Regulation of Dendrite Development and Synapse Formation by Tropomodulin Open Access

Abstract

Neurons of the central nervous system elaborate highly branched dendritic arbors that host numerous dendritic spines, actin-rich protrusions that serve as the postsynaptic platform for excitatory synapses.  The actin cytoskeleton plays an important role in dendrite development and controls the structure and molecular organization of spines during synapse formation. However, the molecules and mechanisms that regulate actin organization and remodeling during postsynaptic development are not fully understood. Tropomodulins (Tmods) are a multi-domain family of proteins that cap the pointed end of actin filaments, thereby regulating the stability, length, and architecture of complex actin networks in diverse cell types. Three members of the Tmod family, Tmod1, Tmod2, and Tmod3 are expressed in the vertebrate CNS, but their function in neuronal development is largely unknown.  In this study, we present evidence that Tmod1 and Tmod2 are expressed in the hippocampus and play an important role in dendrite arborization and synapse formation.  Loss-of-function analysis reveals that Tmod2, but not Tmod1, is required for dendritic branching during postsynaptic development.  Both Tmod1 and Tmod2 localize to a unique sub-spine region, where they regulate F-actin stability in spines. Knockdown of either Tmod1 or Tmod2 disrupts spine morphogenesis and impairs synapse formation. Together, these findings establish an important role for Tmods in postsynaptic development.  Regulation of F-actin stability by Tmod1 and Tmod2 represents a key mechanism underlying the cytoskeletal rearrangements required for spine morphogenesis and synapse development.

Table of Contents

Chapter I:  Introduction............................................................................1

1.1.          The Synapse: Gateway of Neuronal communication …………………………….2

1.2.          Neuronal Morphology and Synaptic Transmission……………………………….4

1.3.          Postsynaptic Dendrite Development and Synapse Formation…………………….2

1.3.1.     Early Postsynaptic Development……………………………………...7

1.3.2.     A coupled Process: Synapse Development and Dendrite Stabilization…………………………………………………………...8

1.3.3.     Synaptotrophic Hypothesis of Dendrite Development……………....10

1.3.4.     Synaptic Refinement: Dendrite and Synapse Uncoupling…………...11

1.3.5.     Adulthood: Stable Structures, Plastic Synapses……………………...13

1.4.          Models of Dendritic Spine Development………………………………………..15

1.4.1.     The ‘Filopodia Elongation Model’………………………………......16

1.4.2.     The Sotelo Model…………………………………………………….17

1.4.3.     The Millers/Peters Model………………………………………….....18

1.5.             Dendritic Spine Structure……………………………………………………….19

1.6.             Ultrastructure of Dendritic Spines: ……………………………………………..22

1.6.1.               The Actin Cytoskeleton: Organization, Kinetic Pools, Plasticity..22

1.6.2.               The Postsynaptic Density: Glutamate Receptors and Scaffolds…25

1.7.                  The Actin Cytoskeleton in Dendritic Spine Morphogenesis………………….28

1.8.                  Modulation of F-actin in Dendritic Spines by Capping Proteins……………..32

1.9.                  Tropomodulin Family of F-actin Pointed End Capping Proteins……………..37

1.9.1.               Tropomodulin Isoforms………………………………………….37

1.9.2.               Structural and Functional Domains of Tropomodulin…………...37

1.9.3.               Tropomyosins…...……………………………………………….38

1.9.4.               Proposed Model of Tropomodulin Attachment to Pointed Ends...39

1.9.5.               Other Actin Regulatory Functions of Tropomodulin…………….40

1.9.6.               Localization and Function in Diverse Cellular Structures ……....40

 

 

1.10.               Summary…………………………………………………………………...…42

Chapter II:    Materials and Methods……………………………….43

Chapter III:   Tmod1 and Tmod2 in Dendrite Development……..62

3.1                      Introduction………………………………………………………………….62

3.2                      Results……………………………………………………………………….63

3.2.1          Expression of Tmod1 and Tmod2 in hippocampal neurons  ..….63

3.2.2          Membrane association of Tmods …………………………….....66

3.2.3          Tmods in Dendrite Development………………………………..68

3.3                      Discussion…………………………………………………………………...69

3.4                      Figures……………………………………………………………………….73

Chapter IV:   Regulation of Dendritic Spine Development and Synapse Formation by Tropomodulin……………………………….84

4.1                      Introduction…………………………………………………………………..84

4.2                      Results………………………………………………………………………..85

Discussion…………………………………………………………………....91

4.3                      Figures……………………………………………………………………......96

Chapter V:    Discussion:  Tropomodulins in the Postsynaptic Compartment…………………………………………………………....105

5.1              Characterizing Tmods at the synapse……………………………………...106

5.2               How does the subspine localization of Tmod extend our understanding of the   spine cytoskeleton?......................................................................................107

5.3              How is the stable pool of F-actin generated?................................................108

5.4              The role of Tmod in dendritic spine development and synapse formation……………………………………………………….………….110

5.5              Tmods and plasticity……………………………………………………….111

5.6               Summary…………………………………………………………………..112

References ………………………………………………………………113

List of Figures

Figure 1.1. Neuronal morphology and synapse function

Figure 1.2. Dendrite and dendrite spine dynamics during development

Figure 1.3. Three models of spinogenesis

Figure 1.4. Dendritic spine structure and morphology

Figure 1.5. Organization of F-actin in mature spines

Figure 1.6. Model for actin cytoskeleton organization during spine morphogenesis 

Figure 1.7. F-actin capping proteins at filament ends

Figure 3.1. Expression profile and subcellular distribution of Tmod1 and Tmod2

Figure 3.2  Membrane association of Tmod1 and Tmod2

Figure 3.3. Distribution of Tmod1 and Tmod2 in dendrites

Figure 3.4  Tmod1 and Tmod2 in Dendrite Development

Figure 3.5 Regulation of dendritic development by Tmod2

Figure 3.6 Cytoskeletal organization in dendrites

Figure 4.1. Expression profile of Tmod1 and Tmod2 in hippocampal neurons                    

Figure 4.2. Subcellular distribution of Tmods in hippocampal neurons

Figure 4.3  Subspine localization of Tmod1 and Tmod2

Figure 4.4. Frap analysis of actin in Tmod1 or Tmod2 knockdown neurons

Figure 4.5. Tmod1 or Tmod2 knockdown impairs spine formation

Figure 4.6. Tmod loss impairs synapse formation/schematic representation of Tmod localization and function in postsynaptic compartment.

 

 

 

List of Tables

Table 1.1: Actin-binding proteins/signaling pathways in spine morphogenesis

 

 

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Biochemistry, Cell & Developmental Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience Biology, Cell
Keyword	Synapse, Actin, Tropomodulin, Dendritic Spine, Dendrite, Pointed end
Committee Chair / Thesis Advisor	Zheng, James, Emory University
Committee Members	Koval, Michael, Emory University Kowalcyzk, Andrew, Emory University Faundez, Victor, Emory University Feng, Yue, Emory University Ono, Shoichiro, Emory University

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