The ocular lens is a unique structure exquisitely designed to focus light onto the retina, a process that requires substantial shape changes of the lens according to the distance of the eye from the object it is focusing on. To accomplish its function, the lens also has to be transparent, and it has to provide a high index of refraction. We are interested in the structure and function of membrane proteins in the lens, which play crucial roles in maintaining lens homeostasis and transparency. A highly specialized array of membrane channels and transporters is the basis for a microcirculation system that supplies deeper-lying fiber cells with nutrients and clears them of waste products. Moreover, membrane proteins mediate the tight packing of the fiber cells, thus reducing spaces between cells to a distance smaller than the wavelength of light, an important prerequisite to avoid light scattering by the lens tissue. This proposal focuses on the two major membrane proteins found in lens fiber cells, the tetraspanin MP20 and the aquaglyceroporin MIP (also referred to as aquaporin-0). Mutations in either one of these two membrane proteins lead to the formation of cataracts, demonstrating their importance for proper lens function. Structural information on MP20 as well as other tetraspanins is still sparse.
Specific Aim 1 is thus to determine the structure of MP20 primarily by electron microscopy but also pursuing an X-ray crystallographic approach. The structural information obtained for MP20 will be useful to model the structure of other members of the tetraspanin family, which are important in many cellular processes, such as cell adhesion, proliferation, activation, migration, and apoptosis.
Specific Aim 2 is directed towards characterizing the function of MP20 as a cell adhesion molecule by further studies of its interaction with galectin-3, a prominent adhesion modulator. We will also determine whether MP20 can bind to lens-specific integrins, as many tetraspanins are known to interact with integrins, especially if these contain a b1 subunit. The function of MIP as a pore for water and small uncharged molecules is well characterized, but unlike other aquaporins MIP also has adhesive properties and can form intercellular junctions, possibly creating continuous water pores between fiber cells. The objective of Specific Aim 3 is to create a pseudo-atomic model for the MIP-mediated membrane junction using a combination of X-ray and electron crystallography. The structure of the membrane junction will reveal the arrangement of the water pores in interacting MIP tetramers, which has important implications for the functioning of the microcirculation system in the lens.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY015107-04
Application #
7171776
Study Section
Special Emphasis Panel (ZRG1-VISA (01))
Program Officer
Araj, Houmam H
Project Start
2004-01-01
Project End
2008-12-31
Budget Start
2007-01-01
Budget End
2007-12-31
Support Year
4
Fiscal Year
2007
Total Cost
$329,179
Indirect Cost
Name
Harvard University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Saboe, Patrick O; Rapisarda, Chiara; Kaptan, Shreyas et al. (2017) Role of Pore-Lining Residues in Defining the Rate of Water Conduction by Aquaporin-0. Biophys J 112:953-965
Hite, Richard Kevin; Chiu, Po-Lin; Schuller, Jan Michael et al. (2015) Effect of lipid head groups on double-layered two-dimensional crystals formed by aquaporin-0. PLoS One 10:e0117371
Schenk, Andreas D; Philippsen, Ansgar; Engel, Andreas et al. (2013) A pipeline for comprehensive and automated processing of electron diffraction data in IPLT. J Struct Biol 182:173-85
Aponte-Santamaria, Camilo; Briones, Rodolfo; Schenk, Andreas D et al. (2012) Molecular driving forces defining lipid positions around aquaporin-0. Proc Natl Acad Sci U S A 109:9887-92
Kumar, Manish; Habel, Joachim E O; Shen, Yue-xiao et al. (2012) High-density reconstitution of functional water channels into vesicular and planar block copolymer membranes. J Am Chem Soc 134:18631-7
Chiu, Po-Lin; Kelly, Deborah F; Walz, Thomas (2011) The use of trehalose in the preparation of specimens for molecular electron microscopy. Micron 42:762-72
Hite, Richard K; Li, Zongli; Walz, Thomas (2010) Principles of membrane protein interactions with annular lipids deduced from aquaporin-0 2D crystals. EMBO J 29:1652-8
Li, Zongli; Hite, Richard K; Cheng, Yifan et al. (2010) Evaluation of imaging plates as recording medium for images of negatively stained single particles and electron diffraction patterns of two-dimensional crystals. J Electron Microsc (Tokyo) 59:53-63
Schenk, Andreas D; Hite, Richard K; Engel, Andreas et al. (2010) Electron crystallography and aquaporins. Methods Enzymol 483:91-119
Hite, Richard K; Schenk, Andreas D; Li, Zongli et al. (2010) Collecting electron crystallographic data of two-dimensional protein crystals. Methods Enzymol 481:251-82

Showing the most recent 10 out of 21 publications