Localization of Heat Shock Proteins HSPA6 (HSP70B') and HSPA1A (HSP70-1) in Cultured Differentiated Human Neuronal Cells Following Thermal Stress

ii ACKNOWLEDGEMENTS . iv LIST OF TABLES . viii LIST OF FIGURES . ix LIST OF ABBREVIATIONS . xi LIST OF PUBLICATIONS . xiv 1 INTRODUCTION . 1 1.1 Heat shock response (HSR) . 1 1.2 Heat shock proteins (Hsps) . 2 1.3 Neurodegenerative diseases . 4 1.3.1 Protective role of Hsps in neurodegenerative diseases . 4 1.3.2 SH-SY5Y human neuronal tissue culture cells . 6 1.4 HSP70 (HSPA) family . 7 1.5 Nomenclature . 8 2 OBJECTIVES . 10 3 MATERIALS AND METHODS . 12 3.1 Storage and maintenance of SH-SY5Y cells . 12 3.2 Preparation of plasmids . 12 3.3 Transformation of bacterial cells and Maxi Prep . 12 3.4 Transfection and selection of SH-SY5Y cells . 13 3.5 Differentiation and heat shock of SH-SY5Y cells . 13 3.6 Western blotting . 13 3.7 α-amanitin and RNase/DNase treatment. 14 3.8 Immunofluorescence . 15 vi 3.9 Quantification and statistical analysis . 16 3.10 Sequence and structural analysis of HSPA6 and HSPA1A proteins . 16 4 RESULTS . 18 4.1 Stress-induced localization of HSPA6 (HSP70B′) and HSPA1A (HSP70-1) proteins to centrioles in cultured differentiated human neuronal cells . 18 4.1.1 Characterization of human neuronal cell lines expressing YFP-tagged HSPA6 and YFP-tagged HSPA1A . 18 4.1.2 YFP-HSPA6 and YFP-HSPA1A at centrioles in differentiated human neuronal cells following thermal stress . 21 4.1.3 Prolonged association of YFP-HSPA6 with the proximal end of centrioles . 21 4.2 Localization of HSPA6 and HSPA1A proteins to nuclear structures following thermal stress in cultured differentiated human neuronal cells . 28 4.2. 1 YFP-HSPA6 and YFP-HSPA1A proteins associate with nuclear components of differentiated human neuronal cells after heat shock . 29 4.2. 2 Transient localization of YFP-HSPA6 and YFP-HSPA1A proteins with nuclear speckles (type I structures) . 32 4.2. 3 YFP-tagged HSPA6 and HSPA1A proteins associate with the granular component (GC) of nucleolus (type II structures) . 32 4.2. 4 Localization of YFP-HSPA6 protein to the periphery of nuclear speckles (type III structures) . 41 4.2. 5 Type III structures at the periphery of nuclear speckles that is enriched in RNA polymerase II . 44 4.2. 6 Effect of α-amanitin on the localization of YFP-HSPA6 protein to type III nuclear structures . 44 4.2. 7 Type III nuclear structures are RNase-sensitive . 49 4.2. 8 Comparison of type III nuclear structures in YFP-HSPA6 and YFP-HSPA1A transfected cells . 49 4.3 Sequence and structural analysis of HSPA6 and HSPA1A proteins . 56 4.3.1 Sequence analysis of HSPA6 and HSPA1A proteins . 56 4.3.2 Mechanism of action of HSPA (HSP70) proteins . 59 4.3.3 Structural analysis of the HSPA6 and HSPA1A proteins . 65 vii 5 DISCUSSION . 80 5.1 Stress-induced localization of HSPA6 and HSPA1A proteins to centrioles in cultured differentiated human neuronal cells . 80 5.2 YFP-tagged HSPA6 and HSPA1A proteins associate with nuclear structures following thermal stress . 83 5.2. 1 YFP-tagged HSPA6 and HSPA1A proteins localize to type I structures at nuclear speckles . 84 5.2. 2 YFP-tagged HSPA6 and HSPA1A proteins associate with type II nuclear structures in the granular component (GC) of nucleolus . 85 5.2. 3 YFP-HSPA6 protein associates with type III nuclear structures at the periphery of nuclear speckles . 87 5.3 Sequence and structural analysis of YFP-HSPA6 and YFP-HSPA1A proteins . 89 5.3.1 Potential effect of the cysteine substitutions on HSPA6 protein disulfide bond formation . 93 5.3.2 Potential effect of the serine to cysteine substitution on HSPA6 protein phosphorylation . 94 5.4 Conclusion . 96 6 REFERENCES . 98 7 Appendix: oneand three-letter codes of amino acids . 124