A Fascin-ating Journey: The Role of an Actin Bundling Protein in Axon Development Restricted; Files Only
Hardin, Katherine (Summer 2025)
Abstract
Proper neural circuit formation relies on precise axon guidance and branching, processes critically dependent on dynamic remodeling of the actin cytoskeleton. Fascin1, a conserved actin-bundling protein, organizes filamentous actin into tightly packed bundles essential for cellular protrusions, such as filopodia, which function in sensing environmental cues and directing axonal growth cone motility. Despite fascin’s established roles in cell motility and cancer metastasis, its function in neuronal development, particularly axon guidance and branching, remains poorly defined.
This thesis investigates fascin1's role in neurodevelopment, hypothesizing that fascin-mediated actin bundling is crucial for proper axon growth and neural circuit formation. Using cultured mammalian neurons, I demonstrate that pharmacological inhibition of fascin significantly reduces growth cone filopodia number and length, resulting in impaired axon extension and branching. These results confirm fascin’s pivotal role in actin-based structural dynamics essential for neuronal outgrowth.
With an in vivo Drosophila melanogaster model, I explored fascin’s (Singed in flies) function in axonal development within the mushroom body (MB), a structure analogous to the mammalian hippocampus involved in learning and memory. singed null flies exhibited pronounced defects in MB axon projection, alongside substantial locomotor impairments. These defects could be selectively rescued by reintroducing fascin specifically in MB neurons, underscoring its cell-autonomous role in axonal wiring and behavioral outputs. Further genetic manipulations revealed distinct effects of fascin phosphorylation state on neuronal morphology and functional rescue.
Additionally, challenges in genetically manipulating fascin1 in primary neuron cultures were systematically explored, highlighting limitations and technical considerations relevant to future investigations.
Collectively, this research establishes fascin1 as a critical regulator of axonal development and neuronal connectivity, emphasizing its role in brain wiring and function. These findings contribute significantly to our understanding of cytoskeletal dynamics in neurodevelopmental processes.
Table of Contents
Chapter 1: Introduction………………………………………………………………………...1
1.1 Dissertation hypothesis and questions…………………………………………...2
1.1 The Actin Cytoskeleton…………………………………………………………….3
1.1.1 Actin Binding Proteins…………………………………………………..4
1.1.1.1 Actin monomer sequesters…………………………………5
1.1.1.2 Nucleating proteins………………………………………….6
1.1.1.3 Severing proteins……………………………………………7
1.1.1.4 Capping proteins…………………………………………….8
1.1.1.5 Crosslinking proteins………………………………………..8
1.1.2 Actin Filopodia…………………………………………………………...9
1.1.2.1 How filopodia form…………………………………………10
1.1.2.2 Importance in cell motility…………………………………12
1.1.2.3 Filopodia in cell signaling…………………………………14
1.2 Axon Guidance……………..……………………………………………………..15
1.2.1 Functional importance of axon guidance……………………………16
1.2.2 The growth cone………………………………………………………17
1.2.3 Guidance cues…………………………………………………………18
1.2.4 Actin cytoskeleton remodeling……………………………………….23
1.3 Axon Branching…………………………………………………………………..24
1.3.1 Types of branching……………………………………………………25
1.3.2 Role of actin in branching……………………………………………27
1.4 Fascin: an actin filament bundling family………………………………………28
1.4.1 Post-translational regulation of fascin1…………………………….29
1.4.2 Fascin in cell motility………………………………………………….31
1.4.3 Fascin in neurodevelopment………………………………………..32
1.5 Use of Drosophila melanogaster as a model system…………………………33
1.5.1 Use as an axon guidance model……………………………………33
1.5.1.1 The ventral nerve cord…………………………………..34
1.5.1.2 The mushroom body…………………………………….35
1.5.2 Fascin in Drosophila…………………………………………………37
1.6 Summary ……………………………….………………………………………..38
1.7 Figures….…………………………………………………………………………39
2 Chapter Two: A Critical Role for the Fascin Family of Actin Bundling Proteins in Axon Development, Brain Wiring and Function………………………………………………49
2.1Summary………………………………………………………………………….50
2.1 Introduction……………………………………………………………………….51
2.2 Methods…………………………………………………………………………..54
2.3 Results……………………………………………………………………………65
2.3.1 Spatial distribution of fascin…………………………………………..65
2.3.2 Inhibition of fascin’s actin bundling activity impairs growth cone filopodia, axon growth, and branching………………………………68
2.3.3 Drosophila melanogaster as an in vivo model to study axon development and brain wiring………………………………………..70
2.4 Discussion……………………………………………………………………...81
2.5 Figures & Tables………………………………………………………………..89
3 Chapter Three: Challenges of genetic manipulation of fascin…………...………….128
3.1 CRISPR-Cas9 gene editing…………………………………………………..129
3.1.1 A brief history………………………………………………………..129
3.1.2 Challenges of editing post-mitotic cells…………………………..130
3.1.3 Targeted Knock-In with Two Guides (TKIT)……………………..131
3.1.4 Homology-independent Universal Genome Editing (HiUGE)….132
3.2 Methods…………………………………………………………………………134
3.3 Genetic manipulation of fascin1……………………………………………….139
3.3.1 TKIT……………………………………………………………………139
3.3.2 HiUGE and 3 guide pair knockout systems……………………….141
3.3.3 shRNA…………………………………………………………………143
3.4 Discussion………………………………………………………………………..145
3.5 Figures & Tables…………………………………………………………………147
4 Chapter Four: Discussion and future directions………………………………………155
4.1 Summary………………………………………………………………………….156
4.2 Fascin in axon development……………………………………………………157
4.3 Genetic manipulation of fascin…………………………………………………159
4.4 Future directions…………………………………………………………………159
References……………………………………………………………………………………162
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