Identification and characterization of tropomodulin and tropomyosin in the adult rat lens
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ABSTRACTThe lens fiber cells express all the major components of the erythrocyte membrane skeleton including spectrin, protein 4.1 and ankyrin. We have used immunoblot and immuno-precipitation analyses, as well as immunofluorescence localization to identify and characterize two additional components of the membrane skeleton in the rat lens: tropomyosin and the tropomyosin-binding protein tropomodulin. In the erythrocyte, tropomyosin and tropomodulin are proposed to stabilize and limit the lengths of the short actin filaments of the spectrin-actin network, thus influencing the organization and mechanical properties of the erythrocyte membrane skeleton.Antibodies directed against erythrocyte tropomodulin specifically recognize a Mr43,000 polypeptide from rat lens that comigrates with erythrocyte tropomodulin on SDS-gels. A non-muscle isoform of tropomyosin is also present in the lens. This tropomyosin isoform migrates on SDS-gels with a Mrof ∼28,000 and is distinct from the two erythrocyte isoforms of tropomyosin (Mr27,000 and 29,000). Indirect immunofluorescence staining of 5 μm cryosections of adult rat lens reveals that both tropomodulin and tropomyosin colocalize with rhodamine phalloidin staining for actin filaments on fiber cell plasma membranes. Lens tropomodulin exhibits many characteristics that are similar to its erythrocyte counterpart. For example, lens tropomodulin binds tropomyosin in a solid-phase blot binding assay, and extraction experiments with Triton X-100, urea and NaOH show that the membrane-bound tropomodulin in the lens is a tightly associated peripheral membrane protein that is a component of the Triton-insoluble cytoskeleton. However, unlike the erythrocyte, there are approximately 2000 actin monomers per tropo-modulin in the lens. This differs greatly from the 16/1 ratio seen in the erythrocyte, where actin, tropomodulin and tropomyosin associate to form the short actin filaments in the membrane skeleton. In addition, we have identified a large soluble pool of actin and tropomodulin in the lens, which does not exist in erythrocytes. These differences imply the presence of distinct pools of cytoskeletal components in the lens. The membrane-bound portion of tropomodulin and tropomyosin may associate with that portion of lens actin that is associated with spectrin and protein 4.1 to form a membrane skeleton in the lens fiber cell, whereas the soluble pools of tropomodulin and actin may serve as precursors necessary for rapid assembly of the membrane skeleton during fiber cell elongation. The membrane skeleton may play a role in the maintenance of cell shape and deformability during visual accommodation as well as in the generation of plasma membrane domains during fiber cell differentiation.
