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Duke University Dissertation Template Novel Regulators of Actin Signaling During the Developmental Stage of Spine Formation and Maturation by Erin Faith Spence Department of Cell Biology Duke University Date: May 23rd, 2018 Approved: ___________________________ Scott Soderling, Supervisor ___________________________ Cagla Eroglu, Chair ___________________________ Marc Caron ___________________________ Richard Mooney ___________________________ Yong-Hui Jiang Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Cell Biology in the Graduate School of Duke University 2018 ABSTRACT Novel Regulators of Actin Signaling During the Developmental Stage of Spine Formation and Maturation by Erin Faith Spence Department of Cell Biology Duke University Date: May 23rd, 2018 Approved: ___________________________ Scott Soderling, Supervisor ___________________________ Cagla Eroglu, Chair ___________________________ Marc Caron ___________________________ Richard Mooney ___________________________ Yong-Hui Jiang An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Cell Biology in the Graduate School of Duke University 2018 Copyright by Erin F Spence 2018 Abstract Excitatory synapse formation during development involves the complex orchestration of both structural and functional alterations at the postsynaptic side, beginning with the formation of transient dendritic filopodia. Abnormalities in synapse development are linked to developmental brain disorders such as autism spectrum disorders, schizophrenia, and intellectual disability. However, the molecular mechanisms that underlie excitatory synaptogenesis remain elusive, in part because the internal machinery of developing synapses is largely unknown. Unlike mature excitatory synapses, there is currently no way to biochemically isolate the dendritic filopodia of nascent synapses. This lack of understanding is a critical barrier to our grasp of synapse development as well as the etiology of many neurodevelopmental disorders. This dissertation work focuses on the detection and analysis of proteins which localize to and are critical for spinogenesis and synaptogenesis. Using state-of-the-art in vivo proteomics, we identified a network of proteins which localize to the receiving end of the developing excitatory synapse, the dendritic filopodia. We then used the CRISPR/Cas9 system to identify candidates which drive the formation and maturation of dendritic filopodia. We finally did careful functional analysis of CARMIL3 and the Arp2/3 complex to identify their critical and diverse roles in synaptogenesis. iv In our analysis, we found that CARMIL3 is expressed in the brain predominately during synaptogenesis, localizes to developing dendritic protrusions, and is important for the morphological and functional maturation of synapses, likely through its role in recruiting capping protein to maturing synapses. Loss of CARMIL3 leads to structurally and functionally immature synapses that are capping protein deficient. Further, we found that the Arp2/3 complex, a critical regulator of the actin cytoskeleton which creates branched actin networks, is required for both the functional and morphological maturation of dendritic spines. In the absence of the Arp2/3 complex, dendritic protrusions make presynaptic contact, recruit key proteins such as MAGUKs, and recruit certain receptors such as NMDA receptors, but lack AMPA receptors which are required for synapse unsilencing. Together, this work demonstrates that the actin cytoskeleton controls the functional maturation of synapses by altering the cytoskeletal dynamics towards the creation of a branched actin network. CARMIL3 contributes to this process by providing capping protein, which biases actin nucleation towards branched actin networks. Arp2/3 creates the branched actin network. Without this network, there is not a sufficient framework to dock AMPA receptors in the post-synaptic density, and without AMPA receptors, dendritic protrusions remain functionally silent. Together, this work shows that the dynamics of the actin cytoskeleton drive synapse unsilencing. v Table of Contents Abstract ......................................................................................................................................... iv Table of Contents ......................................................................................................................... vi List of Tables ................................................................................................................................. xi List of Figures .............................................................................................................................. xii 1 Introduction ................................................................................................................................ 1 1.1 Overview of the Excitatory Glutamatergic Synapse ................................................... 1 1.1.2 Dendritic Spines ...................................................................................................... 1 1.1.2 Excitatory Transmission in Dendritic Spines .......................................................... 4 1.1.3 Dendritic Spine Structural Plasticity......................................................................... 6 1.2 The Cytoskeleton of the Dendritic Spine ...................................................................... 7 1.2.1 Actin Filament Assembly ........................................................................................... 8 1.2.1.1 The Arp2/3 Complex ......................................................................................... 10 1.2.1.3 The Formin Family ............................................................................................ 12 1.2.1.3 Ena/VASP and the Actin Cytoskeleton ........................................................... 14 1.2.1.4 Profilin in Dendritic Spines .............................................................................. 15 1.2.2. Actin Filament Disassembly ................................................................................... 15 1.2.3 Actin Filament Stabilization ..................................................................................... 17 1.2.3.1 The CARMIL Family of Proteins ..................................................................... 18 1.2.4 The Interplay between Actin Stabilization and Assembly .................................. 21 1.2.4.1 The Actin Funneling Hypothesis ..................................................................... 21 vi 1.2.4.2 Monomer Gating ................................................................................................ 21 1.3 Dendritic Spine Formation ............................................................................................ 22 1.3.1 Models of Spinogenesis ............................................................................................ 23 1.3.1.1 The Sotelo Model ............................................................................................... 23 1.3.1.2 The Miller/Peters Model ................................................................................... 24 1.3.1.3 The Filopodia Model ......................................................................................... 25 1.3.2 Regulation of Dendritic Filopodia .......................................................................... 28 1.3.2.1 Regulators of Filopodia Formation and Degradation ................................... 29 1.3.2.2. Regulators of Filopodia Stabilization ............................................................. 31 1.3.2.3 Extracellular Signals which Regulate Dendritic Filopodia .......................... 32 1.3.3 Transformation from a Dendritic Filopodia to a Dendritic Spine ...................... 34 1.3.3.1 Response to Neurotransmitters ....................................................................... 34 1.3.1.2 Binding of Adhesion Molecules ....................................................................... 34 1.3.1.3 Changes in Membrane Tension ....................................................................... 35 1.3.4 Uncovering Novel Regulators of Synaptogenesis ................................................ 36 1.4 The Actin Cytoskeleton and Neurodevelopmental Disorders ................................. 37 1.4.1 Schizophrenia ............................................................................................................. 38 2 Materials and Methods............................................................................................................ 41 2.1 Animals ............................................................................................................................ 41 2.2 Slice Cultures................................................................................................................... 41 2.3 Primary Neuronal Cultures .......................................................................................... 42 2.4 Analysis of Slice Dendritic Spine Morphology .......................................................... 42 vii 2.5 Analysis of Cultured Neuron Dendritic Spine Morphology and Filopodial Dynamics ............................................................................................................................... 43 2.6 FLEx Rescue Strategy ....................................................................................................
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