Eye lens transparency depends to a great extent on the vectorial transport of metabolites, waste products and water between the anterior and posterior lens surfaces. Fiber cells, the principal component of mammalian lens, impose unique demands upon transport; they lack tight junctions, the structures that regulate transport through the extracellular clefts, and contain only small concentrations of Na,K- ATPase, the enzyme that maintains the cell's resting potential by moving Na+ and K+ against their electrochemical gradients. The principal aim of this application is to study the structure and function of the channel for communication between fiber cells in calf lenses (the cell-to-cell channel). The channels form a pathway which is principally responsible for the diffusion of metabolites, waste products and water throughout fiber cells. The application proposes to purify the fiber cell-to-cell channels from bovine calf lenses using non-ionic detergents and column chromatography methods. Experiments are proposed to determine whether fiber cell-to-cell channels are assembled from one connexin (homotypic) or two (i.e., a """"""""mixed"""""""" channel). Purified cell-to-cell channels will be crystallized in two-dimensional sheets and their structure studied by cryo-electron microscopy and computer image processing. The purified channels also will be reconstituted in planar lipid bilayers and characterized electrically at the single and multichannel levels. Another aim of the application is to study the pathways of specialized regions of the lens called sutures which are formed by the interaction of fiber cells from opposite regions of the lens. These sutures are important in maintaining lens transparency because their extracellular clefts constitute a direct pathway into the lens interior. Sutures also contain large number of membranous vesicles and tubules which may function in the transport of molecules into fiber cells via a pinocytotic mechanism. The proposed studies of the structure and function of fiber cell-to-cell channels and sutures will be important for the understanding of the mechanisms involved in the maintenance of lens transparency and in the formation of cataracts, the principal disease of the lens.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY004110-15
Application #
2710845
Study Section
Visual Sciences A Study Section (VISA)
Project Start
1982-08-01
Project End
1999-05-31
Budget Start
1998-06-01
Budget End
1999-05-31
Support Year
15
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Zampighi, Guido A; Serrano, Raul; Vergara, Julio L (2014) A novel synaptic vesicle fusion path in the rat cerebral cortex: the ""saddle"" point hypothesis. PLoS One 9:e100710
Souda, Puneet; Ryan, Christopher M; Cramer, William A et al. (2011) Profiling of integral membrane proteins and their post translational modifications using high-resolution mass spectrometry. Methods 55:330-6
Schietroma, C; Fain, N; Zampighi, L M et al. (2009) The structure of the cytoplasm of lens fibers as determined by conical tomography. Exp Eye Res 88:566-74
Salvi, Eleonora; Cantele, Francesca; Zampighi, Lorenzo et al. (2008) JUST (Java User Segmentation Tool) for semi-automatic segmentation of tomographic maps. J Struct Biol 161:287-97
Zampighi, Guido A; Fain, Nick; Zampighi, Lorenzo M et al. (2008) Conical electron tomography of a chemical synapse: polyhedral cages dock vesicles to the active zone. J Neurosci 28:4151-60
Cantele, Francesca; Zampighi, Lorenzo; Radermacher, Michael et al. (2007) Local refinement: an attempt to correct for shrinkage and distortion in electron tomography. J Struct Biol 158:59-70
Zampighi, G A; Zampighi, L M; Fain, N et al. (2006) Conical electron tomography of a chemical synapse: vesicles docked to the active zone are hemi-fused. Biophys J 91:2910-8
Lin, Dingbo; Barnett, Micheal; Lobell, Samuel et al. (2006) PKCgamma knockout mouse lenses are more susceptible to oxidative stress damage. J Exp Biol 209:4371-8
Hegde, Balachandra G; Isas, J Mario; Zampighi, Guido et al. (2006) A novel calcium-independent peripheral membrane-bound form of annexin B12. Biochemistry 45:934-42
Zampighi, Guido A; Kreman, Michael; Lanzavecchia, Salvatore et al. (2003) Structure of functional single AQP0 channels in phospholipid membranes. J Mol Biol 325:201-10

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