Cytoplasmic microtubule-based motor proteins.

A multitude of microtubule-based motors drives diverse forms of intracellular transport and generates forces for maintaining the dynamic structural organization of cytoplasm. Recent work has illuminated the functions and mechanisms of action of some microtubule motors, and appears to have uncovered unforseen functional interactions between tubulin-based and actin-based systems.

Spatial and temporal colocalization of the Golgi apparatus and microtubules rich in detyrosinated tubulin.

The integrity and intracellular distribution of the Golgi apparatus appear to depend upon microtubules. We have found that the microtubules rich in detyrosinated tubulin are located preferentially in the vicinity of the Golgi. Cells were double-stained with antibodies specific for either tyrosinated or detyrosinated tubulin and an antibody to prolactin or wheat germ agglutinin (Golgi markers). Microtubules rich in detyrosinated tubulin showed a close codistribution with the Golgi in three different cultured cell lines GH3, BS-C-1, and AtT20. Disruption of microtubules with nocodazole in GH3 cells resulted in fragmentation and dispersal of the Golgi apparatus as reported previously. During recovery of the microtubules and the Golgi complex after removal of the nocodazole, there was a spatial and temporal colocalization of the Golgi apparatus and microtubules rich in detyrosinated tubulin. Our results suggest that a functional relationship may exist between the structure and organization of the Golgi complex and the detyrosination of alpha-tubulin in microtubules.

Human survivin is a kinetochore-associated passenger protein.

Survivin, a dimeric baculovirus inhibitor of apoptosis repeat (BIR) motif protein that is principally expressed in G2 and mitosis, has been associated with protection against apoptosis of cells that exit mitosis aberrantly. Mammalian survivin has been reported to associate with centrosomes and with the mitotic spindle. We have expressed a human hemagglutinin-tagged survivin plasmid to determine its localization, and find instead that it clearly acts as a passenger protein. In HeLa cells, survivin first associates with the kinetochores, and then translocates to the spindle midzone during anaphase and, finally, to the midbody during cell cleavage. Its localization is similar to that of TD-60, a known passenger protein. Both a point mutation in the baculovirus IAP repeat motif (C84A) and a COOH-terminal deletion mutant (Delta106) of survivin fail to localize to either kinetochores or midbodies, but neither interferes with cell cleavage. The interphase localization of survivin is cell cycle regulated since in permanently transfected NIH3T3 cells it is excluded from the nuclei until G2, where it localizes with centromeres. Survivin remains associated with mitotic kinetochores when microtubule assembly is disrupted and its localization is thus independent of microtubules. We conclude that human survivin is positioned to have an important function in the mechanism of cell cleavage.

Structural characterization of HBXIP: the protein that interacts with the anti-apoptotic protein survivin and the oncogenic viral protein HBx.

Hepatitis B X-interacting protein (HBXIP) is a ubiquitous protein that was originally identified as a binding partner of the hepatitis B viral protein HBx. HBXIP is also thought to serve as an anti-apoptotic cofactor of survivin, promoting the suppression of pro-caspase-9 activation. Here were port the crystal structure of the shortest isoform of HBXIP (91 aa long,∼11 kDa) at 1.5 Å resolution. HBXIP crystal shows a monomer per asymmetric unit, with a profilin-like fold which is common to a super family of proteins, the Roadblock/LC7 domain family involved in protein-protein interactions. Based on this fold, we propose that HBXIP can form a dimer that can indeed be found in the crystal when symmetric molecules are generated around the asymmetric unit. This dimer shows an extended ß-sheet area formed by 10 anti-parallel ß-strands from both subunits. Another interesting aspect of the proposed HBXIP dimer interface is the presence of a small leucine zipper between the two α2 helices of each monomer. In solution, the scattering curve obtained by small-angle X-ray scattering for the sample used for crystallization indicates that the protein is dimeric form in solution. The fit between the experimental small angle X-ray scattering curve and the back calculated curves for two potential crystal dimers shows a significant preference for the Roadblock/LC7 fold dimer model. Moreover, the HBXIP crystal structure represents a step towards understanding the cellular role of HBXIP.

Mechanism of inhibition of microtubule polymerization by colchicine: inhibitory potencies of unliganded colchicine and tubulin-colchicine complexes.

The tubulin-colchicine binding reaction appears to involve a number of intermediate steps beginning with rapid formation of a transient preequilibrium complex that is followed by one or more slow steps in which conformational changes in tubulin and colchicine lead to formation of a poorly reversible final-state complex. In the present study, we investigated the relative ability of unliganded colchicine and preformed final-stage tubulin-colchicine complex to incorporate at microtubule ends and to inhibit addition of tubulin at the net assembly ends of bovine brain microtubules in vitro. Addition of 0.1 microM final-stage tubulin-colchicine complex to suspensions of microtubules at polymer-mass steady-state resulted in rapid incorporation of one to two molecules of tubulin-colchicine complex per microtubule net assembly end concomitant with approximately 50-60% inhibition of tubulin addition. Incorporation of colchicine-tubulin complex continued slowly with time, without significant additional change in the rate of tubulin addition. In contrast, addition of unliganded colchicine to microtubule suspensions resulted in incorporation of small numbers of colchicine molecules at microtubule ends and inhibition of tubulin addition only after periods of time that varied from several minutes to approximately 20 min depending upon the concentration of colchicine. Inhibition of tubulin addition beginning with unliganded colchicine increased slowly with time, concomitant with increases in the concentration of final-state tubulin-colchicine complex and the amount of colchicine bound per microtubule end. The results indicate that inhibition of tubulin incorporation at microtubule ends is caused by colchicine-liganded tubulin in the form of a final-state complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Assembly and colchicine binding characteristics of tubulin with maximally tyrosinated and detyrosinated alpha-tubulins.

The posttranslational removal and readdition of tyrosine at the C-terminus of alpha-tubulin is associated with generation of microtubule populations that differ in intracellular distributions, turnover rates, and sensitivities to microtubule-depolymerization agents. Here, we compared the in vitro assembly and colchicine binding characteristics of tubulin dimer preparations composed of alpha-tubulin that had been maximally tyrosinated (approximately 40% tyrosinated) by tubulin-tyrosine ligase and maximally detyrosinated (100% detyrosinated) by carboxypeptidase A. Maximally tyrosinated and detyrosinated tubulins had similar critical concentrations for polymerization and similar association constants for colchicine binding. Microtubules polymerized from the two tubulins also had similar steady-state mean lengths and length distributions. The growing and shortening dynamics (dynamic instability parameters) of individual microtubules made from maximally tyrosinated or detyrosinated alpha-tubulin as determined by video-enhanced dark-field microscopy were similar, but subtle differences in the growing and shortening rates were found. On balance, however, the dynamicity and thus the overall kinetic stability of the two microtubule populations were indistinguishable. The results support the idea that detyrosination of alpha-tubulin does not by itself generate stable microtubules.

Colchicine-binding sites of brain tubulins from an antarctic fish and from a mammal are functionally similar, but not identical: implications for microtubule assembly at low temperature.

The tubulins of Antarctic fishes possess adaptations that favor microtubule formation at low body temperatures (Detrich et al.: Biochemistry 28:10085-10093, 1989). To determine whether some of these adaptations may be present in a domain of tubulin that participates directly or indirectly in lateral contact between microtubule protofilaments, we have examined the energetics of the binding of colchicine, a drug thought to bind to such a site, to pure brain tubulins from an Antarctic fish (Notothenia gibberifrons) and from a mammal (the cow, Bos taurus). At temperatures between 0 and 20 degrees C, the affinity constants for colchicine binding to the fish tubulin were slightly smaller (1.5-2.6-fold) than those for bovine tubulin. van't Hoff analysis showed that the standard enthalpy changes for colchicine binding to the two tubulins were comparable (delta H degrees = +10.6 and +7.4 kcal mol-1 for piscine and bovine tubulins, respectively), as were the standard entropy changes (delta S degrees = +61.3 eu for N. gibberifrons tubulin, +51.2 eu for bovine tubulin). At saturating concentrations of the ligand, the maximal binding stoichiometry for each tubulin was approximately 1 mol colchicine/mol tubulin dimer. The data indicate that the colchicine-binding sites of the two tubulins are similar, but probably not identical, in structure. The apparent absence of major structural modifications at the colchicine site suggests that this region of tubulin is not involved in functional adaptation for low-temperature polymerization. Rather, the colchicine site of tubulin may have been conserved evolutionarily to serve in vivo as a receptor for endogenous molecules (i.e., "colchicine-like" molecules or MAPs) that regulate microtubule assembly.

Disruption of the Golgi apparatus with brefeldin A does not destabilize the associated detyrosinated microtubule network.

Stable subsets of microtubules (MTs) are often enriched in detyrosinated alpha-tubulin. Recently it has been found that the Golgi apparatus is associated with a subset of relatively stable MTs and that detyrosinated MTs colocalize spatially and temporally with the Golgi apparatus in several cell lines. To determine whether the Golgi apparatus actively stabilizes associated MTs and thus allows their time-dependent detyrosination, we have used the drug brefeldin A (BFA) to disrupt the Golgi apparatus and have monitored changes in the Golgi apparatus and MT populations using simultaneous immunofluorescence and fluorescent lectin microscopy. We found that although BFA caused the Golgi apparatus to completely redistribute to the endoplasmic reticulum (ER), the detyrosinated MTs were not disrupted and remained in a juxtanuclear region. By Western blot analysis we found that even after 6 h of continuous exposure of cells to BFA, there was no detectable reduction in the level of detyrosinated alpha-tubulin. Simultaneous treatment with nocodazole and BFA led to a complete disruption of all MTs and normal Golgi structure/organization. Upon removal of nocodazole in the continued presence of BFA, we found that the detyrosinated MTs reformed in a compact juxtanuclear location in the absence of an intact Golgi complex. Finally, we found that the detyrosinated MTs colocalized precisely with a BFA-resistant structure that binds to the lectin, wheat germ agglutinin. We conclude that the juxtanuclear detyrosinated MTs are not actively stabilized by association with BFA-sensitive Golgi membranes. However, another closely associated structure which binds wheat germ agglutinin may serve to stabilize the juxtanuclear MTs. Alternatively, the MT organizing center (MTOC) and/or MT-associated proteins (MAPs) may organize and stabilize the juxtanuclear detyrosinated MTs.

Mammalian mad2 and bub1/bubR1 recognize distinct spindle-attachment and kinetochore-tension checkpoints.

Metaphase checkpoint controls sense abnormalities of chromosome alignment during mitosis and prevent progression to anaphase until proper alignment has been attained. A number of proteins, including mad2, bub1, and bubR1, have been implicated in the metaphase checkpoint control in mammalian cells. Metaphase checkpoints have been shown, in various systems, to read loss of either spindle tension or microtubule attachment at the kinetochore. Characteristically, HeLa cells arrest in metaphase in response to low levels of microtubule inhibitors that leave an intact spindle and a metaphase plate. Here we show that the arrest induced by nanomolar vinblastine correlates with loss of tension at the kinetochore, and that in response the checkpoint proteins bub1 and bubR1 are recruited to the kinetochore but mad2 is not. mad2 remains competent to respond and is recruited at higher drug doses that disrupt spindle association with the kinetochores. Further, although mad2 forms a complex with cdc20, it does not associate with bub1 or bubR1. We conclude that mammalian bub1/bubR1 and mad2 operate as elements of distinct pathways sensing tension and attachment, respectively.

The carboxyl-terminal domain of kinesin heavy chain is important for membrane binding.

Sea urchin kinesin is a plus end-directed microtubule-based motor consisting of two heavy chains and two light chains and is proposed to be responsible (a) for the transport of membranous organelles along microtubules in sea urchin mitotic spindles (Wright, B. D., Henson, J. H., Wedaman, K. P., Willy, P. J., Morand, J. N., and Scholey, J. M. (1991) J. Cell Biol. 113, 817-833) and (b) for the radial dispersion of endoplasmic reticulum and endosomal membranes in non-mitotic cultured coelomocytes (Henson, J. H., Nesbitt, D., Wright, B. D., and Scholey, J. M. (1992) J. Cell Sci. 103, 309-320). We report here that sea urchin kinesin is indeed able to bind in a concentration-dependent and saturable manner to microsomal membranes isolated from sea urchin eggs in the presence of MgATP. The kinesin light chains may not be essential for membrane binding since kinesin containing negligible amounts of light chains binds as well as kinesin containing stoichiometric amounts of light chains. Finally, we propose that kinesin binds to membranes with the carboxyl-terminal domain of the heavy chain (amino acid residues 858-1031) since the bacterially expressed and then isolated stalk-tail fragment of kinesin heavy chain, in contrast to the stalk fragment, is able (a) to bind membranes in a concentration-dependent and saturable manner and (b) to compete with native kinesin for membrane binding. Our results support the hypothesis that the carboxyl-terminal domains of the heavy chains attach kinesin molecules to their membranous cargo in mitotic and interphase sea urchin cells.

Isolation of a sea urchin egg kinesin-related protein using peptide antibodies.

To understand the roles of kinesin and its relatives in cell division, it is necessary to identify and characterize multiple members of the kinesin superfamily from mitotic cells. To this end we have raised antisera to peptides corresponding to highly conserved regions of the motor domains of several known members of the kinesin superfamily. These peptide antibodies react specifically with the motor domains of kinesin and ncd protein, as expected, and they also react with several polypeptides (including kinesin heavy chain) that cosediment with microtubules (MTs) precipitated from AMPPNP-treated sea urchin egg cytosol. Subsequent fractionation of ATP eluates of these MTs yields a protein of relative molecular mass 330 x 10(3) that behaves as a complex of three polypeptides that are distinct from conventional kinesin subunits or fragments thereof. This complex contains 85 kDa and 95 kDa polypeptides, which react with our peptide antibodies, and a 115 kDa polypeptide, which does not. This triplet of polypeptides, which we refer to as KRP(85/95), binds to purified sea urchin egg tubulin in an AMPPNP-enhanced, ATP-sensitive manner and induces the formation of microtubule bundles. We therefore propose that the triplet corresponds to a novel sea urchin egg kinesin-related protein.

Crystal structure of human survivin reveals a bow tie-shaped dimer with two unusual alpha-helical extensions.

Survivin is a mitotic spindle-associated protein involved in linking mitotic spindle function to activation of apoptosis in mammalian cells. The structure of the full-length human survivin has been determined by X-ray crystallography to 2.7 A. Strikingly, the structure forms a very unusual bow tie-shaped dimer. It does not dimerize through a C-terminal coiled-coil, contrary to sequence analysis prediction. The C-terminal helices contain hydrophobic clusters with the potential for protein-protein interactions. The unusual shape and dimensions of survivin suggest it serves an adaptor function through its alpha-helical extensions.