Multiprotein Regulation at Actin Barbed ends by Twinfilin, Capping Protein, and Formin Restricted; Files Only
Ulrichs, Heidi (Summer 2025)
Abstract
Actin is an essential protein that is necessary for various eukaryotic processes like embryonic development, muscular movement, and neuronal formation. Cellular actin dynamics are regulated by a multitude of actin filament binding proteins. These actin binding proteins are described via their impact on filament dynamics: polymerases, cappers, depolymerases, and stabilizers. First, I study barbed end dynamics with a capper, capping protein, a polymerase, formin, and a depolymerase, twinfilin. The dynamics of these three proteins at the actin barbed end have been studied individually, but never all together. Second, I study F-actin side binding and stabilization via coactosin-like protein (CLP) and filament severing via cofilin. CLP and cofilin are a part of the same ADF protein family yet have opposing effects on actin filament dynamics. Additionally, CLP has never been studied at the single filament level. In this dissertation, I visualize labeled capping protein, twinfilin, formin, actin, and CLP to biochemically characterize the dynamics of these proteins in vitro. I use microfluidics assisted total internal reflection fluorescence (mf-TIRF) microscopy to elucidate the mechanisms of actin binding proteins on actin filament barbed and pointed ends. Overall, this work reveals fundamental mechanisms governing actin filament, and network, dynamics; ultimately providing a biochemical framework to understand cytoskeletal organization in cellular processes and various disease states.
Table of Contents
Table of Contents
CHAPTER 1: Introduction 1
1.1 Actin 2
History 2
Structure and Dynamics 2
Cellular Processes 4
Genes and Mutants in Disease 5
1.2 Actin binding proteins 5
Actin filament nucleation proteins 6
Actin filament elongation proteins 7
Formin 7
Actin filament capping proteins 11
Capping Protein 11
Actin filament crosslinking and stabilizing proteins 14
Coactosin-Like Protein 14
Actin monomer-pool regulating proteins 16
Profilin 16
Actin Depolymerases 19
Twinfilin 20
1.3 Figures 28
Figure 1: Actin Nucleotide State and Dynamics. 28
Figure 2: Formin Structure and function. 29
Figure 3: Capping protein (CapZ) Structure and function. 30
Figure 4: Coactosin-like protein (CLP) and cofilin structure. 31
Figure 5: Profilin Structure and function. 32
Figure 6: Cellular localization of twinfilin in various cell types. 32
Figure 7: Domain composition and structure of twinfilin. 33
Figure 8: Schematic representation of diverse interactions of twinfilin with actin filaments. 34
1.4 References 35
CHAPTER 2: Aims of the study and Methods 54
2.1 Aims of the Study 55
2.2 Methods 55
Protein Purifications and labeling 55
Actin 55
Twinfilin Polypeptides 57
Formin 58
Capping Protein 59
Profilin 60
Cofilin 61
Coactosin-like Protein 1 61
VCA 63
Coronin 1B 64
Conventional TIRF Microscopy 65
Barbed-End, Formin, Capping protein, Twinfilin Single Molecule Imaging 65
Coactosin-like Protein Single Molecule Imaging 67
Arp2/3 Debranching Assay 67
Cofilin ADP-Pi Uncapping 68
Microfluidics assisted TIRF Microscopy 68
Twinfilin on BFC “decision complexes” 69
Coactosin-like Protein on Actin Depolymerization 69
VopF Pointed end Elongation with 549-Capz 70
Image Analysis 70
Determination of rates 70
CP or formin dissociation from BFC complexes (k’-F and k’-C) 70
Coactosin-like protein-549 Filament binding Kinetics 72
2.3 References 72
CHAPTER 3: Characterization of Twinfilin on both a Capping protein and a Formin bound Actin barbed end 74
3.1 Introduction 75
Abstract 75
Background 75
3.2 Results 76
Single-molecule analysis uncovers twinfilin’s uncapping mechanism 77
Capping protein and twinfilin differentially influence formin’s processivity 78
3.3 Figures 81
Figure 1: Visualization and characterization of twinfilin’s interactions with CP-bound barbed ends. 81
Figure 2: Photobleaching tests of 549-CP molecules. 83
Figure 3: Photobleaching tests of 549-mTwf1 molecules. 83
Figure 4: SNAP tagging of mTwf1 doesn’t influence its uncapping activities. 84
Figure 5: Effect of twinfilin and capping protein (CP) on processivity of formin. 86
Figure 6: Presence of 1 µM mTwf1 does not change the elongation rate of formin mDia1. 87
Figure 7: Working Model for Free Barbed ends. 88
3.4 Discussion 88
3.5 References 90
CHAPTER 4: Mechanism of twinfilin’s action on formin-capping protein barbed end “decision complexes” 93
4.1 Introduction 94
Abstract 94
Background 94
4.2 Results 96
Twinfilin accelerates dissociation of the formin-CP complex at barbed ends 96
Single-molecule visualization of twinfilin’s effects on BFC dynamics 98
Twinfilin’s interaction with actin is essential for its effects on BFC dynamics 101
4.3 Figures 102
Figure 1: Effect of twinfilin on capping protein (CP)—formin decision complex. 103
Figure 2: Concentration dependent effect of mTwf1 on dissociation rate of BFC complexes and appearance of BF and BC. 104
Figure 3: Photobleaching tests of 649-mDia1 molecules. 105
Figure 4: Direct visualization of formin, CP, and mTwf1 at barbed ends. 106
Figure 5: Twinfilin’s direct interaction with actin filament is essential for its effects on formin-CP decision complexes. 108
Figure 6: Working model for regulation of actin dynamics by twinfilin, formin, and CP. 109
4.4 Discussion 110
4.5 References 112
CHAPTER 5: Coactosin-Like Protein 1 protects actin filaments from cofilin-mediated severing 117
5.1 Introduction 118
Abstract 118
Background 118
5.2 Results 120
Coactosin-like Protein 1 slows depolymerization of actin filament ends 120
CLP Protects from Cofilin-mediated Severing 121
Single-molecule visualization of coactosin-like protein on actin filaments 122
5.3 Figures 123
Figure 1: CLP slows barbed and pointed end depolymerization. 123
Figure 2: CLP does not affect barbed-end polymerization. 124
Figure 3: CLP protects filaments from cofilin severing. 125
Figure 4: Photobleaching tests of CLP-549 molecules. 126
Figure 5: CLP-549 single molecule imaging on 488-labeled actin filaments. 127
Figure 6: CLP-549 Observed Binding Kinetics. 128
Figure 7: Cartoon of effects of CLP on F-actin. 129
5.4 Discussion 129
5.4 References 132
CHAPTER 6: Discussion 135
6.1 Introduction 136
6.2 Discussion 136
Twinfilin is an inefficient barbed end uncapper 136
Twinfilin increases dynamics of Formin-CP-bound Barbed end Complex 137
CLP Stabilizes actin filament Ends and sides 139
Future Directions 140
6.3 Final Remarks 141
6.4 References 142
APPENDIX 1: Profilin affects microtubule dynamics via actin 146
1.1 Overview 147
1.2 Abstract 147
1.3 Introduction 147
1.4 Article Spotlight 148
1.5 Conclusions 150
1.6 Figure 152
Figure 1. Schematic representation illustrating changes in microtubule and actin networks resulting from genetic and pharmacological perturbations. 152
1.7 References 153
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