Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.4.4 (kinesin)
5,033 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We determined the crystal structure of the motor domain of the fast fungal kinesin from Neurospora crassa (NcKin). The structure has several unique features. (i) Loop 11 in the switch 2 region is ordered and enables one to describe the complete nucleotide-binding pocket, including three inter-switch salt bridges between switch 1 and 2. (ii) Loop 9 in the switch 1 region bends outwards, making the nucleotide-binding pocket very wide. The displacement in switch 1 resembles that of the G-protein ras complexed with its guanosine nucleotide exchange factor. (iii) Loop 5 in the entrance to the nucleotide-binding pocket is remarkably long and interacts with the ribose of ATP. (iv) The linker and neck region is not well defined, indicating that it is mobile. (v) Image reconstructions of ice-embedded microtubules decorated with NcKin show that it interacts with several tubulin subunits, including a central beta-tubulin monomer and the two flanking alpha-tubulin monomers within the microtubule protofilament. Comparison of NcKin with other kinesins, myosin and G-proteins suggests that the rate-limiting step of ADP release is accelerated in the fungal kinesin and accounts for the unusually high velocity and ATPase activity.
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PMID:Structure of a fast kinesin: implications for ATPase mechanism and interactions with microtubules. 1170 93

We detail here how "free" centrosomes, lacking associated chromosomes, behave during mitosis in PtK(2) homokaryons stably expressing GFP-alpha-tubulin. As free centrosomes separate during prometaphase, their associated astral microtubules (Mts) interact to form a spindle-shaped array that is enriched for cytoplasmic dynein and Eg5. Over the next 30 min, these arrays become progressively depleted of Mts until the two centrosomes are linked by a single bundle, containing 10-20 Mts, that persists for > 60 min. The overlapping astral Mts within this bundle are loosely organized, and their plus ends terminate near its midzone, which is enriched for an ill-defined matrix material. At this time, the distance between the centrosomes is not defined by external forces because these organelles remain stationary when the bundle connecting them is severed by laser microsurgery. However, since the centrosomes move towards one another in response to monastrol treatment, the kinesin-like motor protein Eg5 is involved. From these results, we conclude that separating asters interact during prometaphase of mitosis to form a spindle-shaped Mt array, but that in the absence of chromosomes this array is unstable. An analysis of the existing data suggests that the stabilization of spindle Mts during mitosis in vertebrates does not involve the chromatin (i.e., the RCC1/RanGTP pathway), but instead some other chromosomal component, e.g., kinetochores.
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PMID:Separating centrosomes interact in the absence of associated chromosomes during mitosis in cultured vertebrate cells. 1211 53

When mammalian somatic cells enter mitosis, a fundamental reorganization of the Mt cytoskeleton occurs that is characterized by the loss of the extensive interphase Mt array and the formation of a bipolar mitotic spindle. Microtubules in cells stably expressing GFP-alpha-tubulin were directly observed from prophase to just after nuclear envelope breakdown (NEBD) in early prometaphase. Our results demonstrate a transient stimulation of individual Mt dynamic turnover and the formation and inward motion of microtubule bundles in these cells. Motion of microtubule bundles was inhibited after antibody-mediated inhibition of cytoplasmic dynein/dynactin, but was not inhibited after inhibition of the kinesin-related motor Eg5 or myosin II. In metaphase cells, assembly of small foci of Mts was detected at sites distant from the spindle; these Mts were also moved inward. We propose that cytoplasmic dynein-dependent inward motion of Mts functions to remove Mts from the cytoplasm at prophase and from the peripheral cytoplasm through metaphase. The data demonstrate that dynamic astral Mts search the cytoplasm for other Mts, as well as chromosomes, in mitotic cells.
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PMID:Reorganization of the microtubule array in prophase/prometaphase requires cytoplasmic dynein-dependent microtubule transport. 1223 19

The tau family of microtubule-associated proteins has a microtubule-binding domain which includes three or four conserved sequence repeats. Pelleting assays show that when tubulin and tau are co- assembled into microtubules, the presence of taxol reduces the amount of tau incorporated. In the absence of taxol, strong binding sites for tau are filled by one repeat motif per tubulin dimer; additional tau molecules bind more weakly. We have labelled a repeat motif with nanogold and used three-dimensional electron cryomicroscopy to compare images of microtubules assembled with labelled or unlabelled tau. With kinesin motor domains bound to the microtubule outer surface to distinguish between alpha- and beta-tubulin, we show that the gold label lies on the inner surface close to the taxol binding site on beta-tubulin. Loops within the repeat motifs of tau have sequence similarity to an extended loop which occupies a site in alpha-tubulin equivalent to the taxol-binding pocket in beta-tubulin. We propose that loops in bound tau stabilize microtubules in a similar way to taxol, although with lower affinity so that assembly is reversible.
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PMID:Repeat motifs of tau bind to the insides of microtubules in the absence of taxol. 1250 85

Near-simultaneous three-dimensional fluorescence/differential interference contrast microscopy was used to follow the behavior of microtubules and chromosomes in living alpha-tubulin/GFP-expressing cells after inhibition of the mitotic kinesin Eg5 with monastrol. Kinetochore fibers (K-fibers) were frequently observed forming in association with chromosomes both during monastrol treatment and after monastrol removal. Surprisingly, these K-fibers were oriented away from, and not directly connected to, centrosomes and incorporated into the spindle by the sliding of their distal ends toward centrosomes via a NuMA-dependent mechanism. Similar preformed K-fibers were also observed during spindle formation in untreated cells. In addition, upon monastrol removal, centrosomes established a transient chromosome-free bipolar array whose orientation specified the axis along which chromosomes segregated. We propose that the capture and incorporation of preformed K-fibers complements the microtubule plus-end capture mechanism and contributes to spindle formation in vertebrates.
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PMID:Minus-end capture of preformed kinetochore fibers contributes to spindle morphogenesis. 1260 91

Microtubules are highly dynamic components of the cytoskeleton. They are important for cell movement and they are involved in a variety of transport processes together with motor proteins, such as kinesin. The exact mechanism of these transport processes is not known and so far the focus has been on structural changes within the motor domains, but not within the underlying microtubule structure. Here we investigated the interaction between kinesin and tubulin and our experimental data show that microtubules themselves are changing structure during that process. We studied unstained, vitrified samples of microtubules composed of 15 protofilaments using cryo electron microscopy and helical image analysis. 3D maps of plain microtubules and microtubules decorated with kinesin have been reconstructed to approximately 17A resolution. The alphabeta-tubulin dimer could be identified and, according to our data, alpha- and beta-tubulin adopt different conformations in plain microtubules. Significant differences were detected between maps of plain microtubules and microtubule-kinesin complexes. Most pronounced is the continuous axial inter-dimer contact in the microtubule-kinesin complex, suggesting stabilized protofilaments along the microtubule axis. It seems, that mainly structural changes within alpha-tubulin are responsible for this observation. Lateral effects are less pronounced. Following our data, we believe, that microtubules play an active role in intracellular transport processes through modulations of their core structure.
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PMID:Complex formation with kinesin motor domains affects the structure of microtubules. 1465 46

In fission yeast, gamma-tubulin (encoded by the gtb1+ gene), Alp4 (Spc97/GCP2), and Alp6 (Spc98/GCP3) are essential components of the gamma-tubulin complex. We isolated gtb1 mutants as allele-specific suppressors of temperature-sensitive alp4 mutations. Mutation sites in gtb1 mutants and in several alp4 alleles were determined. The majority of substituted amino acids were mapped to a small area on the predicted surface of the gamma-tubulin molecule that might directly interact with the Alp4 protein. The cold sensitivity of gamma-tubulin mutants was almost completely suppressed by an alpha-tubulin mutation and partially suppressed by a low concentration of thiabendazole, a microtubule assembly inhibitor. Other gtb1 mutants had increased resistance to this drug. Gel-filtration and immunoprecipitation analyses suggested that the mutant gamma-tubulin formed an altered gamma-tubulin complex with increased stability compared to wild-type gamma-tubulin. In most gtb1 mutants, sexual development was impaired, and aberrant asci that contained an irregular spore shape and number were produced. In contrast, spore formation was not appreciably damaged in some alp4 and alp6 mutants, even at temperatures where vegetative proliferation was substantially defective. These results suggested that the function of the gamma-tubulin complex or the requirement of each component of the complex is differentially regulated between the vegetative and sexual phases of the life cycle in fission yeast. In addition, genetic data indicated intimate functional connections of gamma-tubulin with several kinesin-like proteins.
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PMID:Functional dissection of the gamma-tubulin complex by suppressor analysis of gtb1 and alp4 mutations in Schizosaccharomyces pombe. 1528 Feb 26

The 26S proteasome plays essential roles in cell cycle progression in various types of cell. We previously reported that the inhibition of 26S proteasome activities by a proteasome inhibitor, MG-132, exclusively caused cell cycle arrest in synchronized tobacco BY-2 cells. Here we report a further observation of 26S proteasome involvement during M/G1 transition utilizing a transgenetic BY-2 cell line that stably expresses a GFP-alpha-tubulin fusion protein (BY-GT16). Interestingly, MG-132 treatment caused the arrest of cell cycle progression prior to entering the G1 phase. Indeed, phragmoplast-like structures were formed and cortical microtubules were not organized after the collapse of the original phragmoplasts. Additionally, actin microfilaments showed irregular rearrangements when further incubated with MG-132 and as the phragmoplast-like structures developed. Since these phragmoplast-like structures had a similar configuration and ability to form cell plates to that of the original phragmoplasts, we designated these phragmoplast-like structures as extra phragmoplasts. Furthermore, we showed that a tobacco kinesin-related polypeptide of 125 kDa (TKRP125) localized in the extra phragmoplasts and that its protein level remained unchanged during MG-132 treatment. We propose that TKRP125 might be one of the possible targets of the ubiquitin-proteasome degradation pathway during M/G1 transition.
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PMID:Inhibition of proteasome by MG-132 treatment causes extra phragmoplast formation and cortical microtubule disorganization during M/G1 transition in synchronized tobacco cells. 1557 38

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that cleave and degrade a wide spectrum of extracellular matrix components. By enhancing turnover of extracellular matrix, MMP activity is also known to play a key role in tumor cell invasion. Because extracellular protease activity requires efficient release of these proteases to the cellular surface, we investigated storage, transport, and exocytosis of MMP-2 and MMP-9 in human melanoma cells using immunofluorescence, electrical, and biochemical techniques. Immunolabeling of melanoma cells with antibodies specific for MMP-2 and MMP-9 led to the identification of two distinct populations of small cytoplasmatic vesicles containing MMP-2 or MMP-9, respectively. In combination with alpha-tubulin-specific antibodies, both vesicle populations were found to be aligned along the microtubular network. Moreover, the molecular motor protein kinesin is shown to be localized on most of these vesicles, providing evidence that the identified vesicles are actively propelled along microtubules toward the plasma membrane. The functional relevance of these findings is demonstrated using low dosage (5.9 nmol/L) of paclitaxel to affect the microtubular function of melanoma cells. Although cell proliferation is not altered, paclitaxel treatment impairs secretion of MMP-2/MMP-9 and significantly reduces invasive activity in our new cell invasion assay. In conclusion, we demonstrate in melanoma cells that microtubule-dependent traffic of MMP-containing vesicles and exocytosis are critical steps for invasive behavior and therefore are potential targets for specific antitumor drugs.
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PMID:Microtubule-dependent matrix metalloproteinase-2/matrix metalloproteinase-9 exocytosis: prerequisite in human melanoma cell invasion. 1560 54

Ocsyn, a syntaxin-interacting protein characterized by Safieddine et al. [Safieddine, S., Ly, C.D., Wang, Y.-X., Kachar, B., Petralia, R.S., Wenthold, R.J., 2002. Ocsyn, a novel syntaxin-interacting protein enriched in the subapical region of inner hair cells. Mol. Cell. Neurosci., 20, 343-353] in the guinea pig organ of Corti was primarily identified in organelles located at the subapical region of inner hair cells. They proposed that in cochlear inner hair cells, ocsyn was involved in protein trafficking associated to recycling endosomes. Ocsyn happens to be highly homologous to syntabulin with an almost identical syntaxin-binding domain. Syntabulin is believed to attach syntaxin-containing vesicles to kinesin for their axonal transport along microtubules. The present study shows the distribution of ocsyn in guinea pig and rat vestibular hair cells using immunocytochemistry and confocal microscopy. Ocsyn was characterized by intense immunolabeled spots distributed exclusively in type I and II vestibular hair cells. The subcuticular region under the cuticular plate exhibited particularly densely packed spots. In the neck region of the sensory cells, where microtubules are abundant, there was no colocalization of ocsyn and alpha-tubulin. Ocsyn labeled spots were also present in the medial and basal hair cell regions, particularly in the supranuclear and infranuclear regions. Mitochondria are particularly numerous in these three regions (subcuticular, supranuclear and infranuclear). Double labeling of ocsyn and cytochrome c showed that ocsyn was present in the same zones that mitochondria. This, together with the great similarity of ocsyn and syntabulin, suggest that, akin to syntabulin, ocsyn is involved in addressing organelles. We propose that ocsyn is involved in the formation of the canalicular-mitochondrial complexes depicted by Spicer et al. [Spicer, S.S., Thomopoulos, G.N., Schulte, B.A., 1999. Novel membranous structures in apical and basal compartments of inner hear cells. J. Comp. Neurol., 409, 424-437].
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PMID:Ocsyn and mitochondrial-canalicular complexes in vestibular hair cells. 1704 36


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