Membrane skeleton regulation of cell shape and interactions in lens development

Summary

Principal Investigator: Velia M Fowler
Abstract: DESCRIPTION (provided by applicant): The transparency of the vertebrate lens depends on assembly of connexins into gap junctions that are required for lens ionic homeostasis and metabolite circulation. Mutations in fiber cell connexins (Cx46, Cx50) in humans and mice result in loss of lens transparency and cataracts, which are a major cause of blindness worldwide. In differentiating fiber (DF) cells, connexins assemble into large plaques on fiber cell broad sides, whereas in organelle-free mature fiber (MF) cells, the plaques fragment and disperse around the membranes. A cross-linked network of spectrin and actin filaments (F-actin), attaches to NrCAM and N-cadherin, creating micron-scale membrane subdomains in lens fiber cells. The F-actin linkers are capped by tropomodulin1 (Tmod1), and coated with tropomyosin (?TM), stabilizing spectrin-actin network integrity. We have shown that absence of Tmod1 in the mouse lens leads to F-actin disassembly and spectrin-actin network disruption, along with abnormal cell shapes. Tmod1-/- lenses are transparent, but Tmod2 levels are elevated, suggesting partial compensation. Recently, in collaboration with Rick Mathias, we found that gap junction coupling conductance in Tmod1-/- lenses is reduced to about half normal, consistent with ~2-fold elevations in hydrostatic pressure and intracellular Na+. Total levels of Cx46 and Cx50 are unchanged, but confocal microscopy reveals that the large Cx46 and Cx50 plaques on the broad sides of DF cels are dispersed to smaller puncta in Tmod1-/- lenses. We hypothesize that assembly of a long-range spectrin-actin network linked to its attachment proteins (NrCAM, N-cadherin) forms a fiber cell membrane subdomain that excludes connexins, thereby 'corralling'the connexins into the large gap junction plaques required for optimal fiber cell coupling in DF. We will test this hypothesis using mouse knock-out models to destabilize F-actin and weaken spectrin-actin network linkages (Tmod1-/-, Tmod2-/-, ?TM-/-), and a knock-in model to strengthen network linkages (calpain/caspase-resistant [unreadable]II-spectrin mutant). The Specific Aims are: 1) to investigate the roles of Tmods in Cx46 and Cx50 assembly and function in gap junction plaques of DF cells by analysis of Tmod1-/- and Tmod1-/-;Tmod2-/- lenses. 2) To investigate TM regulation of spectrin-actin network stability, connexin assembly, and gap junction coupling in DF by analysis of ?TM?exon9d-/- lenses. 3) To investigate gap junctions in MF cells with a calpain/caspase- resistant [unreadable]II-spectrin mutant that is expected to prevent normal spectrin-actin network disassembly in MF (with J. Morrow). Fiber cell membrane subdomains containing connexins or the spectrin-actin network will be defined by confocal and TIRF microscopy, protein associations by biochemical approaches, gap junction plaque morphology by freeze-fracture TEM (with W.-K. Lo) and lens ionic homeostasis and coupling by electrophysiology of whole lenses (with R. Mathias). These studies will establish basic mechanisms for gap junction control of lens transparency, and provide new insights for therapies in cataract prevention.
Funding Period: 2006-07-01 - 2016-06-30
more information: NIH RePORT

Top Publications

  1. pmc Tropomodulin1 is required for membrane skeleton organization and hexagonal geometry of fiber cells in the mouse lens
    Roberta B Nowak
    Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
    J Cell Biol 186:915-28. 2009
  2. pmc Tropomodulin 1 constrains fiber cell geometry during elongation and maturation in the lens cortex
    Roberta B Nowak
    The Scripps Research Institute, La Jolla, CA 92037, USA
    J Histochem Cytochem 60:414-27. 2012
  3. pmc Tropomodulins: pointed-end capping proteins that regulate actin filament architecture in diverse cell types
    Sawako Yamashiro
    Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA
    Cytoskeleton (Hoboken) 69:337-70. 2012
  4. pmc Tmod1 and CP49 synergize to control the fiber cell geometry, transparency, and mechanical stiffness of the mouse lens
    David S Gokhin
    Department of Cell Biology, The Scripps Research Institute, La Jolla, California, United States of America
    PLoS ONE 7:e48734. 2012

Research Grants

  1. Regulation of Lens Fiber Cell Organization
    Renping Zhou; Fiscal Year: 2013
  2. Determinants for the assembly and maturation of clathrin coated vesicles.
    THERESA JOYCE O'HALLORAN; Fiscal Year: 2013
  3. Thrombus Formation and Antithrombotic Intervention
    John H Griffin; Fiscal Year: 2013
  4. Transcriptional control of the mouse aA-crystallin locus
    Ales Cvekl; Fiscal Year: 2013
  5. INTERCELLULAR COMMUNICATION IN THE LENS
    Miduturu Srinivas; Fiscal Year: 2013
  6. BIOLOGY OF LENS INTERCELLULAR COMMUNICATION
    Eric C Beyer; Fiscal Year: 2013
  7. Strategies for Improved Shock Wave Lithotripsy
    JAMES ALEXANDER MCATEER; Fiscal Year: 2013
  8. Mechanisms of lens growth regulation
    Xiaohua Gong; Fiscal Year: 2013
  9. Vermont Center on Behavior and Health
    Stephen T Higgins; Fiscal Year: 2013
  10. Signaling of Endothelial Permeability and Lung Vascular Injury
    Asrar B Malik; Fiscal Year: 2013

Detail Information

Publications4

  1. pmc Tropomodulin1 is required for membrane skeleton organization and hexagonal geometry of fiber cells in the mouse lens
    Roberta B Nowak
    Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
    J Cell Biol 186:915-28. 2009
    ....
  2. pmc Tropomodulin 1 constrains fiber cell geometry during elongation and maturation in the lens cortex
    Roberta B Nowak
    The Scripps Research Institute, La Jolla, CA 92037, USA
    J Histochem Cytochem 60:414-27. 2012
    ..An unstable spectrin-actin network without Tmod1 may result in imbalanced forces along membranes, leading to fiber cell rearrangements during elongation, followed by propagation of disorder as fiber cells mature...
  3. pmc Tropomodulins: pointed-end capping proteins that regulate actin filament architecture in diverse cell types
    Sawako Yamashiro
    Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA
    Cytoskeleton (Hoboken) 69:337-70. 2012
    ..Tmod-based stabilization and organization of intracellular actin filament networks provide key insights into how the emergent properties of the actin cytoskeleton drive tissue morphogenesis and physiology...
  4. pmc Tmod1 and CP49 synergize to control the fiber cell geometry, transparency, and mechanical stiffness of the mouse lens
    David S Gokhin
    Department of Cell Biology, The Scripps Research Institute, La Jolla, California, United States of America
    PLoS ONE 7:e48734. 2012
    ..These experiments reveal that the spectrin-actin membrane skeleton and beaded filament cytoskeleton establish a novel functional synergy critical for regulating lens fiber cell geometry, transparency, and mechanical stiffness...

Research Grants30

  1. Regulation of Lens Fiber Cell Organization
    Renping Zhou; Fiscal Year: 2013
    ..These studies will enhance our understanding of how lens cell interaction is regulated to ensure lens transparency and provide insights into the mechanisms of cataractogenesis. ..
  2. Determinants for the assembly and maturation of clathrin coated vesicles.
    THERESA JOYCE O'HALLORAN; Fiscal Year: 2013
    ..Collectively, the outcome of the three specific aims will advance an understanding of how dynamic and functional clathrin coated vesicles emerge from the plasma membrane and couple with a tightly focused band of actin filaments. ..
  3. Thrombus Formation and Antithrombotic Intervention
    John H Griffin; Fiscal Year: 2013
    ..New knowledge will contribute to improving prevention, diagnosis and treatment of relevant diseases related to thrombosis. ..
  4. Transcriptional control of the mouse aA-crystallin locus
    Ales Cvekl; Fiscal Year: 2013
    ....
  5. INTERCELLULAR COMMUNICATION IN THE LENS
    Miduturu Srinivas; Fiscal Year: 2013
    ..The effect of modifications on conductance and permeability of gap junctions will be assessed using electrophysiological methods. ..
  6. BIOLOGY OF LENS INTERCELLULAR COMMUNICATION
    Eric C Beyer; Fiscal Year: 2013
    ..abstract_text> ..
  7. Strategies for Improved Shock Wave Lithotripsy
    JAMES ALEXANDER MCATEER; Fiscal Year: 2013
    ..and the session can be ended * Determine the mechanism by which cavitation within a vessel causes hemorrhage * Develop numerical models to understand the role of cavitation and non-cavitational mechanisms in causing tissue damage ..
  8. Mechanisms of lens growth regulation
    Xiaohua Gong; Fiscal Year: 2013
    ..Anticipated results may be useful for developing a new strategy to inhibit lens size increase, thus delaying or preventing presbyopia and age-related cataract. ..
  9. Vermont Center on Behavior and Health
    Stephen T Higgins; Fiscal Year: 2013
    ..S. public health. ..
  10. Signaling of Endothelial Permeability and Lung Vascular Injury
    Asrar B Malik; Fiscal Year: 2013
    ..Moreover the depth of understanding to be gained of the signaling pathways mediating the increase in lung vascular permeability will provide novel insights into the mechanisms of protein-rich pulmonary edema and ARDS. ..
  11. STRUCTURE AND DYNAMICS OF CONNEXIN26 GAP JUNCTIONS
    Gina E Sosinsky; Fiscal Year: 2013
    ..The proposed research is significant because results will be useful in defining better drugs and other therapeutics that potentially ameliorate connexin-related diseases. ..
  12. POST TRANSCRIPTIONAL CONTROL OF GENE EXPRESSION IN THE LENS (LENS GENE EXPRESSION
    SALIL LACHKE; Fiscal Year: 2013
    ..Since mutation of one lens-specific RG component, TDRD7, causes human cataracts, these studies will also provide fundamental information about cataract formation. ..
  13. MITOCHONDRIAL ENCEPHALOMYOPATHIES AND MENTAL RETARDATION
    Salvatore DiMauro; Fiscal Year: 2013
    ....
  14. Cataractogenesis, Connexin Mutants, and Genetic Modifiers
    Xiaohua Gong; Fiscal Year: 2013
    ..The information will be important for developing a new strategy to effectively prevent or delay nuclear cataracts, which account for about one-third of age-related cataracts. ..
  15. Capping Actin Growth in Erythroid and Nonerythroid Cells
    Velia M Fowler; Fiscal Year: 2013
    ....
  16. New Approaches To Cardiothoracic Tolerance Induction
    Joren C Madsen; Fiscal Year: 2013
    ..We anticipate ongoing progress will continue to contribute to a reduction in the morbidity and mortality associated with solid organ transplantation. ..
  17. PPG - Mechanisms of Cardiovascular Protection and Disease
    Donald D Heistad; Fiscal Year: 2013
    ..abstract_text> ..