LIM and SH3 Protein 1 in Actin-Based Cellular Motility and Axon Guidance Open Access

Pollitt, Stephanie (Summer 2020)

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The actin cytoskeleton drives cellular motility and is essential for neuronal development and function. LIM and SH3 Protein 1 (LASP1) is a unique actin-binding protein that is expressed in a wide range of cells including neurons, but its roles in cellular motility and neuronal development are not well understood. In this thesis work, I have investigated these outstanding questions in the contexts of lamellipodial actin structures and axonal development. I first show that LASP1 is expressed in rat hippocampus early in development, and this expression is maintained through adulthood, which supports the notion that LASP1 may play a role in early axonal growth. High-resolution imaging reveals that LASP1 is selectively concentrated at the leading edge of lamellipodia in migrating cells and axonal growth cones. This local enrichment of LASP1 is dynamically associated with the protrusive activity of lamellipodia, depends on the barbed ends of actin filaments, and requires both the LIM domain and nebulin repeats of LASP1. To understand the function of LASP1 in actin-based axon motility, I performed loss-of-function experiments. I found that knockdown of LASP1 in cultured rat hippocampal neurons resulted in a substantial reduction in axonal outgrowth and arborization. Furthermore, knockdown of the Drosophila homolog Lasp from a subset of commissural neurons in the developing ventral nerve cord produced defasciculated axon bundles that do not reach their targets. These data support a novel role for LASP1 in actin-based lamellipodial protrusion and establish LASP1 as a positive regulator of both in vitro and in vivo axon development. In the second part of my thesis work, I have developed an innovative approach that will enable immunolabeling of selected cells in multi-cell culture and tissue. This approach takes advantage of lipid oxidation-induced membrane permeability by reactive oxygen species (ROS) for selective labeling of single cells, termed RASCL (ROS-Assisted Single Cell Labeling). I have successfully utilized RASCL to label the actin cytoskeleton in selected cells of a multi-cell culture. Future optimization and use in brain slices will enable this technique to significantly advance our understanding of the subcellular distribution of specific proteins in a selected set of cells of intact tissues.

Table of Contents

Table of Contents

Chapter 1: Introduction1

1.1. Mechanisms of Actin-Based Cellular Motility 3

1.1.1. Actin and Actin Polymerization 3

1.1.2. Actin-Binding Proteins and Higher Order Actin Structures 5

1.2. Axon Guidance, Signaling, and Mechanisms 6

1.2.1. A Brief History of the Axon Guidance Field 7

1.2.2. Drosophila melanogaster as a Model System for Neurodevelopment 8

1.2.3. Axon Guidance Cues and their Detection: Navigating the Extracellular Space 10

1.2.4. The Development of Axon Collaterals 13

1.2.5. Regulation of Actin Remodeling Downstream of Axon Guidance Signaling 14 Actin Depolymerizing Factor and Cofilin 14 The Rho GTPases 17 GEFs and GAPs: Key Regulators of Actin Motility 18

1.3. LIM and SH3 Protein 1 (LASP1): a Review of the Literature and Outstanding Questions 19

1.3.1. The Nebulin Family of Actin-Binding Proteins 19

1.3.2. LASP1: a Review 21

1.4. Dissertation Hypothesis and Questions 23

1.5. Figures 24

Chapter 2: LIM and SH3 Protein 1 Localizes to the Leading Edge of Protruding Lamellipodia and Regulates Axon Development 26

2.1. Summary 27

2.2. Introduction 27

2.3. Methods 30

2.4. Results 36

2.4.1. LASP1 Expression in Neurons and its Dynamic Localization to the Leading Edge of Actin-Based Membrane Protrusions 36

2.4.2. Mechanisms of LASP1 Localization to the Leading Edge 39

2.4.3. LASP1 Promotes Axon Elongation and Branching 42

2.4.4. Lasp Promotes Axon Commissure Development and Fasciculation in vivo 44

2.5. Discussion 46

2.6. Figures 53

2.7. Supplemental Tables 67

Chapter 3: RASCL: A New Method for Labeling Optically- and Genetically-Selected Intracellular Protein Targets 71

3.1. Summary 72

3.2. Introduction 72

3.3. Methodology 73

3.4. Description of Materials 75

3.5. Procedure 76

3.6. Results 77

3.6.1. Efficacy of the RASCL Technique with Small Fluorescent Probes 78

3.6.2. RASCL-Induced Antibody Labeling 79

3.7. Future Troubleshooting for RASCL 80

3.8. Discussion and Future Directions 81

3.9. Figures 83

Chapter 4: Discussion and Concluding Remarks 86

4.1. Summary 87

4.1.1. Evidence for LASP1 as a Possible Regulator of Actin Polymerization or Anti-Capping 87

4.1.2. RASCL: A New Method for Labeling Optically- and Genetically-Selected Intracellular Protein Targets 90

4.2. Future Directions 90

References 93

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