A tau-like protein interacts with stress fibers and microtubules in human and rodent cultured cell lines.
The cytoskeletal integrity of human and rodent cell lines was analyzed using site-directed monoclonal antibodies prepared from hybridomas. Secreting hybridomas were produced by immunizing mice with synthetic peptides from the C-terminal domain of the beta II-tubulin isotype, beta II(422-434), YQQYQDATADEQG, and the first imperfect repeat from brain tau, Tau-I(187-204), VRSKIGSTENLKHQPGGG. Two hybridomas were selected for this work: MTB6.22, an anti-idiotypic monoclonal antibody, which was obtained from a mouse immunized with the beta II-peptide and recognizes specific tubulin-binding domains on MAP-2 and tau; and Tau-I/1, which recognizes the repetitive binding sequences on tau and MAP-2. Immunoblots of cytoskeletal protein preparations from the five different tumor cell lines studied, showed the interaction of the site-directed antibodies MTB6.22 and Tau-I/1 with a group of proteins that co-migrate with brain tau. Immunoreactive tau components were also identified using an anti-tau monoclonal antibody (clone Tau-2), and several polyclonal anti-tau antibodies that interact with tau epitopes, other than those of the tubulin-binding domains. These tau-like proteins bound to a calmodulin-Sepharose affinity column and were eluted using 2 mM EGTA. Interestingly, washing the extracted cytoskeleton pellet with 5 x 10(-3) M Ca2+ for short periods of time selectively released the tau-like protein components, whilst most of the other cytoskeletal proteins remained in the pellet. On the other hand, immunofluorescence microscopy of detergent-extracted cells showed immunostaining of MAP components that appear to be co-localized in a discrete dot-like distribution along the stress fibers, which were revealed using rhodamine-phallacidin. Further support for the specificity of tau interaction with sites on tubulin and actin polymers was obtained with double-immunofluorescence, using the MAP-reactive monoclonal antibody MTB6.22 and a polyclonal antibody to a tubulin peptide containing part of the tau-binding domain on tubulin. Considering the anti-idiotypic nature of the MTB6.22 monoclonal antibody, our studies indicate that, in all the cell lines analyzed, a tau-like protein component is involved in mediating the interaction of both actin and tubulin polymers.
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International Center for Cancer and Developmental Biology, ICC, Santiago, Chile.
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Maccioni
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D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D000939:Epitopes / Q000032:analysis; D006801:Humans; D006825:Hybridomas; D007150:Immunohistochemistry; D008377:Maps as Topic; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D051381:Rats; D016875:tau Proteins / Q000032:analysis / Q000378:metabolism*
D000199:Actins; D000939:Epitopes; D016875:tau Proteins