Scripps Research Joint Appointments

Faculty, Kellogg School of Science and Technology

Research Focus

Actin Cytoskeleton Regulation of Cell Architecture and Physiology

Our research program aims to elucidate how temporal and spatial control of actin dynamics creates the diverse cytoskeletal structures that influence cell and tissue architecture and physiology. We focus on the tropomodulin (Tmod) family of actin-capping proteins, which bind tropomyosins (TMs), inhibit actin monomer association and dissociation from pointed ends, regulate actin dynamics and turnover, and stabilize actin filament (F-actin) lengths and cytoskeletal architecture. There are four canonical, ~40 kD Tmod isoforms (Tmods 1-4) in vertebrates, and three larger variants (Leiomodins 1-3), with tissue-specific and developmentally regulated patterns of expression. By understanding Tmods' functions in the context of their molecular structure, actin regulation, and binding partners, our research aims to explain the diverse phenotypes arising from Tmod perturbation experiments and the mechanisms of disease pathogenesis involving the actin cytoskeleton. Our studies use in vitro approaches with purified proteins, in vivo approaches with transgenic mouse models, and translational studies in human tissues, to focus on three systems: red blood cells (RBCs), striated muscle, and eye lens fiber cells.

In RBCs, absence of Tmod1 leads to F-actin length mis-regulation and a disrupted spectrin-actin network, resulting in anemia with RBC membrane instability and shortened cell lifespan. However, the mouse phenotype is relatively mild, due to compensation by Tmod3 and retention of TMs, necessitating study of RBCs from mice with targeted deletions in Tmod3, Tmod1/3 and/or TMs. RBC membrane structure is examined by biochemistry, confocal and total internal reflection fluorescence (TIRF) microscopy and electron microscopy, and RBC mechanics is tested by osmotic fragility and shearing assays. RBC production and survival are determined from blood hematology as well as studies of progenitors in hematopoietic organs (fetal liver, bone marrow, spleen). These experiments will elucidate how actin regulation contributes to the pathogenesis of human hemolytic anemias due to RBC instability and altered lifespan, and to congenital anemias due to defects in RBC biogenesis and production.

In skeletal muscle, Tmod1 and Tmod4 control thin filament lengths in sarcomeres, determining sarcomere length-tension relationships and muscle biomechanics, which vary across muscle types. A key unsolved question is how thin filament lengths are coordinated to be uniform within each sarcomere and muscle, yet precisely regulated to determine different muscle-specific thin filament lengths. We take in vitro and in vivo approaches, utilizing actin polymerization assays, proteomics, muscle physiology, fluorescence microscopy, and quantitative imaging to measure thin filament lengths and actin dynamics at pointed ends. These experiments are relevant to understanding a group of congenital human muscle diseases termed nemaline myopathies, which are characterized by thin filament length dysregulation and aberrant extrasarcomeric protein aggregates. Another Tmod isoform in skeletal muscle, Tmod3, stabilizes a γ-actin-based linking system essential for sarcoplasmic reticulum (SR) architecture, calcium handling, and linkage to myofibrils. Current studies are testing the hypothesis that Tmod3 stabilization of SR membrane domains contributes to the pathogenesis of congenital muscular dystrophies.

In the eye lens, Tmod1 stabilizes TM-coated F-actin in the spectrin-actin network on fiber cell membranes, controlling fiber cell morphology and hexagonal packing while contributing to lens mechanical resilience. The goal of our work is to define the molecular basis for spectrin-actin network control of membrane subdomain assembly, mechanical stability, and cell coupling during lens development and aging. Our work utilizes lenses from mice with targeted mutations or deletions in components of the spectrin-actin network, with confocal fluorescence microscopy, image analysis and proteomics approaches to define interactions and structural relationships. For functional analysis, optical, electrophysiological, and biomechanical experiments are performed. Our studies will provide insight into how membrane domain dysfunction leads to loss of lens transparency, cataracts, and blindness in humans.

In summary, central questions we are addressing are: (1) To what extent are common molecular mechanisms of actin dynamics utilized to specify F-actin architecture and function in diverse cell and tissue contexts? (2) To what extent can Tmod-null phenotypes in mice provide insights into normal tissue development and physiology, and into pathological mechanisms of human disease? Approaches used in our laboratory span from protein binding assays, to biophysical studies of F-actin polymerization in vitro, to fluorescence microscopy of actin dynamics in living cells, to development and physiology of transgenic mouse models, to translational studies in human cells and tissues. Together, these complementary approaches allow us to connect the dots -- from actin dynamics regulation, to assembly and organization of cytoskeletal structures in cells, to morphogenetic differentiation during development, to cell and tissue physiology in both health and disease.

Education

B.A., Oberlin College, 1974
Ph.D., Harvard University, 1980

Professional Experience

1980-1982 Jane Coffin Childs Postdoctoral Fellow NIADDK, NIH, and Dept Cell Biology and Anatomy, Johns Hopkins University School of Medicine; 1983-1984 Research Associate, Dept Cell Biology and Anatomy, Johns Hopkins University School of Medicine; 1984-1987 Assistant Professor, Dept Anatomy and Cell Biology, Harvard Medical School; 1987-1993 Assistant Professor, Depts Molecular and Cell Biology, The Scripps Research Institute; 1993-2000 Associate Professor, Dept Cell Biology, The Scripps Research Institute; 2000 Professor, Dept Cell Biology, The Scripps Research Institute.

Awards & Professional Activities

1975-1978 National Science Foundation Predoctoral Fellowship Award; 1980-1982 Jane Coffin Childs Foundation Postdoctoral Fellowship Award; 1983-1984 NIH New Investigator Research Grant Award; 1990-1995 American Heart Association Established Investigator Award; 2001-2003 Chair, Cell and Developmental Function 6 (CDF6) Study Section for Postdoctoral Fellowships and AREA grants, NIH; 2003 Chair, “Motile and Contractile Systems” Gordon Research Conference, Colby-Sawyer College, NH (Vice-Chair in 2001); 2009-2010 Erythrocyte and Leukocyte Biology (ELB) NIH Study Section; 2010- Molecular and Cellular Hematology (MCH) NIH Study Section; 2010 ASCB Program Committee for 2011 Annual Meeting; 2011 Chair, “Red Cells” Gordon Research Conference, Proctor Academy, Andover, NH (Vice-Chair in 2009); 2011 Lens and Cataract Program Planning Panel, National Eye Institute, NIH; 2011-pres. Associate Program Director & Imaging Core Director, San Diego Skeletal Muscle Research Center (NIAMS/NIH), a P30 Core of UCSD, TSRI, Sanford-Burnham, Salk and San Diego State University. Editorial Boards and Consulting: 2005- Cytoskeleton; 2012- Journal of Clinical Investigation; 2012- Journal of Biological Chemistry.

Selected References

For a complete list of publications: http://www.scripps.edu/fowler/publications.html

Yamashiro S, Gokhin DS, Kimura S, Nowak RB, Fowler VM. Tropomodulins: Pointed-end capping proteins that regulate actin filament architecture in diverse cell types. Cytoskeleton (Hoboken). 2012 Apr 4. doi: 10.1002/cm.21031 [Epub ahead of print]

Nowak RB, Fowler VM. Tropomodulin1 Constrains Fiber Cell Geometry During Elongation and Maturation in the Lens Cortex. J Histochem Cytochem. 2012 Apr 3. [Epub ahead of print]

Damani S, Bacconi A, Libiger O, Chourasia AH, Serry R, Gollapudi R, Goldberg R. Rapeport K, Haaser S,K, Kuhn P, Wood M, Carragher B, Schork NJ, Jiang J, Rao C, Connelly M, Fowler VM, Topol EJ. Characterization of Circulating Endothelial Cells in Acute Myocardial Infarction. Science Translational Medicine. 2012;4(126):126ra33.

Gokhin DS, Kim NE, Lewis SA, Hoenecke HR, D'Lima DD, Fowler VM. Thin-filament length correlates with fiber type in human skeletal muscle. Am J Physiol Cell Physiol. 2012;302(3):C555-65.

Gokhin DS, Fowler VM. The sarcoplasmic reticulum: actin and tropomodulin hit the links. BioArchitecture. 2011;1(4):175-9.

Gokhin DS, Fowler VM. Tropomodulin Capping of Actin Filaments in Striated Muscle Development and Physiology. Journal of Biomedicine and Biotechnology. 2011;2011:103069.

Gokhin DS, Fowler VM. Cytoplasmic γ-actin and tropomodulin isoforms link to the sarcoplasmic reticulum in skeletal muscle fibers. J Cell Biol. 2011 July 11; 194(1):105-20.

Fath T, Fischer RS, Dehmelt L, Halpain S, Fowler VM. Tropomodulins are negative regulators of neurite outgrowth. Eur J Cell Biol. 2011 Apr; 90(4):291-300.

Links

Biobytes from Journal of Cell Biology

Team Develops a Simple Test to Predict Heart Attacks

Dynamic Actin