Stephanie L. Gupton, PhD - US grants

DESCRIPTION (provided by applicant): Damage to connections within the adult Central Nervous System (CNS) by injury or disease is often irreparable. To design therapies to repair CNS damage requires a detailed understanding of the cellular mechanisms underlying CNS development. As the brain develops, neurotrophic cues, such as netrin, guide axons to their postsynaptic targets and induce axon branching to increase synaptic capacity. Both the guided locomotion of axonal growth cones, and their ramification into multiple axon branches, require the same fundamental cellular machinery. F-actin and microtubule (MT) dynamics initiate and steer membrane protrusions. Exocytosis delivers phospholipids and membrane proteins required to supply material to the expanding plasma membrane. Coordination of cytoskeletal dynamics and vesicle trafficking likely plays key roles in axon guidance and branching. However, the molecular mechanisms that mediate such interactions during axon guidance and axon branching are not understood. Our findings place TRIM9 at the junction of netrin/DCC signaling to both the cytoskeletal and vesicle trafficking machinery. Using a combination of mouse genetics, primary cell culture, live cell imaging and neuroanatomical studies, my lab found that TRIM9-deficient cortical neurons show misregulated exocytosis and defective actin and MT dynamics. Furthermore, we found that cortical neurons devoid of TRIM9 have constitutive branching defects, fail to form from branches in response to netrin, and are defective in netrin-based axon guidance. In vivo, we have found that loss of TRIM9 is associated with defective cortical axon fiber tracts. Our findings that TRIM9 interacts with and regulates the exocytic tSNARE, SNAP25, lead us to hypothesis that TRIM9 spatially and temporally regulates exocytosis in the growing axon. Novel interactions identified with multiple cytoskeletal regulators, including Ena/VASP proteins, Lamellipodin, and MAP1B lead us to hypothesize that TRIM9 participates in protein networks that play key roles in F-actin and MTs dynamics. As we have found that TRIM9 binds directly to the netrin receptor, DCC, and is required for functions downstream of the axon guidance cue netrin, we hypothesize that TRIM9 is essential for the coordinated activities of the cytoskeleton and exocytosis that dictate axon branching and guidance in response to netrin/DCC. My lab is in a unique position to determine the molecular mechanism that link guidance cues to local changes in cytoskeletal dynamics and axon branching through live-cell imaging, quantitative image analysis, biochemistry, and mouse models. Our long-term goal is to understand how neurons integrate environmental cues to orchestrate changes in their morphology and movement necessary to establish a functional nervous system. A better understanding of the mechanistic basis of axon guidance and axon branching will provide fundamental insight into how connections in the nervous system are established and how they are remodeled during plasticity. The results of our research plan should be of great value to the development of therapeutic approaches to repair these connections subsequent to disease or injury.

ABSTRACT The overall goal of this proposal is to understand the mechanism by which Rho GTPases, Rab GTPases, tethering agents, and SNAREs contribute to direct polarized trafficking and growth at the cell surface. Previous work from our laboratories and others has implicated members of the Rho/Cdc42 and Sro7/Tomosyn/Lgl protein families as factors that have important roles in both polarity and membrane trafficking to the cell surface in yeast and neurons. Spatial regulation of trafficking in yeast requires specific patterns of Rho/Cdc42 localization as well as tight regulation of vesicle tethering and fusion by the exocyst complex and Lgl/Sro7 protein. In this proposal, we will examine the mechanism by which Sro7 acts as a Rab-dependent tethering agent in exocytosis, and how gain-of-function mutants in the exocyst which mimic Rho GTPase activation of the exocyst complex, structurally change in order to upregulate its activity as a Rab-dependent tethering agent. Additionally, we will explore the hypothesis that a novel schizophrenia-linked protein with homology to an endocytic SNARE protein, acts to regulate exo/endocytic trafficking in the neuron.

PROJECT SUMMARY Cellular shape change is a fundamental characteristic of metazoan cells that is key to development, physiology, and pathology. The formation and plasticity of neural networks are key examples of cell shape change during development and physiology, whereas cell shape and motility goes awry in cancers such as melanoma. The active control of the cytoskeleton is acknowledged as critical to cellular shape change, whereas the concurrent remodeling of the plasma membrane is perhaps less well appreciated. Although many cytoskeletal and membrane remodeling components are known and their biochemical and structural characteristics described, we lack a systematic understanding of how these disparate systems are regulated and coordinated to orchestrate cellular shape change. Perhaps the most important problem in cell morphogenesis is understanding how cells perceive cues in their environment and convert this extracellular information into shape changes through coordinated cytoskeletal dynamics and plasma membrane remodeling. Functions of small GTPases and kinases are well studied in regulating cytoskeletal dynamics and membrane remodeling. Work from my lab identified an emerging role for E3 ubiquitin ligases in regulated cellular shape change. We identified two E3 ubiquitin ligases, TRIM9 and TRIM67, which regulate cytoskeletal and exocytic proteins and cellular shape changes in response to netrin. Netrin is an extracellular morphogen that promotes neuronal morphogenesis and the progression of cancers, such as melanoma. TRIM9 and TRIM67 thus provided an excellent entry point for the lab to investigate how cytoskeletal and membrane remodeling are coordinated during netrin triggered morphogenesis and motility. TRIM9 and TRIM67 share similar sequences, localization, and interaction partners, however our studies identified distinct functions of these related proteins and antagonistic phenotypes associated with their deletion. The overarching goal of this program is to test the hypothesis that TRIM9 and TRIM67 coordinate cytoskeletal dynamics and exocytosis during netrin-dependent morphogenesis in neurons and migrating melanoma cells. Our work will provide fundamental mechanistic understanding of the regulation of the cytoskeleton and membrane trafficking during development and metastasis.

PROGRAM DESCRIPTION The overarching research goal of my lab is to define cellular and molecular mechanisms mediating cellular shape change. Cellular shape change is a fundamental characteristic of metazoan cells, key to development, physiology, and pathology. The formation and plasticity of neural networks are key examples of cell shape change during development and physiology, whereas cell shape and motility goes awry in pathological conditions, such as melanoma. We study two main themes during cellular shape change: The active control of the cytoskeleton, which is acknowledged as critical to cellular shape change, and the concurrent remodeling of the plasma membrane, which is perhaps less well appreciated. Although many cytoskeletal and membrane remodeling components are known and their biochemical and structural characteristics described, we lack a systematic understanding of how these disparate systems are regulated and coordinated to orchestrate cellular shape change. Perhaps the most important problem in cell morphogenesis is understanding how cells perceive cues in their environment and convert this extracellular information into shape changes through coordinated cytoskeletal dynamics and plasma membrane remodeling; this is the focus of this proposal. Functions of small GTPases and kinases have been extensively studied in regulating cytoskeletal dynamics and membrane remodeling. Work from my lab identified an emerging role for E3 ubiquitin ligases in regulated cellular shape change. We identified two E3 ubiquitin ligases, TRIM9 and TRIM67, which regulate cytoskeletal and exocytic proteins and cellular shape changes in response to netrin. The extracellular morphogen netrin promotes neuronal morphogenesis and cancer progression. Despite these important consequences, we know little about how cells interpret netrin into shape changes. TRIM9 and TRIM67 provide an excellent opportunity to investigate the function of ubiquitination in cytoskeletal and membrane remodeling, and how these functions are coordinated during netrin triggered cell shape change and motility. TRIM9 and TRIM67 share similar sequences, localization, and interaction partners, however our studies identified distinct functions of these related proteins and antagonistic phenotypes associated with their deletion. The overarching goal of this program is to test the hypothesis that TRIM9 and TRIM67 coordinate cytoskeletal dynamics and exocytosis during netrin-dependent morphogenesis in multiple cell types. Since netrin plays roles in both neuronal development and cancer pathogenesis, our work will exploit developing neurons and migrating melanoma cells as model systems. Our preliminary and published data indicate both cell types respond to netrin and express TRIM9 and TRIM67. Our work will illuminate fundamental generalities and cell type specific mechanisms of shape change, providing mechanistic understanding of the coordination of the cytoskeleton and membrane trafficking during development and metastasis.