Marginal band protein components and microtubule bundling.
Item
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Title
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Marginal band protein components and microtubule bundling.
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Identifier
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AAI9325141
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identifier
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9325141
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Creator
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Sanchez, Ivelisse.
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Contributor
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Adviser: William D. Cohen
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Date
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1993
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Cell | Biology, Animal Physiology
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Abstract
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The marginal band (MB) is a mechanically functional continuous bundle of microtubules (MTs) found in non-mammalian vertebrate erythrocytes. The mechanical properties of MBs may be determined by proteins involved in MT stability and interactions at the interface between MB and membrane skeleton (MS). To identify proteins in the MB, the structure and protein composition of isolated MBs were examined. A new MB isolation method was developed which avoids use of proteases, based on the differential solubilization of the membrane skeleton (MS) by detergents.;Dogfish erythrocyte cytoskeletons and isolated MBs contained proteins in the 50-67kD range located along the length of MBs which exhibited reactivity with mammalian brain tau antibodies as shown by immunoblotting and immunofluorescence. Two dimensional SDS-PAGE of cytoskeleton and isolated MB proteins revealed in addition to tubulin (pI {dollar}\sim{dollar} 5.3), proteins of pI {dollar}\sim{dollar} 6.8 as would be expected for brain tau. Isolated chicken MBs also contained tau as shown by anti-mammalian brain tau immunofluorescence. Spectrin, F-actin, and a 290kD protein immunologically related to syncolin were minor components of isolated MBs, comprising MS remnant patches which may represent a specialized region of MB/MS interface.;Rewarming of low temperature extracts of dogfish cytoskeletons induced MT assembly and the formation of MT bundles with mechanical properties reminiscent of MBs, as visualized by video enhanced light microscopy and TEM (negative staining). The cold-labile fraction of cytoskeletons and assembled MT bundles consisted of tubulin plus proteins of the tau family as shown by immunoblotting. Inclusion of antibodies against brain tau during temperature-induced MT assembly inhibited tubulin polymerization and thus bundling. Immunoprecipitation of MB tau prior to MT assembly had the same effect.;Dogfish isolated MBs and assembled MT bundles were unbundled by exposure to either subtilisin or high salt. Heat stable proteins obtained from salt-extracts of MT bundles exhibited bundling activity when mixed with tubulin prior to temperature-induced reassembly. However, no bundling activity was observed with pre-assembled MTs.;The results indicate that tau is an intrinsic component of isolated MBs and assembled MT bundles, and that it plays a role in MT bundling.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
