The lens of the eye has the unusual biological requirement to be optically clear. How that is achieved is not fully understood, but a very high degree of structural order and uniformity is considered essential.
We have identified two proteins that are very divergent members of the intermediate filament family of proteins that assemble into a unique cytoskeletal element called the beaded filament. Both of these proteins and the beaded filament are expressed only in the lens.
Using genetic engineering approaches we created mice that lack the beaded filament proteins, in an effort to determine beaded filament function. The lenses of these animals were able to develop and differentiate normally, achieving the high degree of structural order that characterizes the lens, but they were unable to maintain that order with age. Thus the structural order seen in the lens is inherently unstable, and the beaded filament serves to confer resistance to the age-dependent loss of structure.
We are now focused on determining how the beaded filament accomplishes its mission:
- What is the structure of the filament?
- What proteins link it to the plasma membrane?
- What membrane proteins is it linked to?
- What membrane domains are formed at these sites?
The study of beaded filament structure has lead to a more general consideration of intermediate filament structure. Toward this end we have adapted site directed spin labeling and electron paramagnetic resonance to the study of its structure.
Research Support - Principal Investigator
- NIH R01 EY08747-19 Molecular Analysis of Ocular Lens Beaded Filaments. Through July 31, 2011.
- NIH R01 EY015560-3 SDSL EPR Study of Intermediate Filament Structure. Through March 31, 2011.
- NIH P30 EY12576-01-05 Vision Research Core Grant. Through July 1, 2009 (Renewal Submitted). PI: Leo Chalupa, Microscopy Module Director: Paul FitzGerald
2014 Jacquelyn Gerhart, Marvin Greenbaum, Victoria Scheinfeld, Paul FitzGerald. Mitchell Crawford, Arturo Bravo-Nuevo, Meghan Pitts, and Mindy George-Weinstein. Myo/Nog Cells: Targets for Preventing the Accumulation of Skeletal 1 Muscle-Like Cells in the Human Lens PLoS (in Press March 26, 2014)
2013 Abby L Manthey; Salil A Lachke; Paul G FitzGerald; Robert W Mason; David A Scheiblin; John H McDonald; Melinda K Duncan, Ph.D. Loss of Sip1 leads to migration defects and retention of ectodermal markers during lens development. Mechanisms in Development
2013 Stewart DN, Lango J, Nambiar KP, Falso MJ, FitzGerald PG, Rocke DM, Hammock BD, Buchholz BA Carbon turnover in the water-soluble protein of the adult human lens. Mol Vis. 2013;19:463-75. Epub 2013 Feb 25. PMC # 3580966
2012 Hess, J. F., Budamagunta, M., S., Aziz, A., FitzGerald, P. G*., & Voss, J. C. Electron paramagnetic resonance analysis of the vimentin tail domain reveals points of order in a largely disordered region and conformational adaptation upon filament assembly. Protein Science, 22(1), 47-55. PMID: 23109052. PMCID: PMC3575859. *Corresponding Author
2012* Aziz, A., Hess, J. F., Budamagunta, M. S., Voss, J. C., Kuzin, A. P., Huang, Y. J., Xiao, R., Montelione, G. T., FitzGerald, P**., Hunt, J. F. (2012). The structure of vimentin linker 1 and rod 1B domains characterized by site-directed spin-labeling electron paramagnetic resonance (SDSL-EPR) and x-ray crystallography. Journal of Biological Chemistry, 287, 28349-28361. PMCID: PMC3436525. * Selected by PSI Nature Structural Biology as a featured advance in structural biology ** Corresponding Author
2010 Fudge, D.S., McCuaig, J.V.,Van Stralen, S., Hess, J.F., Wang, H., Mathias, R., and FitzGerald, P.G. Intermediate filaments regulate tissue size and stiffness in the murine lens. Investigative Ophthalmology and Visual Science. In submission.
2010 John J. Castorino, Shannon M. Gallagher-Colombo, Alex Levin, Paul G. FitzGerald, Jessica Polishook1, Barbara Kloeckener-Gruissem, Eric Ostertag, and Nancy J. Philp. Truncation Mutation of solute carrier SLC16A12 associated juvenile cataract impairs trafficking of the protein to the plasma membrane. Human Molecular Genetics. In submission
2010 Daniel N. Stewart, Krishnan P. Nambiar, Jozsef Lango, Miranda J. Sarachine, Paul G. FitzGerald, David M.Rocke, Bruce D. Hammock, and Bruce A. Buchholz. Protein Turnover in the Adult Human Lens. Experimental Eye Research. In submission.
2010 Atya Aziz, John F. Hess, Madhu S. Budamagunta, John C. Voss and Paul G. J. SDSL EPR Determination of Vimentin Head Domain Structure. Biological Chemistry 285:15278-85.
2009 Lindstrom SH, Nacsa N, Blankenship T, Fitzgerald PG, Weller C, Vaney DI, Wilson M. Distribution and structure of efferent synapses in the chicken retina. Vis Neurosci. 2009 Mar-Apr;26(2):215-26.
2009 Atya Aziz, John F. Hess, Madhu S. Budamagunta, Paul G. FitzGerald* and John C. Voss. Head and Rod 1 Interactions in Vimentin: Identification of contact sites, structure and changes with phosphorylation using Site Directed Spin Labeling and Electron Paramagnetic Resonance. J. Biological Chemistry, 284:7330-8. *Corresponding Author.
2009 Fitzgerald, Paul G. Lens Intermediate Filaments. Experimental Eye Research, 8:165–172. Review Article, with original data, Peer Reviewed.
2008 Yoon, Kyoung-hye and FitzGerald, P.G. Periplakin serves as a linker for both vimentin and the highly divergent beaded filament proteins in the ocular lens. Investigative Opthalmology and Visual Science. 50:1283-9.
2008 Josh Pittenger, John Hess, Madhu Budamagunta, John Voss and Paul FitzGerald. SDSL EPR characterization of phosphorylation-induced structural changes in vimentin. Intermediate Filaments Biochemistry. 47:10863-70.
2008 Yoon, Kyoung-hye, Tom Blankenship, Bradley Shibata and Paul FitzGerald. Resisting the effects of aging: A function for the fiber cell beaded filament. Investigative Ophthalmology and Visual Science. 49:130-6.
2007 Pittenger, J.T., Hess, J. F. and FitzGerald, P.G. Identifying the role of specific motifs in the lens fiber cell-specific intermediate filament protein phakosin. Investigative Opthalmology and Visual Science. 48:5132-41.
2007 Lagerstedt, Jens; Cavigiolio, Giorgio; Roberts, Linda; Hong, Hyun-Seok; Jin, Lee-Way; FitzGerald, Paul; Oda, Michael; Voss, John. Mapping the Structural Transition in an amyloidogenic apolipoprotien A-I. Biochemistry. 46:9693-9699
2007 Madhu Budamagunta, John Hess, Paul FitzGerald and John Voss. Describing the structure and assembly of protein filaments by EPR spectroscopy of spin labeled side chains. Cell Biochem and Biophys. 48:45-53
2007 Blankenship, T, Bradshaw, L, Shibata, B, and FitzGerald P. Structural specializations emerging late in mouse lens fiber cell differentiation. Investigative Ophthalmology and Visual Science 48: 3269-3276.
2006 John F. Hess, Madhu S. Budamagunta, Rebecca L. Shipman, Paul G. FitzGerald, John C. Voss. Characterization of the linker 1-2 region in human vimentin using site directed spin labeling and electron paramagnetic resonance. Biochemistry 45:11737-43.
2006 Hess, J. F., Voss, J. C. and FitzGerald, P.G. Vimentin production, purification , assembly and sudy by EPR. In "Cell Biology Protocols", edited by Harris Graham and Rickwood, published by John Wiley and sons, London, UK.
2005 Hess, John., Madhu S. Budamagunta, Paul FitzGerald, John C. Voss. Characterization of structural changes in vimentin bearing an EBS-like mutation using site directed spin labeling and electron paramagnetic resonance. Journal of Biological Chemistry 280:2141-6