May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
FOXC1 Interacts with Components of the Nucleo/cytoskeleton
Author Affiliations & Notes
  • F.B. Berry
    Ophthalmology, University of Alberta, Edmonton, AB, Canada
  • F. Mirzayans
    Ophthalmology, University of Alberta, Edmonton, AB, Canada
  • M. Coca-Prados
    Ophthalmology and Visual Sciences, Yale University Medical School, New Haven, CT, United States
  • M.A. Walter
    Ophthalmology and Visual Sciences, Yale University Medical School, New Haven, CT, United States
  • Footnotes
    Commercial Relationships  F.B. Berry, None; F. Mirzayans, None; M. Coca-Prados, None; M.A. Walter, None.
  • Footnotes
    Support  CIHR
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 421. doi:
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      F.B. Berry, F. Mirzayans, M. Coca-Prados, M.A. Walter; FOXC1 Interacts with Components of the Nucleo/cytoskeleton . Invest. Ophthalmol. Vis. Sci. 2003;44(13):421.

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

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Abstract

Abstract: : Purpose:FOXC1 mutations result in anterior segment malformations and an increased susceptibility of glaucoma. To further understand the role of FOXC1 in anterior segment function, proteins interacting with FOXC1 were identified. Methods: The FOXC1 protein was expressed in bacteria and immobilized on a Ni2+-NTA agarose support. Nonpigmented cilliary epithelial whole cell or nuclear extracts were incubated with the immobilized FOXC1 protein, washed with an increasing concentration gradient of NaCl and finally eluted with imidazole. Fractions were collected and analyzed by polyacrylamide gel electrophoresis. Ni2+-NTA agarose lacking FOXC1 was used as a control. Proteins that were enriched in FOXC1 containing fractions were isolated from the gel and identified by mass spectrometry (MS). Results: Using this affinity chromatography/pulldown coupled-MS assay a number of potential FOXC1 interacting proteins were identified. Two proteins visualized as prominent bands were the actin binding proteins filamin A and non-muscle myosin. Indirect immunofluoresence revealed an overlapping nuclear expression pattern between FOXC1 and filamin A. Immunoprecipitation experiments also confirmed the interaction between FOXC1 and filamin A. FOXC1 and filamin A deletion constructs are being used to map the interacting regions of both proteins. The biological significance of this interaction will be examined using M2 melanoma cells which lack filamin A protein. Similar analysis of the FOXC1-non-muscle myosin interactions are underway. Conclusions: FOXC1 interacts with the actin-binding proteins filamin A and non-muscle myosin. Extracellular stimuli that can alter cytoskeletal organization of a cell, such as stretch and contraction of trabecular meshwork cells, may carry over to the nucleus and disrupt proper localization of transcription factors such as FOXC1. Recompartmentalization of FOXC1 may impair or inappropriately stimulate FOXC1 activity, resulting in a cascade of gene expression events that may contribute to the glaucoma phenotype. Supported by the CIHR. F.B.B. is the recipient of a CIHR Postdoctoral Fellowship.

Keywords: proteins encoded by disease genes • protein purification and characterization • gene/expression 
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