Fiber cells in the avascular mammalian lens are coupled extensively by gap (communicating) junctions (GJS) which facilitate the flow of ions and small molecules throughout the lens. A second type of junction characterized by a 6.6 nm square array of protein in the membrane plane is also a prominent feature of fiber cells, especially of older cells in the lens nucleus. The function of the square array junction (SAJ) is not known, although it has been noted that SAJs are intimately associated and probably produce the undulating membranes of the tongue- and-groove interdigitations. We propose to quantitate the distribution of junctions, from the younger fibers in the cortex to the older fibers in the nucleus, by the use of electron microscopy and a new procedure to preserve lens tissue. Fresh lenses will be sectioned with a vibrotome (oscillating knife microtome) to give 50-200 pm thick sections which will be chemically fixed of cryofixed. Cryofixed thick sections will be processed by freeze- substitution/thin sectioning or by freeze-fracture. Junctional profiles in thin sections and intramembrane particles in freeze- fracture images will be used to distinguish GJs and SAJs and to correlate the distributions of functions with the age of the cells. We propose to evaluate the hypothesis that the major membrane protein (MP26), or its degradation product (MP22), forms both GJs and SAJs by locating MP26 and MP22 with antibodies in intact membranes and in membranes isolated from different regions. Low-dose electron microscopic images of frozen-hydrated square arrays, which give 1.0 nm resolution in x-ray diffraction patterns, and high resolution tantalum replicas of SAJs will be obtained to determine whether the substructure within square arrays is consistent with channel formation. Furthermore, we propose to test the hypothesis that, in undulating membranes, the SAJs minimize the extracellular space between cells through electrostatic interactions. Preliminary results indicate that membrane junctions are structurally abnormal in certain types of cataracts. Therefore, we propose to determine the structure of junctions in three types of cataractous lenses: bovine cortical cataracts induced by exposure to low pH; sugar cataracts from drug induced diabetic rabbits; and surgically removed human senile cataracts.
