Specific association of STOP protein with microtubules in vitro and with stable microtubules in mitotic spindles of cultured cells.

STOP (Stable Tubule Only Polypeptide) is a neuronal microtubule associated protein of 145 kd that stabilizes microtubules indefinitely to in vitro disassembly induced by cold temperature, millimolar calcium or by drugs. We have produced monoclonal antibodies against STOP. Using an antibody affinity column, we have produced a homogeneously pure 145 kd protein which has STOP activity as defined by its ability to induce cold stability and resistance to dilution induced disassembly in microtubules in vitro. Western blot analysis, using a specific monoclonal antibody, demonstrates that STOP recycles quantitatively with microtubules through three assembly cycles in vitro. Immunofluorescence analysis demonstrates that STOP is specifically associated with microtubules of mitotic spindles in neuronal cells. Further, and most interestingly, STOP at physiological temperature appears to be preferentially distributed on the distinct microtubule subpopulations that display cold stability; kinetochore-to-pole microtubules and telophase midbody microtubules. The observed distribution suggests that STOP induces the observed cold stability of these microtubule subpopulations in vivo.     

Axonal tubulin and microtubules: morphologic evidence for stable regions on axonal microtubules

Biochemical studies indicate that axonal tubulin is composed of at least two distinct pools that differ in cold solubility and biochemical composition [Brady et al: J. Cell Biol. 99:1716-1724]. To determine the morphologic correlate of cold-insoluble tubulin, segments of rat optic nerves were exposed to a series of in vitro experimental conditions that affect microtubules (MTs), including cold, podophyllotoxin (PT), triflupromazine (TFP), and taxol, and then examined by electron microscopy. Longitudinal sections of control axons showed MTs oriented parallel to the long axis of the axons. Axons exposed to cold, PT, and TFP showed short segments of MTs in association with cytoskeletal disarray. Morphometric studies were used to distinguish between a simple malorientation of MTs (undulation or zigzags in their course) and the loss of labile segments of MTs, leaving the stable portions behind. The lengths of MT segments were measured in longitudinal sections, and the numbers of MTs were determined in the cross sections. All MT segment-length histograms showed a unimodal distribution. Cold and PT produced a simple shift of the control histogram to the shorter length MTs. In cross sections the numbers of MTs in cold- and PT-exposed axons were significantly decreased, indicating that the presence of short segments of MTs in the longitudinal plane of sections was due to a loss of portions of MTs. Taxol, an agent that promotes MT assembly, reversed the cold effect partially and resulted in increases in both MT segment length and number. These studies indicate that stable MT segments are portions of longer MTs containing both stable and labile regions. Furthermore, these findings are consistent with the hypothesis that cold-insoluble tubulin functions as a transportable MT-organizing complex in the axon.     

Sliding of STOP proteins on microtubules

Microtubules are stabilized against cold temperature disassembly by 145-kilodalton proteins [stable tubule only polypeptides (STOPs)] that block the end-wise dissociation of subunits from the polymers. We describe here several kinetic parameters of the interaction of STOPs with microtubules. STOPs will bind to microtubules either during assembly of the polymer or at steady state. The addition appears random on the polymers and does not require the mediation of tubulin subunits. Tubulin subunits compete with microtubules for STOP binding, but binding to the polymers is apparently irreversible. We demonstrate that STOPs do not exchange measurably between polymers at steady state. Nonetheless, a displacement of STOPs within a single polymer is readily demonstrable. We have determined that the displacement is apparently due to a surface translocation, or "sliding", of STOPs on microtubules.     

Polarity orientation of axonal microtubules

The polarity orientation of cellular microtubules is widely regarded to be important in understanding the control of microtubule assembly and microtubule-based motility in vivo. We have used a modification of the method of Heidemann and McIntosh (Nature (Lond.). 286:517-519) to determine the polarity orientation of axonal microtubules in postganglionic sympathetic fibers of the cat. In fibers from three cats we were able to visualize the polarity of 68% of the axonal microtubules; of these, 96% showed the same polarity orientation. Our interpretation is that the rapidly growing end of all axonal microtubules is distal to the cell body. We support Kirschner's hypothesis on microtubule organizing centers. (J. Cell Biol. 86:330- 334), although this interpretation raises questions about the continuity of axonal microtubules. Our results are inconsistent with a number of models for axonal transport based on force production on the surface of microtubules in which the direction of force is determined by the polarity of microtubules.     

P34(cdc2) kinase is associated with cortical microtubules from higher plant protoplasts.

The cell cycle regulatory enzyme p34(cdc2) kinase is known to be localized to the preprophase band, the spindle and the phragmoplast, but not to interphase cortical microtubules. This was investigated further by mechanically cleaving substrate-attached protoplasts to leave plasma membrane disks bearing microtubules freed of nuclear and cytosolic signal. Antibodies to PSTAIRE and to specific C-terminal peptides of cdc2a, were used in immunofluorescence, protein blotting and immunogold electron microscopy to demonstrate that antigen is located on the cortical microtubules of carrot, tobacco BY-2 and Arabidopsis cells.     

Heat-shock protein 90 is associated with microtubules in tobacco cells

Summary The localization of HSP90 (heat-shock protein 90) was analyzed with respect to the microtubular cytoskeleton by double immunofluorescence and confocal laser microscopy in tobacco VBI-O cells during axial cell division and elongation. HSP90 was observed to be colocalized with cortical and radial microtubules and the nuclear envelope in premitotic cells, with the preprophase band, and with the phragmoplast. The HSP90 epitope could not be detected in mature division spindles. The association of the HSP90 epitope with radial and cortical microtubules was not continuous in space. HSP90 was organized in discrete foci that were found to be aligned with microtubules, and the distance between these foci increased, when the cells entered the elongation phase. Elimination of microtubules by drugs resulted in a loss of cell axiality and alignment of the HSP90 epitope. Together with biochemical data demonstrating binding of tobacco HSP90 to tubulin dimers these data indicate a possible role of HSP90 in the organization of microtubules.Abbreviations EPC ethyl-N-phenylcarbamate - FITC fluorescein isothiocyanate - HSP90 heat-shock protein 90 - MAP microtubuleassociated protein - TRITC tetramethylrhodamine B isothiocyanate     

Activated murine alpha-globin gene is not preferentially associated with the nuclear matrix

The association of the murine alpha-globin gene with the nuclear matrix was studied in three different states of the gene: inactive (EAT cells), potentially active (MEL cells) and active (induced MEL cells). When "native" nuclei were digested with DNase I it was found that the nuclear matrix was not enriched in alpha-globin DNA sequences in all three different types of cells. A nuclease-hypersensitive site in the 5'-flanking region of the alpha-globin gene was detected in the induced MEL-cells.     

p53 is associated with cellular microtubules and is transported to the nucleus by dynein

Here we show that p53 protein is physically associated with tubulin in vivo and in vitro, and that it localizes to cellular microtubules. Treatment with vincristine or paclitaxel before DNA-damage or before leptomycin B treatment reduces nuclear accumulation of p53 and expression of mdm2 and p21. Overexpression of dynamitin or microinjection of anti-dynein antibody before DNA damage abrogates nuclear accumulation of p53. Our results indicate that transport of p53 along microtubules is dynein-dependent. The first 25 amino acids of p53 contain the residues that are essential for binding to microtubules. We propose that functional microtubules and the dynein motor protein participate in transport of p53 and facilitate its accumulation in the nucleus after DNA damage.     

The DNA-binding activity of protein disulfide isomerase ERp57 is associated with the a(') domain

ERp57 belongs to the protein disulfide isomerases, a family of homologous proteins mainly localized in the endoplasmic reticulum and characterized by the presence of a thioredoxin-like folding domain. ERp57 is a protein chaperone with thiol-dependent protein disulfide isomerase and additional activities and recently it has been shown to be involved, in cooperation with calnexin or with calreticulin, in the correct folding of glycoproteins. However, we have demonstrated that the same protein is also present in the nucleus, mainly associated with the internal nuclear matrix fraction. In vitro studies have shown that ERp57 has DNA-binding properties which are strongly dependent on its redox state, the oxidized form being the competent one. A comparison study on a recombinant form of ERp57 and several deletion mutants, obtained as fusion proteins and expressed in Escherichia coli, allowed us to identify the C-terminal a(') domain as directly involved in the DNA-binding activity of ERp57.     

DNA associated with hyperacetylated histone is preferentially digested by DNase I.

Butyrate-treated cells give rise to massive hyperacetylation of histones and have been used to test the idea that regions of DNA in association with hyperacetylated histones are preferentially solubilized upon digestion with DNase I. Such hyperacetylated histones can be derived from both pre-existing histones or from histone newly synthesized in the presence of butyrate which leads to extreme modification. The DNA in association with both types of hypermodified histone is equally and selectively digested.     

Phosphorylated human keratinocyte ornithine decarboxylase is preferentially associated with insoluble cellular proteins

Ornithine decarboxylase (ODC), the first enzyme in polyamine biosynthesis, is highly regulated by many trophic stimuli, and changes in its levels and organization correlate with cytoskeletal changes in normal human epidermal keratinocytes (NHEK). NHEK ODC exhibits a filamentous perinuclear/nuclear localization that becomes more diffuse under conditions that alter actin architecture. We have thus asked whether ODC colocalizes with a component of the NHEK cytoskeleton. Confocal immunofluorescence showed that ODC distribution in NHEK was primarily perinuclear; upon disruption of the actin cytoskeleton with cytochalasin D, ODC distribution was diffuse. The ODC distribution in untreated NHEK overlapped with that of keratin in the perinuclear but not cytoplasmic area; after treatment with cytochalasin D, overlap between staining for ODC and for keratin was extensive. No significant overlap with actin and minimal overlap with tubulin filament systems were observed. Subcellular fractionation by sequential homogenizations and centrifugations of NHEK lysates or detergent and salt extractions of NHEK in situ revealed that ODC protein and activity were detectable in both soluble and insoluble fractions, with mechanical disruption causing additional solubilization of ODC activity (three- to sevenfold above controls). Fractionation and ODC immunoprecipitation from [(32)P]orthophosphate-labeled NHEK lysates showed that a phosphorylated form of ODC was present in the insoluble fractions. Taken together, these data suggest that two pools of ODC exist in NHEK. The first is the previously described soluble pool, and the second is enriched in phospho-ODC and associated with insoluble cellular material that by immunohistochemistry appears to be organized in conjunction with the keratin cytoskeleton.     

Nucleotide-dependent interaction of the N-terminal domain of MukB with microtubules

The MukB protein from Escherichia coli has a domain structure that is reminiscent of the eukaryotic motor proteins kinesin and myosin: N-terminal globular domains, a region of coiled-coil, and a specialised C-terminal domain. Sequence alignment of the N-terminal domain of MukB with the kinesin motor domain indicated an approximately 22% sequence identity. These observations raised the possibility that MukB might be a prokaryotic motor protein and, due to the sequence homology shared with kinesin, might bind to microtubules (Mts). We found that a construct encoding the first 342 residues of MukB (Muk342) binds specifically to Mts and shares a number of properties with the motor domain of kinesin. Visualisation of the Muk342 decorated Mt complexes using negative stain electron microscopy indicated that the Muk342 smoothly decorates the outside of Mts. Biochemical data demonstrate that Muk342 decorates Mts with a binding stoichiometry of one Muk342 monomer per tubulin monomer. These findings strongly suggest that MukB has a role in force generation and that it is a prokaryotic homologue of kinesin and myosin.     

Polysomes are associated with microtubules in fertilized eggs of Chinese pine (Pinus tabulaeformis)

Summary. Transmission electron microscopy of immunogold-labeled Chinese pine egg cells before and after fertilization revealed that polysomes are associated with microtubules (MTs) from fertilization to the 2-nucleate embryo stage. Ribosome aggregates of various size and shape were randomly distributed in the cytoplasm of the eggs before fertilization. Single MTs or clusters were observed to be free of polysomes at this stage. Upon fertilization, all polysomes were attached to MTs, and this association persisted until the formation of the polarized embryo. Thereafter, the polysomes spread into the cytoplasm and no polysome-MT association was observed in the embryo. Some of the polysomes were attached to one end of the MTs, while others appeared to form contacts along their entire length. No polysome-microfilament association was observed at any stage of the development. The polysome-MT association may provide a mechanism for MT-dependent mRNA localization in early embryo development of this plant. Correspondence and reprints: Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, U.S.A.     

Dissociation of the inhibition of fast axonal transport by chlorimipramine from an effect on axonal microtubules

The effect of in vitro exposure of bullfrog spinal nerves to 0.2 mM chlorimipramine on the density of axonal microtubules was studied in an attempt to clarify the mechanism by which chlorimipramine inhibits fast axonal transport. A 17-h exposure to chlorimipramine reduced the density of microtubules in unmyelinated axons by only 18%; this microtubular loss does not reach the upper limit of the range of microtubule reduction associated with inhibition of fast axonal transport. A 23-h exposure to chlorimipramine, which had decreased microtubular density in unmyelinated axons by 40% in a previous study, did not decrease microtubular density in myelinated axons in the present study. These results rule out microtubular destruction as the mechanism responsible for inhibition of fast orthograde axonal transport by chlorimipramine, and greatly reduce the likelihood that microtubular destruction plays a significant role in the inhibition of fast retrograde transport by chlorimipramine.     

G protein β subunit is closely associated with microtubules

Previously, we have identified the association of G protein β subunit (Gβ) with mitotic spindles in various mammalian cells. Since microtubules are the main component of mitotic spindles, here we have isolated bovine brain microtubules and purified Gβ subunit to identify the close association of Gβ subunit with purified brain microtubules and have shown the direct incorporation of Gβ subunit into the microtubules both in vitro and in vivo. It was found that: (1) microtubular fraction isolated from bovine brain contained Gβ subunit, (2) coimmunoprecipitation demonstrated that Gβ subunit could be coprecipitated with tubulin, (3) addition of purified Gβ subunit into cytosolic extract for microtubule assembly caused direct incorporation of Gβ subunit into assembled microtubules and increased the association of microtubule-associated proteins with microtubules, and (4) incubation of exogenous Gβ subunit with detergent-permeabilized cells resulted in direct incorporation of Gβ subunit into microtubule fibers and depolymerized tubulin molecules. We conclude that G protein β subunit is closely associated with microtubules and may play an important role in the regulation of microtubule formation in addition to its regulatory role in cellular signal transduction. J. Cell. Biochem. 70:553–562, 1998. © 1998 Wiley-Liss, Inc.     

Yeast Kar3 is a minus-end microtubule motor protein that destabilizes microtubules preferentially at the minus ends.

Mutants of the yeast Kar3 protein are defective in nuclear fusion, or karyogamy, during mating and show slow mitotic growth, indicating a requirement for the protein both during mating and in mitosis. DNA sequence analysis predicts that Kar3 is a microtubule motor protein related to kinesin, but with the motor domain at the C-terminus of the protein rather than the N-terminus as in kinesin heavy chain. We have expressed Kar3 as a fusion protein with glutathione S-transferase (GST) and determined the in vitro motility properties of the bacterially expressed protein. The GST-Kar3 fusion protein bound to a coverslip translocates microtubules in gliding assays with a velocity of 1-2 microns/min and moves towards microtubule minus ends, unlike kinesin but like kinesin-related Drosophila ncd. Taxol-stabilized microtubules bound to GST-Kar3 on a coverslip shorten as they glide, resulting in faster lagging end, than leading end, velocities. Comparison of lagging and leading end velocities with velocities of asymmetrical axoneme-microtubule complexes indicates that microtubules shorten preferentially from the lagging or minus ends. The minus end-directed translocation and microtubule bundling of GST-Kar3 is consistent with models in which the Kar3 protein crosslinks internuclear microtubules and mediates nuclear fusion by moving towards microtubule minus ends, pulling the two nuclei together. In mitotic cells, the minus end motility of Kar3 could move chromosomes polewards, either by attaching to kinetochores and moving them polewards along microtubules, or by attaching to kinetochore microtubules and pulling them polewards along other polar microtubules.(ABSTRACT TRUNCATED AT 250 WORDS)     

The pool of map kinase associated with microtubules is small but constitutively active.

Mitogen-activated protein kinase (MAPK) is activated by many kinds of stimuli and plays an important role in integrating signal transduction cascades. MAPK is present abundantly in brain, where we have studied its association with microtubules. Immunofluorescence of primary hippocampal neurons revealed that MAPK staining co-localized with microtubules and biochemical analyses showed that MAPK co-purified with microtubules. Approximately 4% of MAPK in cytosolic extracts was associated with microtubules, where it was associated with both tubulin and microtubule-associated proteins (MAPs) fractions. Further fractionation of MAPs suggested that a portion of MAPK is associated with MAP2. An association with MAP2 was also demonstrated by co-immunoprecipitation and in vitro binding experiments. A similar association was shown for the juvenile MAP2 isoform, MAP2C. The pool of MAPK associated with microtubules had a higher activity relative to the nonassociated pool in both brain and proliferating PC12 cells. Although MAPK was activated by nerve growth factor in PC12 cells, the activity of microtubule-associated MAPK did not further increase. These results raise the possibility that microtubule-associated MAPK operates through constitutive phosphorylation activity to regulate microtubule function in neurons.