Fiber cells in the outer cortical zones of the vertebrate lens have an extensive complement of cytoskeletal elements. These elements are markedly reduced, if not completely lost, during the process of differentiation/aging, whereby the cells achieve positions in the deeper cortex and nucleus. In contrast to most elements, intermediate filaments (IF) are distributed throughout the cytoplasm, as well as paramembranally. The identification of proteins homologous to those in the red blood cell membrane skeleton suggest many parallels between the two cell types, including the presence of a membrane skeleton. Some other systems show possible interaction between the membrane skeleton and the deeper cytoskeleton in the form of IF-spectrin associations. If so, this could have important implications for lens function, ranging from membrane-cytoplasm coupling (transport) to force/support systems important for cell deformation (accommodation). Examination of non-cytoskeletal roles for IF warrants attention in view of the claim that IF are inactive forms of nucleic acid-binding proteins. This may have significance for the lens due to the close relationship between cell nucleus dissolution and dimunition of IF. The present proposal is addressed to: 1) ascertaining the presence and nature of IF-spectrin interaction in the lens; 2) identifying proteins ancillary to this interaction; 3) investigating the presence of IF-associated proteins (IFAPs) which may influence IF-IF interaction in cytoplasmic (non-membranal) regions of the fiber cell; 4) attempting to generate in vitro models of the cytoskeleton using purified proteins; and 5) investigating the possible presence of soluble IF particles which may serve as nucleic acid-binding proteins during nuclear dissolution. The methods to be employed include preparative purification of the proteins of interest and in vitro binding studies between various ratios of lens IF and lens spectrin under a variety of solvent and temperature conditions in the presence and absence of intrinsic ancillary proteins or IFAPs. Assays will consist of centrifugational, electrophoretic, chromatographic, overlay, microscopic and cross-linking studies. Some in situ determinations will also be made. The generation of a model integrating the various protein interactions revealed above will be undertaken. Soluble IF subunits and nucleic acids will be sought in mid/deep cortical zones under several extraction conditions using precautions for DNase and RNase activities. Assessments will be via electrophoretic, chromatographic and spectrophotometric techniques.
