Analyses of aging and heterogeneity in nuclei of multinucleated skeletal muscle cells Open Access
Cutler, Alicia (Fall 2017)
Abstract
The primary cell type of skeletal muscle is the myofiber. Myofibers are multinucleated containing thousands of nuclei all sharing a single, common cytoplasm. Despite all nuclei sharing the same cytoplasm, individual nuclei differ in key characteristics. Although the nucleus is the major site of gene regulation, the muscle nucleus has been underexamined because of technical limitations including contamination with non-myofiber derived nuclei and inability to selectively analyze individual nuclei. In this dissertation, we overcome these difficulties and demonstrate that nuclei in a single myofiber differ in nuclear import as a potential mechanism for achieving variation in nuclear content and function among nuclei in a single cell. We also present an approach to selectively isolate myonuclei, the first detailed myonuclear proteome, and changes that occur in the myonuclear proteome with aging. This work reinforces the significance of variation among myonuclei and emphasizes the importance of examining nuclear activity on the level of individual nuclei. Additionally, we show that we have developed the technical approach needed to pursue myonuclear-specific studies. Together our findings lay the foundation for careful examination of the myonuclear proteome, transcriptome, and epigenome in response to muscle growth, regeneration, exercise, and disease.
Table of Contents
Chapter 1: Background and Significance. 1
Introduction. 2
1.1 Skeletal muscle. 3
1.1.1 Myogenesis. 4
1.1.2 Aging Muscle. 6
1.2 Nucleocytoplasmic transport 8
1.2.1 Nuclear envelope. 9
1.2.2 Nuclear pore complex. 11
1.2.3 Nuclear transport receptors. 17
1.3 Multinucleated cells. 20
1.3.1 Multinucleated single-celled organisms. 21
1.3.2 Multinucleated cells in multicellular organisms. 23
1.4 Summary. 30
1.5 Figures. 33
Chapter 2: Non-equivalence of nuclear import among nuclei in multinucleated skeletal muscle cells 38
2.1 Summary. 39
2.2 Introduction. 40
2.3 Results. 44
2.3.1 Nuclear import varies among nuclei within single cultured multinucleated myotubes 44
2.3.2 Independence of nuclear import pathways in cultured myotubes. 48
2.3.3 Variation in cNLS import among myofiber nuclei in situ. 51
2.3.4 Nuclear import varies with muscle differentiation. 54
2.4 Discussion. 55
2.5 Methods. 66
2.6 Tables. 75
2.7 Figures. 76
Chapter 3: Biochemical isolation of myonuclei employed to define changes to the myonuclear proteome that occur with aging. 92
3.1 Summary. 93
3.2 Introduction. 94
3.3 Results. 96
3.3.1 Isolation of pure intact nuclei 96
3.3.2 Isolated nuclei are predominantly myonuclei 97
3.3.3 Increased depth of proteomic detection in myonuclear proteome. 99
3.3.4 The myonuclear proteome changes with age. 100
3.3.5 Myonuclei share common aging pathways with brain nuclei 102
3.4 Discussion. 104
3.5 Experimental Procedures. 108
3.6 Tables. 107
3.7 Figures. 112
Chapter 4: Discussion and future directions. 121
Appendix 1: Biochemical isolation of myonuclei from mouse skeletal muscle tissue. 134
Appendix 1.1 Abstract 135
Appendix 1.2 Background. 135
Appendix 1.3 Materials and Reagents. 136
Appendix 1.4 Equipment 138
Appendix 1.5 Procedure. 139
Appendix 1.6 Optional alternative approach. 146
Appendix 1.7 Data analysis. 147
Appendix 1.8 Notes. 148
Appendix 1.9 Recipes. 148
References. 153
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