification ______________________________________________________ 26 1.3. Identification of importin α7 binding partners from fibroblast cells __________________________ 27 1.4. Involvement of importin α7 binding partners from fibroblast cells in RNA processing, chromosome organization and chromatin modification ______________________________________________________ 28 1.5. 36 % overlap of importin α7 binding partners identified by co-immunoprecipitation from fibroblast cells with GST pull-down results from ovary ____________________________________________________ 28 1.6. Nuclear localization of candidate proteins despite the absence of importin α7 _________________ 36 1.7. Differences between importin α7 binding partners and importin α2 and α3 substrates __________ 47 1.8. Limited availability of importin α proteins in murine oocytes and zygotes _____________________ 52 1.9. Brg1 bound importin α7, but not other maternally expressed importin α subtypes in vitro _______ 53 1.10. Normal Brg1 nuclear localization in importin α7 -/- oocytes ________________________________ 54 1.11. Proteomic analysis of importin α7 depleted MEFs – SILAC approach _________________________ 55 1.12. Summary and Discussion Part A _______________________________________________________ 61 3 1.12.1. Importin α7 binding partners play a role in RNA processing, chromosome organization and chromatin remodeling ___________________________________________________________________ 61 1.12.2. Brg1 is one representative cargo in pre-implantation embryos that preferentially binds importin α7 63 1.12.3. Importin α7 cargo sets differ from that of other importin α subtypes ____________________ 64 1.12.4. Loss of importin α7 leads to disturbed nuclear import of cell-cycle regulators and chromosome organization factors in MEFs ______________________________________________________________ 65 1.12.5. Conclusion and Future Perspectives _______________________________________________ 68 2. Analysis of published importin α substrate specificities (Part B) ________________________ 69 2.1. Nuclear localization of Keap1 despite the absence of importin α7 ___________________________ 70 2.2. Decreased nuclear RCC1 levels in importin α3 depleted MEFs ______________________________ 71 2.3. Normal IFN-γ stimulated P-STAT1 nuclear import in importin α5-/- and α7-/- MEFs _____________ 74 2.4. Summary and Discussion Part B _______________________________________________________ 78 3. Characterization of importin α3 knockout mice (Part C) ______________________________ 80 3.1. Tissue specific expression pattern of importin α3 ________________________________________ 80 3.2. Depletion of importin α3 led to partially embryonic lethality in mice _________________________ 83 3.3. Fertility problems of importin α3-/- mice _______________________________________________ 83 3.4. Histological analysis by HE staining ____________________________________________________ 84 3.5. Metabolic analysis _________________________________________________________________ 88 3.6. Echocardiographic analysis ___________________________________________________________ 90 3.7. Respiratory frequency analysis _______________________________________________________ 94 3.8. Blood cell count ___________________________________________________________________ 94 3.9. Blood gas analysis __________________________________________________________________ 97 3.10. Blood pressure and heart rate measurements ___________________________________________ 98 3.11. Analysis of vasoconstriction response upon Angiotensin II treatment ________________________ 99 3.12. Summary and Discussion Part C ______________________________________________________ 103 3.12.1. Depletion of importin α3 caused embryonic death and fertility problems in mice _________ 103 3.12.2. Importin α3-/- mice show signs of right ventricular hypertrophy _______________________ 105 3.12.3. Importin α3-/- mice have misshaped glomeruli _____________________________________ 106 3.12.4. Conclusion and Future perspectives ______________________________________________ 107 4. MATERIAL AND METHODS ________________________________________________ 108 4.1. MATERIALS _______________________________________________________________ 108 4.1.1. Chemicals _____________________________________________________________________ 108 4.1.2. Kits and Enzymes _______________________________________________________________ 109 4.1.3. Antibodies _____________________________________________________________________ 110 4.1.4. Plasmids ______________________________________________________________________ 110 4.1.5. Primer ________________________________________________________________________ 110 4.1.6. Equipment and Expendable Material _______________________________________________ 111 4.2. METHODS ________________________________________________________________ 113 4.2.1. Cell culture ____________________________________________________________________ 113 4.2.1.1. Generation and cultivation of murine embryonic fibroblast cell lines ___________________ 113 4.2.1.2. Stimulation of cells____________________________________________________________ 113 4 4.2.1.3. Transfection of cells ___________________________________________________________ 113 4.2.1.4. Isotope labeling by amino acids in cell culture (SILAC) _______________________________ 114 4.2.2. Molecular biology _______________________________________________________________ 114 4.2.2.1. Cloning of importin expression constructs _________________________________________ 114 4.2.3. Protein expression and analysis ____________________________________________________ 115 4.2.3.1. Production of GST- and His-fusion proteins in E. coli cells _____________________________ 115 4.2.3.2. Binding studies of in vitro transcribed and translated (IVTT) proteins ___________________ 115 4.2.3.3. Protein preparation from tissues ________________________________________________ 116 4.2.3.4. Nuclear-cytoplasmic fractionation of cells and organs _______________________________ 116 4.2.3.5. SDS PAGE and Western Blot ____________________________________________________ 116 4.2.3.6. Co-immunoprecipitation of importin α binding partners _____________________________ 117 4.2.3.7. GST pull-down of importin α binding partners ______________________________________ 117 4.2.4. Mass spectrometry and analysis of result lists ________________________________________ 118 4.2.5. Stainings of murine cells and tissues ________________________________________________ 119 4.2.5.1. Tissue fixation, embedding and sectioning _________________________________________ 119 4.2.5.2. HE-staining of tissue sections ___________________________________________________ 119 4.2.5.3. Immunocytochemical stainings of MEFs ___________________________________________ 120 4.2.6.