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

---

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

Individual microtubules can be visualised by confocal microscopy in reflection mode; when associated with a glass surface, they show up as black lines against the bright reflection from the surface. The high contrast imaging allows details of the behaviour of sliding microtubules to be studied easily. Taxol-stabilised microtubules sliding over kinesin-coated surfaces are normally straight, but can bend into tight loops if the leading end sticks to the surface. Some remain curved after release and move in circles. In such cases, the microtubule lattice must have become stably deformed. Electron microscopy of microtubules fixed during sliding shows no gross rearrangement of the subunit lattice and indicates that microtubule bending is mainly achieved by increased twisting of the longitudinal protofilaments around the microtubule.
...
PMID:The bending of sliding microtubules imaged by confocal light microscopy and negative stain electron microscopy. 171 72

Taxol is a plant alkaloid that binds to and strongly stabilizes microtubules. Taxol-treated microtubules resist depolymerization under a variety of conditions that readily disassemble untreated microtubules. We report here that taxol-treated microtubules can be induced to disassemble by a combination of depolymerizating conditions. Reversible cycles of disassembly and reassembly were carried out using taxol-containing microtubules from calf brain and sea urchin eggs by shifting temperature in the presence of millimolar levels of Ca2+. Microtubules depolymerized completely, yielding dimers and ring-shaped oligomers as revealed by negative stain electron microscopy and Bio-Gel A-15m chromatography, and reassembled into well-formed microtubule polymer structures. Microtubule-associated proteins (MAPs), including species previously identified only by taxol-based purification such as MAP 1B and kinesin, were found to copurify with tubulin through reversible assembly cycles. To determine whether taxol remained bound to tubulin subunits, we subjected depolymerized taxol-treated microtubule protein to Sephadex G-25 chromatography, and the fractions were assayed for taxol content by reverse-phase HPLC. Taxol was found to be dissociated from the depolymerized microtubules. Protein treated in this way was found to be competent to reassemble, but now required conditions comparable with those for protein that had never been exposed to taxol. Thus, the binding of taxol to tubulin can be reversed. This has implications for the mechanism of taxol action and for the purification of microtubules from a wide variety of sources for use in self-assembly experiments.
...
PMID:Temperature-dependent reversible assembly of taxol-treated microtubules. 289 14

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)
...
PMID:Yeast Kar3 is a minus-end microtubule motor protein that destabilizes microtubules preferentially at the minus ends. 791 93

Glutaraldehyde-cross-linked microtubules were investigated as substrates for kinesin motility. Microtubules, formed in vitro from chicken brain tubulin, were stabilized with Taxol and chemically fixed with glutaraldehyde. The degree of tubulin monomer cross-linking as a function of time and glutaraldehyde concentration was characterized using polyacrylamide gel electrophoresis. Atomic force microscopy of fixed microtubules indicated that the cross-linking is sufficient to stabilize the gross structure of the microtubules against air drying or a distilled water challenge. Kinesin movement on immobilized, fixed microtubules was determined using a kinesin-coated bead motility assay observed with differential interference contrast microscopy. Within measurement error, kinesin bead movement velocities were independent of the degree of microtubule cross-linking. Binding affinity, however, decreased with increased cross-linking. Although air- and water-challenged microtubules did not support kinesin motility, a dilute suspension of glutaraldehyde-fixed microtubules in buffer supported kinesin motility for at least 2 days without any substantial degradation of activity. Fixed microtubules may be useful for several applications, including affinity purification of microtubule-associated proteins and motility measurements under extreme conditions of temperature and other variables.
...
PMID:Kinesin movement on glutaraldehyde-fixed microtubules. 892 59

The single-cell trichomes in wild-type Arabidopsis are either unbranched or have two to five branches. Using transgenic Arabidopsis plants expressing a green fluorescent protein-microtubule-associated protein4 fusion protein, which decorates the microtubular cytoskeleton, we observed that during trichome branching, microtubules reorient with respect to the longitudinal growth axis. Considering branching to be a localized microtubule-dependent growth reorientation event, we investigated the effects of microtubule-interacting drugs on branch induction in trichomes. In unbranched trichomes of the mutant stichel, a change in growth directionality, closely simulating branch initiation, could be elicited by a short treatment with paclitaxel, a microtubule-stabilizing drug, but not with microtubule-disrupting drugs. The growth reorientation appeared to be linked to increased microtubule stabilization and to aster formation in the treated trichomes. Taxol-induced microtubule stabilization also led to the initiation of new branch points in the zwichel mutant of Arabidopsis, which is defective in a kinesin-like microtubule motor protein and possesses trichomes that are less branched. Our observations suggest that trichome cell branching in Arabidopsis might be mediated by transiently stabilized microtubular structures, which may form a component of a multiprotein complex required to reorient freshly polymerizing microtubules into new growth directions.
...
PMID:Microtubule stabilization leads to growth reorientation in Arabidopsis trichomes. 1076 Feb 37

Taxanes are powerful chemotherapy agents that target the microtubule cytoskeleton, leading to mitotic arrest and cell death; however, their clinical efficacy has been hampered due to the development of drug resistance. Therefore, other proteins involved in spindle assembly are being examined as potential targets for anticancer therapy. The mitotic kinesin, Eg5 is critical for proper spindle assembly; as such, inhibition of Eg5 leads to mitotic arrest making it a potential anticancer target. We wanted to validate Eg5 as a therapeutic target and determine if Eg5 inhibitors retain activity in Taxol-resistant cells. Using affinity chromatography we first show that the compound HR22C16 is an Eg5 inhibitor and does not interact with other microtubule motor proteins tested. Furthermore, HR22C16 along with its analogs, inhibit cell survival in both Taxol-sensitive and -resistant ovarian cancer cells with at least 15-fold greater efficacy than monastrol, the first generation Eg5 inhibitor. Further analysis with HR22C16-A1, the most potent HR22C16 analog, showed that it retains efficacy in PgP-overexpressing cells, suggesting that it is not a PgP substrate. We further show that HR22C16-A1 induces cell death following mitotic arrest via the intrinsic apoptotic pathway. Interestingly, the combination of HR22C16-A1 with Taxol results in an antagonistic antiproliferative and antimitotic effect, possibly due to the abrogation of Taxol-induced mitotic spindles by HR22C16-A1. Taken together, our results show that Eg5 inhibitors have promising anticancer activity and can be potentially used to overcome Taxol resistance in the clinical setting.
...
PMID:Mitotic kinesin inhibitors induce mitotic arrest and cell death in Taxol-resistant and -sensitive cancer cells. 1565 76

Taxol functions to suppress the dynamic behavior of individual microtubules, and induces multipolar mitotic spindles. However, little is known about the mechanisms by which taxol disrupts normal bipolar spindle assembly in vivo. Using live imaging of GFP-alpha tubulin expressing cells, we examined spindle assembly after taxol treatment. We find that as taxol-treated cells enter mitosis, there is a dramatic re-distribution of the microtubule network from the centrosomes to the cell cortex. As they align there, the cortical microtubules recruit NuMA to their embedded ends, followed by the kinesin motor HSET. These cortical microtubules then bud off to form cytasters, which fuse into multipolar spindles. Cytoplasmic dynein and dynactin do not re-localize to cortical microtubules, and disruption of dynein/dynactin interactions by over-expression of p50 "dynamitin" does not prevent cytaster formation. Taxol added well before spindle poles begin to form induces multipolarity, but taxol added after nascent spindle poles are visible-but before NEB is complete-results in bipolar spindles. Our results suggest that taxol prevents rapid transport of key components, such as NuMA, to the nascent spindle poles. The net result is loss of mitotic spindle pole cohesion, microtubule re-distribution, and cytaster formation.
...
PMID:Live-cell analysis of mitotic spindle formation in taxol-treated cells. 1848 5

Microtubule interfering agents (MIAs) are anti-tumor drugs that inhibit microtubule dynamics, while kinesin spindle protein (KSP) inhibitors are substances that block the formation of the bipolar spindle during mitosis. All these compounds cause G2/M arrest and cell death. Using 2D-PAGE followed by Nano-LC-ESI-Q-ToF analysis, we found that MIAs such as vincristine (Oncovin) or paclitaxel (Taxol) and KSP inhibitors such as S-tritil-l-cysteine induce the phosphorylation of the nuclear protein p54(nrb) in HeLa cells. Furthermore, we demonstrate that cisplatin (Platinol), an anti-tumor drug that does not cause M arrest, does not induce this modification. We show that the G2/M arrest induced by MIAs is required for p54(nrb) phosphorylation. Finally, we demonstrate that CDK activity is required for MIA-induced phosphorylation of p54(nrb).
...
PMID:Microtubule interfering agents and KSP inhibitors induce the phosphorylation of the nuclear protein p54(nrb), an event linked to G2/M arrest. 1883 53

We performed in vitro assays to visualize the effects of pressure on the filamentous structure of microtubules. Taxol-stabilized microtubules were tethered to kinesin motors on the observation window of a high-pressure chamber. When pressure was applied to the sample solution, all of the microtubules started to shorten from both ends. The length changes were constant over time, irrespective of the microtubule polarity. The shortening rate of microtubules increased exponentially with pressure, and the activation volume was -100 mL/mol, consistent with in vivo studies. These results show that application of pressure works directly to weaken the intermolecular interactions between tubulin molecules.
...
PMID:Microtubule depolymerization at high pressure. 2023 72

Microtubule interfering agents (MIAs) are antitumor drugs that inhibit microtubule dynamics, while kinesin spindle protein (KSP) inhibitors are substances that block the formation of the bipolar spindle during mitosis. All these compounds cause the accumulation of mitotic cells and subsequently cell death. We used two-dimensional gel electrophoresis (2DE) followed by MALDI-MS analysis to demonstrate that the MIAs vinblastine (Velban) and paclitaxel (Taxol), as well as the KSP inhibitor S-tritil-L-cysteine, induce the phosphorylation of annexin A2 in human lung carcinoma A549 cells. Further tandem mass spectrometry analysis using a combination of peptide fragmentation methods (CID and ETD) and multiple reaction monitoring (MRM) analysis determined that this modification occurs mainly at threonine 19. We show that MIAs and KSP inhibitors only induce this phosphorylation in cells capable of reaching the M phase. Furthermore, we demonstrate that CDK activity is required for the phosphorylation of annexin A2 induced by MIAs and KSP inhibitors. Finally, we have used double thymidine block synchronization to demonstrate that annexin A2 is not phosphorylated during a normal mitosis, indicating that this phosphorylation of annexin A2 is a specific response to these drugs.
...
PMID:Proteomic analysis of annexin A2 phosphorylation induced by microtubule interfering agents and kinesin spindle protein inhibitors. 2059 53


1 2 Next >>

---