Localization of tau and other proteins of isolated marginal bands.
To determine which proteins were associated with and intrinsic to the marginal band (MB) of microtubules (MTs), we studied protein components of MBs isolated from nucleated erythrocytes by differential detergent solubilization of the membrane skeleton (MS). MBs isolated from dogfish erythrocytes contained major proteins in the tubulin M(r) range. A high molecular weight protein of approximately 290 kD that bound antibody to syncolin and to heat-stable brain MAPs was present in the whole cytoskeleton. However, most of it was solubilized by the MB isolation medium, together with the MS. Dogfish erythrocyte cytoskeletons and isolated MBs were examined with polyclonal and monoclonal antibodies against mammalian brain tau and chicken erythrocyte tau. As shown by immunofluorescence and immunoblotting, these antibodies bound to proteins in the 50 to 67 kD range, located along the length of isolated MBs. Two-dimensional SDS-PAGE revealed isolated MB proteins of pI approximately 6.8 in the same molecular weight range, as well as alpha- and beta-tubulin with pI approximately 5.4. Subtilisin or high-salt treatment of isolated MBs resulted in unbundling of MTs, indicating involvement of MAPs. MBs isolated from chicken erythrocyte cytoskeletons also contained tau as shown by anti-mammalian brain tau immunofluorescence. Both chicken and dogfish isolated MBs also bound phalloidin, but the binding was usually discontinuous and, for any given MB, matched the pattern of anti-syncolin binding. Both syncolin and F-actin were part of the MS remnant remaining after MT disassembly, supporting their assignment to a specialized MS region at the MB/MS interface. In contrast, tau protein appears to be intrinsic to the MB, where it may have an MT stabilizing and bundling function.
Sanchez
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Department of Biological Sciences, Hunter College of CUNY, New York 10021.
Cohen
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D000199:Actins / Q000032:analysis; D000818:Animals; D003598:Cytoskeletal Proteins / Q000032:analysis; D004284:Dogfish / Q000378:metabolism*; D066298:In Vitro Techniques; D008868:Microtubule Proteins / Q000032:analysis*; D008870:Microtubules / Q000737:chemistry*; D016875:tau Proteins / Q000032:analysis*
D000199:Actins; D003598:Cytoskeletal Proteins; D008868:Microtubule Proteins; D016875:tau Proteins