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MTs (metallothioneins) increase the resistance of cells to exposure to high Cu (copper) levels. Characterization of the MT–Cu complex suggests that MT has an important role in the cellular storage and/or delivery of Cu ions to cuproenzymes. In this work we investigate how these properties contribute to Cu homoeostasis by evaluating the uptake, accumulation and efflux of Cu in wild-type and MT I/II null rat fibroblast cell lines. We also assessed changes in the expression of Cu metabolism-related genes in response to Cu exposure. At sub-physiological Cu levels (0.4 µM), the metal content was not dependent on MT; however, when extracellular Cu was increased to physiological levels (10 µM), MTs were required for the cell's ability to accumulate the metal. The subcellular localization of the accumulated metal in the cytoplasm was MT-dependent. Following supra-physiological Cu exposure (>50 µM), MT null cells had a decreased capacity for Cu storage and an elevated sensitivity to a minor increment in intracellular metal levels, suggesting that intracellular Cu toxicity is due not to the metal content but to the interactions of the metal with cellular components. Moreover, MT null cells failed to show increased levels of mRNAs encoding MT I, SOD1 (superoxide dismutase 1) and Ccs1 (Cu chaperone for SOD) in response to Cu exposure. These results support a role for MT in the storage of Cu in a safe compartment and in sequestering an intracellular excess of Cu in response to supra-physiological Cu exposure. Gene expression analysis suggests the necessity of having MT as part of the signalling pathway that induces gene expression in response to Cu.

With this work, we have determined the cellular content of Cu, Fe and Zn in different cell lines, by using total reflection X-ray fluorescence spectrometry (TXRF). In addition, we examined whether cellular exposure to 100 μmoles l−1of Cu-His modifies the intracellular content and distribution of these trace metals. Our results indicate that all the cell lines displayed the same pattern of relative intracellular abundance of trace metals (Cu

The phosphorylation of microtubule‐associated proteins (MAPs) is thought to be a key factor in the regulation of microtubule (MT) stability. Previously we isolated DMAP‐85, a DrosophilaMAP shown to be associated with stable MTs. In this work we show that DMAP‐85 phosphorylated in cell‐free early embryo extracts is released from MTs. MPM‐2 antibodies recognize the phosphorylated protein. In vitro, DMAP‐85 can be phosphorylated by the mitotic kinase Polo affecting its binding to MTs and creating MPM‐2 epitopes on the protein. The results suggest that phosphorylation of DMAP‐85 might affect its MT stabilizing activity during early mitotic cycles.

Through major research advances in the study of cytoskeletal organization, an integrated view of the complexity of this system has emerged. Recent findings on the microtubule‐interacting protein Mip‐90, which associates with microtubules and actin filaments in different cell domains, have shed light on its roles in cytoskeletal regulation. In order to study structural features of Mip‐90, we sequenced several peptide fragments. A comparative sequence analysis revealed a high degree of similarity between the primary structure of this protein and the human heat shock protein of 90 kDa (hsp‐90). Taken together, the present studies indicate the identity between Mip‐90 and the the β‐isoform of hsp‐90 (hsp‐90β). Western blot assays with an anti‐hsp‐90 monoclonal antibody showed cross‐reactivity of hsp‐90 and Mip‐90 affinity purified from HeLa cells. Furthermore, the observed structural identity of Mip‐90 with the hsp‐90β was sustained by immunoblot assays using monoclonal antibodies that specifically recognize the α‐ and β‐forms of hsp‐90. Comparative fingerprinting analysis, along with the evidence of a remarkably similar biochemical behavior of both hsp‐90 and Mip‐90 in different affinity chromatographic systems, supported these observations. These studies, along with previous investigations, provide new data to elucidate the functional significance of these interesting cellular components and its relationships with other proteins linked to the cell architecture.

Through major research advances in the study of cytoskeletal organization, an integrated view of the complexity of this system has emerged. Recent findings on the microtubule-interacting protein Mip-90, which associates with microtubules and actin filaments in different cell domains, have shed light on its roles in cytoskeletal regulation. In order to study structural features of Mip-90, we sequenced several peptide fragments. A comparative sequence analysis revealed a high degree of similarity between the primary structure of this protein and the human heat shock protein of 90 kDa (hsp-90). Taken together, the present studies indicate the identity between Mip-90 and the the β-isoform of hsp-90 (hsp-90β). Western blot assays with an anti-hsp-90 monoclonal antibody showed cross-reactivity of hsp-90 and Mip-90 affinity purified from HeLa cells. Furthermore, the observed structural identity of Mip-90 with the hsp-90β was sustained by immunoblot assays using monoclonal antibodies that specifically recognize the α- and β-forms of hsp-90. Comparative fingerprinting analysis, along with the evidence of a remarkably similar biochemical behavior of both hsp-90 and Mip-90 in different affinity chromatographic systems, supported these observations. These studies, along with previous investigations, provide new data to elucidate the functional significance of these interesting cellular components and its relationships with other proteins linked to the cell architecture.

Bone is the most frequent site of metastasis in breast cancer. This causes destructive osteolytic lesions. To achieve metastasis to bone, breast cancer cells must proliferate in a new microenvironment, arrest on extracellular matrix and invade. Breast cancer cells progress in the invasive processes only if they destroy bone with the assistance of osteoclasts. In this work, we present data suggesting that MCF-7 cells, an estradiol receptor–positive cell line that exhibits modest invasive capacity, proliferate in the presence of soluble factors secreted by the osteogenic cell line SaOS-2. The cells acquire a more aggressive phenotype when cultured on an extracellular matrix produced by the same osseous cell line. Acquisition of the invasive phenotype appears to be related to the capacity of bone extracellular matrix to induce the expression of urokinase-like plasminogen activator by MCF-7 cells, which is specific for MCF-7 cells, given that MDA-231 cells, an estradiol receptor–negative and more aggressive cell line, did not show significant changes when cultured in the presence of soluble and insoluble bone factors. Int. J. Cancer 83:278–282, 1999. © 1999 Wiley-Liss, Inc.

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 β-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 supportedin vitroby cosedimentation and immunoprecipitation experiments. The cosedimentation analysis indicated that Mip-90 bound to actin filaments with an association constantKa= 1 × 106M−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.

The interaction of microtubule associated proteins (MAPs) with the microtubule system has been characterized in depth in neuronal cells from various mammalian species. These proteins interact with well-defined domains within the acidic tubulin carboxyl-terminal regulatory region. However, there is little information on the mechanisms of MAPs-tubulin interactions in nonmammalian systems. Recently, a novel tau-like protein designated as DMAP-85 has been identified in Drosophila melanogaster, and the regulation of its interactions with cytoskeletal elements was analyzed throughout different developmental stages of this organism. In this report, the topographic domains involved in the binding of DMAP-85 with tubulin heterodimer were investigated. Affinity chromatography of DMAP-85 in matrixes of taxol-stabilized microtubules showed the reversible interaction of DMAP-85 with domains on the microtubular surface. Co-sedimentation studies using the subtilisin-treated tubulin (S-tubulin) indicated the lack of association of DMAP-85 to this tubulin moiety. Moreover, studies on affinity chromatography of the purified 4 kDa C-terminal tubulin peptide bound to an affinity column, confirmed that DMAP-85 interacts directly with this regulatory domain on tubulin subunits. Further studies on sequencial affinity chromatography using a calmodulin affinity column followed by the microtubule column confirmed the similarities in the interaction behavior of DMAP-85 with that of tau. DMAP-85 associated to both calmodulin and the microtubular polymer. These studies support the idea that the carboxyl-terminal region on tubulin constitutes a common binding domain for most microtubule-interacting proteins.