The interaction of Mip-90 with microtubules and actin filaments in human fibroblasts.
The novel microtubule-interacting protein Mip-90 was originally isolated from HeLa cells by using affinity columns of agarose derivatized with peptides from the C-terminal regulatory domain on beta-tubulin. Biochemical and immunocytochemical data have suggested that the association of Mip-90 with the microtubule system contributes to its cellular organization. Here we report the interaction patterns of Mip-90 with microtubules and actin filaments in interphase human fibroblasts. A polyclonal monospecific antibody against Mip-90 was used for immunofluorescence microscopy analysis to compare the distribution patterns of this protein with tubulin and actin. A detailed observation of fibroblasts revealed the colocalization of Mip-90 with microtubules and actin filaments. These studies were complemented with experiments using cytoskeleton-disrupting drugs which showed that colocalization patterns of Mip-90 with microtubules and actin filaments requires the integrity of these cytoskeletal components. Interestingly, a colocalization of Mip-90 with actin at the leading edge of fibroblasts grown under subconfluency was observed, suggesting that Mip-90 could play a role in actin organization, particularly at this cellular domain. Mip-90 interaction with actin polymers was further supported in vitro by cosedimentation and immunoprecipitation experiments. The cosedimentation analysis indicated that Mip-90 bound to actin filaments with an association constant Ka = 1 x 10(6) M-1, while an stoichiometry Mip-90/actin of 1:12 mol/mol was calculated. Western blots of the immunoprecipitates revealed that Mip-90 associated to both actin and tubulin in fibroblasts extracts. These studies indicate that Mip-90, described as a microtubule-interacting protein, also bears the capacity to interact with the microfilament network, suggesting that it may play a role in modulating the interactions between these cytoskeletal filaments in nonneuronal cells.
González
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Department of Biology, Faculty of Sciences and University of Chile, Santiago, Chile.
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D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002648:Child; D015638:Cytochalasin D / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism* / Q000648:ultrastructure; D019084:Fluorescent Antibody Technique, Indirect; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D015739:Nocodazole / Q000494:pharmacology; D011485:Protein Binding; D014404:Tubulin / Q000378:metabolism
D000199:Actins; D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins; C111403:Mip-90 protein, human; D014404:Tubulin; D015638:Cytochalasin D; D015739:Nocodazole