May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Characterization of Lens Fiber Cell Triton Cytoskeleton Reveals ERM (Ezrin, Radixin, Moesin) Proteins as the Major Cytoskeletal-Associated Proteins
Author Affiliations & Notes
  • R. Maddala
    Duke University School of Medicine, Durham, North Carolina
    Ophthalmology,
  • T. Ho
    Duke University School of Medicine, Durham, North Carolina
    Ophthalmology,
  • N. Skiba
    Duke University School of Medicine, Durham, North Carolina
    Ophthalmology,
  • P. V. Rao
    Duke University School of Medicine, Durham, North Carolina
    Ophthalmology/Pharmacology and Cancer Biology,
  • Footnotes
    Commercial Relationships  R. Maddala, None; T. Ho, None; N. Skiba, None; P.V. Rao, None.
  • Footnotes
    Support  NIH grant EY012201, Research to Prevent Blindness
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 4081. doi:https://doi.org/
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      R. Maddala, T. Ho, N. Skiba, P. V. Rao; Characterization of Lens Fiber Cell Triton Cytoskeleton Reveals ERM (Ezrin, Radixin, Moesin) Proteins as the Major Cytoskeletal-Associated Proteins. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4081. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : To gain better understanding of the cytoskeletal and membrane remodeling that occurs during lens fiber cell elongation and differentiation, this study characterizes lens fiber cell triton cytoskeleton-associated proteins.

Methods: : Fiber cell triton X-100 insoluble fractions isolated from the mouse and porcine lenses were separated by SDS-PAGE and resolved protein bands were cut out and subjected to in-gel trypsin digestion. Trypsin digested protein samples were identified by MALDI-TOF mass spectrometry. The distribution of activated ERM proteins in the neonatal mouse lens and primary epithelial cells was evaluated by immunoflourescence and confocal imaging. Lenses obtained from transgenic mice in which Rho-GTPase function was targeted via expression of either C3-exoenzyme or RhoGDI were also analyzed for phosphorylation status of ERM proteins. Growth factor-mediated regulation of ERM protein phosphorylation in lens epithelial cells was also evaluated using immunoblot analysis.

Results: : MALDI-TOF mass spectrometry analysis of lens fiber cell triton insoluble fractions identified spectrin, actin, vimentin, tubulin, filensin and phakinin as the major proteins in both mouse and porcine specimens. Importantly, in addition to these major cytoskeletal proteins, ERM proteins (crosslinkers of the cytoskeleton and plasma membrane), heat-shock cognate 70 (the clathrin uncoating ATPase) and β/γ-crystallins were identified as the major cytoskeleton-associated proteins. ERM proteins were confirmed to exist in their active phosphorylated form, displaying a preferential abundance in the organelle free-zone fibers and at the lens sutures in neonatal mouse lenses. ERM protein phosphorylation was dramatically reduced in Rho GTPase targeted transgenic lenses and in epithelial cells treated with Rho kinase inhibitors. Growth factors including EGF, FGF, and serum elicited significant increases in ERM protein phosphorylation in lens epithelial cells.

Conclusions: : These data reveal that ERM proteins, which regulate cortical actin complexes, membrane morphogenesis and Rho signaling, are among the major cytoskeleton-associated proteins in lens fiber cells. Importantly, the intense localization pattern of ERM proteins to the lens organelle-free zone fibers and lens sutures, together with their regulation by growth factors and Rho GTPases, indicate a pivotal role for these proteins in lens fiber cell actin cytoskeletal remodeling and membrane organization during differentiation.

Keywords: cytoskeleton • signal transduction • differentiation 
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