Regulation of Myoblast Fusion by Creatine Kinase B and its Interacting Partners Open Access

Bankston, Adriana (2013)

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Myoblast fusion is critical for proper muscle growth and regeneration. During myoblast fusion, the localization of some molecules is spatially restricted, however the exact reason for such localization is unknown. Creatine kinase B (CKB), which replenishes local ATP pools, localizes near myotube ends. To gain insights into the function of CKB at the ends of myotubes, we sought to identify CKB interacting proteins using a yeast-two hybrid screen. We identified molecules with a broad diversity of roles, including actin polymerization. Given the importance of actin polymerization for myoblast fusion, we focused on the interaction between CKB and various actin isoforms, as well as actin regulatory proteins. Using co-immunoprecipitation, we identified α-skeletal-actin and α-cardiac-actin, two predominant skeletal muscle actin isoforms, as novel CKB interacting partners, which also colocalized with CKB in cultured mouse myotubes. Importantly, inhibition of CK activity by cyclocreatine treatment led to depolymerized F-actin in myotubes, as well as reduced myotube size and number, suggesting that CKB may be an essential factor for myotube formation by interacting with and modulating the actin cytoskeleton. However, CKB did not directly interact with α-skeletal-actin by co-sedimentation, indicating that intermediary proteins likely mediate the interaction between CKB and the actin cytoskeleton. CKB could therefore regulate actin dynamics indirectly via actin regulatory proteins, such as the N-BAR domain protein, Bridging integrator 3 (Bin3), which we also identified as a CKB interacting partner. We found that Bin3 regulated myofiber size in vitro and in vivo, as well as migration of differentiated muscle cells, where it colocalized with F-actin in lamellipodia. In addition, Bin3 formed a complex with Rac1 and Cdc42, Rho GTPases involved in actin polymerization, and regulated their activity in differentiated muscle cells. Overall, these results suggests a Bin3-dependent pathway is a major regulator of actin dynamics in differentiated muscle cells, which may, in turn, be modulated by the ability of CKB to provide ATP for actin polymerization.

Table of Contents

Chapter 1: Introduction 1

Chapter 2: Background and Significance 5

2.1 Skeletal Muscle 6

2.1.1 Skeletal Muscle Structure and Function 6

2.1.2 Skeletal Muscle Regeneration 8 Branched Myofibers During Myogenesis 10

2.2 Migration and Fusion During Myogenesis 11

2.2.1 Factors Controlling Muscle Cell Migration 12

2.2.2 Regulation of Myoblast Fusion 14

2.2.3 Actin Dynamics During Myogenesis 17

2.3 Creatine Kinase 19

2.3.1 Roles of Creatine Kinase in ATP Production and Actin Regulation 20

2.3.2 Creatine Kinase Functions During Myotube Formation 22

2.4 BAR Domain Proteins 23

2.4.1 BAR Domain Proteins as Membrane Modulating Proteins 24

2.4.2 BAR Domain Proteins Implicated in Actin Regulation 26

2.4.3 Mammalian Studies of the Amphiphysin/Bin Family of Proteins 29

2.4.4 Yeast Studies of the Amphiphysin/Bin Family of Proteins 32

2.4.5 BAR Domain Proteins Involved in Skeletal Muscle Myogenesis 35

2.5 Summary 37

2.6 Figures and Tables 38

Chapter 3: Materials and Methods 54

Chapter 4: Creatine Kinase B Interacts With and Modulates the Actin Cytoskeleton During Myoblast Fusion 70

4.1 Introduction 71

4.2 Results 74

4.3 Discussion 81

4.4 Figures and Tables 85

Chapter 5: The N-BAR Domain Protein, Bridging Integrator 3, Regulates Rac1- and Cdc42-Dependent Processes in Myogenesis 107

5.1 Introduction 108

5.2 Results 111

5.3 Discussion 118

5.4 Figures and Tables 122

Chapter 6: Discussion 148

6.1 Introduction 149

6.2 Specificity of CKB Binding to Muscle-Specific Actin Isoforms 150

6.3 Mechanisms of CKB and CKM Localization in Differentiated Muscle Cells 151

6.4 Interactions of CKB with Actin Regulatory Proteins 154

6.5 Roles of Bin3 in Myogenesis 155

6.6 Molecular Mechanisms of Bin3 Action in Muscle Cells 159

6.7 Bin3 Is Involved in Myofiber Branching 161

6.8 Perspectives and Therapeutic Strategies 164

6.9 Figures 168

References 180

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