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PJ Donaldson, AC Grey, BR Merriman-Smith, J Kistler, MB Cannell, C Soeller, MD Jacobs; Changes In The Distribution Of Lens Membrane Proteins As A Function Of Fiber Cell Differentiation: Functional Implications For Lens Transparency . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3999.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Functional models of lens transparency predict that the spatial distribution of membrane proteins is vital to the generation of an internal microcirculation. To investigate this hypothesis the distributions of four membrane proteins were quantitatively mapped and correlated to fiber cell differentiation. Method: Lenses were cryosectioned in the equatorial and axial planes. Sections were labeled with MP20, GLUT3, MIP26 and Cx46 antibodies. Cellular morphology was highlighted using the membrane label wheatgerm agglutinin and changes in fiber cell differentiation were monitored by labeling cell nuclei with propidium iodide. A series of overlapping image stacks were collected using a two-photon microscope. Individual image stacks were tiled together to produce a single extended focus image that covered a large area of the lens with sub-cellular resolution. The distributions of individual membrane proteins were quantified by imaging processing and correlated to changes in fiber cell differentiation. Results: The distributions of all membrane proteins changed dramatically as a function of fiber cell differentiation. In peripheral fiber cells MP20 was predominantly found in cytoplasmic vesicles and was inserted into the membrane when fiber cell nuclei were lost. GLUT3 was also initially found in cytoplasmic vesicles, and was inserted into the narrow sides of fibre cell membranes before becoming uniformly distributed around fiber cell membranes prior to the degradation of nuclei. In contrast MIP26 and Cx46 were predominately found in the membrane although their distribution patterns differed. The density of the MIP26 signal dramatically increased upon the loss of cell nuclei, while the Cx46 labelling density declined with distance into the lens. Analysis of the Cx46 signal identified 3 distinct zones of Cx46 distribution, which coincided with changes in plaque alignment, plaque size, and cell morphology. These changes were followed by a secondary loss of signal most probably due to cleavage of the cytoplasmic tail of Cx46. Conclusion: As fiber cells differentiate precise and specific changes in the distribution of individual membrane proteins occur. These changes include: the differentiation-dependent insertion of proteins into membrane; the loss of discrete broad and narrow side membrane domains and the concomitant dispersal of membrane proteins; and the age-dependent (cleavage) processing of membrane proteins.
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