Student Projects

Structural and biophysical studies of Tmod regulation of actin dynamics

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Tmod is a master regulator of actin dynamics, exhibiting various activities such as filament binding and stabilization of the pointed-ends, sequestration of actin monomers, and nucleation of new filaments.  The versatility of Tmod explains its involvement in a wide range of cellular processes such as epithelial cell polarity, endothelial cell motility, muscle thin-filament formation, and tissue morphogenesis in embryonic development.  This project aims to obtain mechanistic insights into these functions of Tmod and provides an opportunity to learn how to employ a rational strategy toward this goal, using a combination of structural tools, biochemistry, and biophysics.  Initial work will involve identification and optimization of a minimal actin monomer-Tmod complex for successful crystallization using actin biochemistry and NMR analysis, followed by X-ray crystallographic structure determination.  At later stages, the study will be expanded toward elucidation of the mechanism of filament binding and stabilization of the ends based on the new crystal structure, computational analyses, and potentially EM studies. The structure from this work will be the first visualization of the interaction involving the actin pointed-end at a high resolution, which current textbooks lack.  This project will be a collaboration between the Fowler Lab (http://www.scripps.edu/fowler/), the Otomo Lab (http://www.scripps.edu/mb/otomo/) and the superb structural biology groups at TSRI, including the National Resource for Automated Molecular Microscopy (http://nramm.scripps.edu/) and the Joint Center for Structural Genomics (http://www.jcsg.org/).

Actin dynamics in the red blood cell membrane skeleton

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The red blood cell (RBC) membrane contains the canonical example of a membrane skeleton—a protein meshwork that underlies the plasma membrane and imparts the membrane with the robust mechanical properties required for survival in the circulation.  The short actin filaments in the RBC membrane skeleton are crosslinked by spectrin strands, stabilized along their sides by tropomyosins (TMs), and capped at their barbed and pointed ends by adducin and tropomodulin (Tmod), respectively.  This project will explore the hypothesis that TM and Tmod regulate actin dynamics, spectrin network integrity and membrane mechanical properties in RBCs, using transgenic mice with targeted deletions in TMs or Tmods.  RBC membrane structure will be examined by biochemistry, high-resolution fluorescence microscopy and electron microscopy, and RBC mechanics will be tested by osmotic fragility and shearing assays.  RBC production and survival in vivo will be determined from blood hematology and studies of hematopoietic organs (fetal liver, bone marrow, spleen).  At later stages, the study will be expanded to set up a human CD34+ stem cell culture system to study the role of actin dynamics in erythroid terminal differentiation, using shRNA knockdowns and overexpression approaches.  These experiments will define the origin of the impressive resilience and deformability of RBCs and explain how altered actin dynamics may lead to hereditary anemias due to impaired RBC production or survival.

Spectrin-actin network function in eye lens transparency

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The fiber cells that comprise the eye lens are terminally differentiated cells with a sophisticated 3D architecture and biochemical composition that provides optical clarity and mechanical resilience.  Lens fiber cells contain a spectrin/actin-based membrane skeleton, that is structurally and functionally similar to that found in red blood cells, and is important for maintaining fiber cell shapes and lens stiffness.  Recently, we have shown that spectrin-actin network integrity creates membrane subdomains specialized either for fiber cell adhesions or gap junction-mediated cell coupling.  The goal of this project is to define the molecular basis for spectrin-actin network control of membrane subdomain assembly, stability and function during lens development and aging.  The project will involve studies of lenses from mice with targeted mutations or deletions in Tmods, tropomyosins or spectrins, and will use confocal immunofluorescence imaging and proteomics approaches to define interacting partners and structural relationships.  For functional analysis, lens optical properties, electrophysiology and biomechanical compression testing will be performed.  These studies on lens fiber cell membrane subdomains will provide insight into how domain dysfunction leads to loss of lens transparency, cataracts, and blindness in humans.

Gamma-actin dynamics and regulation in the sarcoplasmic reticulum

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The sarcoplasmic reticulum (SR) of skeletal muscle is a critical membrane system because it serves as the calcium reservoir of muscle contraction.  Our laboratory has recently shown that nonmuscle (gamma) actin filaments, capped by tropomodulin (Tmod) isoform 3 and stabilized by nonmuscle tropomyosins (TMs), form a novel membrane skeleton-like structure that undergirds the SR, regulates calcium handling, and provides mechanical fortification to the intermyofibrillar linkage provided by the SR.  This project will use a variety of biochemical approaches, including F-actin binding and pyrene-actin polymerization, pull-down, blot overlay, and co-immunoprecipitation assays, to understand the molecular basis for the assembly of SR-associated gamma-actin filaments.  To study the functional significance of these SR-associated cytoskeletal proteins, muscle structure and physiology will be studied in mice with targeted perturbations in Tmod3 or gamma-actin.  Later in this research project, SR-associated gamma-actin organization and function will be studied in validated mouse models of Duchenne and Becker muscular dystrophy, which are muscle-wasting disorders associated with compensatory gamma-actin upregulation.  Ultimately, these experiments will elucidate the mechanism of disease progression in various muscular dystrophies.

Actin dynamics in nemaline myopathy

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Nemaline myopathy (NM) is a skeletal muscle disease that results in muscle weakness and pathological protein aggregation within muscle cells.  NM is caused by mutations in proteins that encode thin filament and thin filament-associated proteins.  In this project, confocal immunofluorescence imaging and quantitative image analysis will be used to measure thin filament lengths in muscle biopsies from NM patients.  To understand the cellular mechanisms of NM pathogenesis, NM mutations that have effects on thin filament lengths in humans will be expressed in mouse muscle cells, where myofibril assembly and homeostasis will be examined.  Mutant proteins will also be tested for functionality in actin binding and pyrene-actin polymerization assays in vitro.  These studies will have broad relevance to the NM community and suggest novel therapeutic interventions for this debilitating disorder.

Pointed-end dynamics and thin filament length regulation in skeletal muscle

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The thin filaments in skeletal muscle sarcomeres have lengths that are highly uniform within a muscle fiber but vary substantially on a muscle-by-muscle basis, yielding muscle-specific length-tension relationships.  Thin filament lengths are regulated by actin monomer exchange at the pointed (free) ends of the thin filaments at the center of the sarcomere, which are dynamically capped by tropomodulin (Tmod) isoforms 1 and 4.  This project will use Tmod-knockout mice, in vivo RNAi in wild-type mice, and muscle cell cultures (all in combination with high-resolution fluorescent microscopy and quantitative imaging) to elucidate how Tmods determine skeletal muscle-specific thin filament lengths.  Thin filament pointed-end dynamics during myofibril assembly and homeostasis will be studied by introduction of fluorescent-tagged proteins and fluorescence recovery after photobleaching.  Later studies will provide opportunities to examine thin filament pointed-end dynamics in response to altered use patterns (i.e., muscle adaptation after chronic stretch or electrical stimulation).  The resulting data will provide important insights into the biochemical and cellular basis for how particular muscles acquire their uniquely suited functional contractile properties.