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Individual Functions and Substrate Specificities of Importin Alpha

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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.
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  • (P -Value<0.05, Fold Change≥1.4), 4 Vs. 0 Gy Irradiation

    (P -Value<0.05, Fold Change≥1.4), 4 Vs. 0 Gy Irradiation

    Table S1: Significant differentially expressed genes (P -Value<0.05, Fold Change≥1.4), 4 vs. 0 Gy irradiation Genbank Fold Change P -Value Gene Symbol Description Accession Q9F8M7_CARHY (Q9F8M7) DTDP-glucose 4,6-dehydratase (Fragment), partial (9%) 6.70 0.017399678 THC2699065 [THC2719287] 5.53 0.003379195 BC013657 BC013657 Homo sapiens cDNA clone IMAGE:4152983, partial cds. [BC013657] 5.10 0.024641735 THC2750781 Ciliary dynein heavy chain 5 (Axonemal beta dynein heavy chain 5) (HL1). 4.07 0.04353262 DNAH5 [Source:Uniprot/SWISSPROT;Acc:Q8TE73] [ENST00000382416] 3.81 0.002855909 NM_145263 SPATA18 Homo sapiens spermatogenesis associated 18 homolog (rat) (SPATA18), mRNA [NM_145263] AA418814 zw01a02.s1 Soares_NhHMPu_S1 Homo sapiens cDNA clone IMAGE:767978 3', 3.69 0.03203913 AA418814 AA418814 mRNA sequence [AA418814] AL356953 leucine-rich repeat-containing G protein-coupled receptor 6 {Homo sapiens} (exp=0; 3.63 0.0277936 THC2705989 wgp=1; cg=0), partial (4%) [THC2752981] AA484677 ne64a07.s1 NCI_CGAP_Alv1 Homo sapiens cDNA clone IMAGE:909012, mRNA 3.63 0.027098073 AA484677 AA484677 sequence [AA484677] oe06h09.s1 NCI_CGAP_Ov2 Homo sapiens cDNA clone IMAGE:1385153, mRNA sequence 3.48 0.04468495 AA837799 AA837799 [AA837799] Homo sapiens hypothetical protein LOC340109, mRNA (cDNA clone IMAGE:5578073), partial 3.27 0.031178378 BC039509 LOC643401 cds. [BC039509] Homo sapiens Fas (TNF receptor superfamily, member 6) (FAS), transcript variant 1, mRNA 3.24 0.022156298 NM_000043 FAS [NM_000043] 3.20 0.021043295 A_32_P125056 BF803942 CM2-CI0135-021100-477-g08 CI0135 Homo sapiens cDNA, mRNA sequence 3.04 0.043389246 BF803942 BF803942 [BF803942] 3.03 0.002430239 NM_015920 RPS27L Homo sapiens ribosomal protein S27-like (RPS27L), mRNA [NM_015920] Homo sapiens tumor necrosis factor receptor superfamily, member 10c, decoy without an 2.98 0.021202829 NM_003841 TNFRSF10C intracellular domain (TNFRSF10C), mRNA [NM_003841] 2.97 0.03243901 AB002384 C6orf32 Homo sapiens mRNA for KIAA0386 gene, partial cds.