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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)

Two microtubule-stimulated ATPases, cytoplasmic dynein, and kinesin, are believed to be responsible for the intracellular movement of membrane-bound organelles in opposite directions along microtubules. An unresolved component of this model is the mechanism by which cells regulate these two motors to direct various membrane-bound organelles to their proper locations. To determine if phosphorylation may play a role in the regulation of cytoplasmic dynein, the in vivo phosphorylation state of cytoplasmic dynein from two cellular pools was examined. The entire cellular pool of brain cytoplasmic dynein was metabolically labeled by the infusion of [32P]orthophosphate into the cerebrospinal fluid of rat brain ventricles. To characterize the phosphorylation of dynein associated with anterograde membrane-bound organelles, the optic nerve fast axonal transport system was used. Using a monoclonal antibody to the 74-kD polypeptide of brain cytoplasmic dynein, the native dynein complex was immunoprecipitated from the radiolabled tissue extracts. Autoradiographs of one and two dimensional gels showed labeling of nearly all of the polypeptide isoforms of cytoplasmic dynein from rat brain. These polypeptides are phosphorylated on serine residues. Comparison of the amount of 32P incorporated into the dynein polypeptides revealed differences in the phosphorylation of dynein polypeptides from the anterograde and the cellular pools. Most interestingly, the 530-kD heavy chain of dynein appears to be phosphorylated to a lesser extent in the anterograde pool than in the cellular pool. Since the anterograde pool contains inactive dynein, while the entire cellular pool contains both inactive and active dynein, these results are consistent with the hypothesis that phosphorylation regulates the functional activity of cytoplasmic dynein.
J Cell Biol 1994 Dec
PMID:Differential phosphorylation in vivo of cytoplasmic dynein associated with anterogradely moving organelles. 752 20

The CHO1 antigen is a mitosis-specific kinesin-like motor located at the interzonal region of the spindle. The human cDNA coding for the antigen contains a domain with sequence similarity to the motor domain of kinesin-like protein (Nislow et al., Nature 359, 543, 1992). Here we cloned cDNAs encoding the CHO1 antigen by immunoscreening of a CHO Uni-Zap expression library, the same species in which the original monoclonal antibody was raised. cDNAs of CHO cells encode a 953 amino acid polypeptide with a calculated molecular mass of 109 kDa. The N-terminal 73% of the antigen was 87% identical to the human clone, whereas the remaining 27% of the coding region showed only 48% homology. Insect Sf9 cells infected with baculovirus containing the full-length insert produced 105 and 95 kDa polypeptides, the same doublet identified as the original antigen in CHO cells. Truncated polypeptides corresponding to the N-terminal motor and C-terminal tail produced a 56 and 54 kDa polypeptide in Sf9 cells, respectively. Full and N-terminal proteins co-sedimented with, and caused bundling of, brain microtubules in vitro, whereas the C-terminal polypeptide did not. Cells expressing the N terminus formed one or more cytoplasmic processes. Immunofluorescence as well as electron microscopic observations revealed the presence of thick bundles of microtubules, which were closely packed, forming a marginal ring just beneath the cell membrane and a core in the processes. The diffusion coefficient and sedimentation coefficient were determined for the native CHO1 antigen by gel filtration and sucrose density gradient centrifugation, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
J Cell Sci 1994 Dec
PMID:Heterogeneity and microtubule interaction of the CHO1 antigen, a mitosis-specific kinesin-like protein. Analysis of subdomains expressed in insect sf9 cells. 770

Microtubules are constructed from alpha- and beta-tubulin heterodimers that are arranged into protofilaments. Most commonly there are 13 or 14 protofilaments. A series of structural investigations using both electron microscopy and x-ray diffraction have indicated that there are two potential lattices (A and B) in which the tubulin subunits can be arranged. Electron microscopy has shown that kinesin heads, which bind only to beta-tubulin, follow a helical path with a 12-nm pitch in which subunits repeat every 8-nm axially, implying a primarily B-type lattice. However, these helical symmetry parameters are not consistent with a closed lattice and imply that there must be a discontinuity or "seam" along the microtubule. We have used quick-freeze deep-etch electron microscopy to obtain the first direct evidence for the presence of this seam in microtubules formed either in vivo or in vitro. In addition to a conventional single seam, we have also rarely found microtubules in which there is more than one seam. Overall our data indicates that microtubules have a predominantly B lattice, but that A lattice bonds between tubulin subunits are found at the seam. The cytoplasmic microtubules in mouse nerve cells also have predominantly B lattice structure and A lattice bonds at the seam. These observations have important implications for the interaction of microtubules with MAPs and with motor proteins, and for example, suggest that kinesin motors may follow a single protofilament track.
J Cell Biol 1994 Dec
PMID:Direct visualization of the microtubule lattice seam both in vitro and in vivo. 780 74

We studied fluctuations in the displacement of silica beads driven by single molecules of the motor protein kinesin, moving under low mechanical loads at saturating ATP concentrations. The variance in position was significantly smaller than expected for the case of stepwise movement along a regular lattice of positions with exponentially distributed intervals. The small variance suggests that two or more sequential processes with comparable reaction rates dominate the biochemical cycle. The low value is inconsistent with certain recently proposed thermal ratchet models for motor movement as well as with scenarios where the hydrolysis of a single ATP molecule leads to a cluster of several steps. Fluctuation analysis is a potential powerful tool for studying kinetic behavior whenever the output of a single enzyme can be monitored.
Proc Natl Acad Sci U S A 1994 Dec 06
PMID:Fluctuation analysis of motor protein movement and single enzyme kinetics. 799 36

To study the possible involvement of kinesin-like molecules in mitosis a polyclonal antibody against the head domain of Drosophila kinesin heavy chain (HD antibody) was microinjected into PtK1 cells at the prophase-prometaphase transition. Progress of the cell through mitosis was recorded for subsequent detailed analysis. Cells injected with pre-immune IgG progressed through mitosis at rates similar to those for noninjected cells. After HD antibody injections, chromosomes failed to congress to an equatorial plane and cells failed to form a bipolar spindle. Rather, the spindle poles came together, resulting in a monopolar-like configuration with chromosomes arranged about the poles in a rosette. Sometimes the monopolar array moved to the margin of the cell in a way similar to anaphase B movement in normal cells. Antibody-injected cells progressed into the next cell cycle as evidenced by chromosome decondensation and nuclear envelope reformation. Anti-tubulin immunofluorescence confirmed the presence of a radial monopolar array of microtubules in injected cells. HD antibody stained in a punctate pattern in interphase and the spindle region in mitotic PtK1 cells. The antibody also reacted with spindle fibers of isolated mitotic CHO spindles and with kinetochores of isolated CHO chromosomes. Immunoblotting indicated that the major component recognized by the antibody is the 120 kDa kinesin heavy chain. At higher protein loads the antibody recognized also a 34 kDa polypeptide in PtK1 cell extracts, a 135 kDa polypeptide in a preparation of CHO spindles and a 300 kDa polypeptide in a preparation of CHO mitotic chromosomes. We conclude that a kinesin-like molecule is important for the formation and/or maintenance of the structure of mitotic spindle.
J Cell Sci 1993 Dec
PMID:Kinesin-like molecules involved in spindle formation. 812 99

We have used an in vitro fusion assay to study the mechanisms of transport from early to late endosomes. Our data show that the late endosomes share with the early endosomes a high capacity to undergo homotypic fusion in vitro. However, direct fusion of early with late endosomes does not occur. We have purified vesicles which are intermediates during transport from early to late endosomes in vivo, and analyzed their protein composition in two-dimensional gels. In contrast to either early or late endosomes, these vesicles do not appear to contain unique proteins. Moreover, these vesicles undergo fusion with late endosomes in vitro, but not with each other or back with early endosomes. In vitro, fusion of these endosomal vesicles with late endosomes is stimulated by polymerized microtubules, consistent with the known role of microtubules during early to late endosome transport in vivo. In contrast, homotypic fusion of early or late endosomes is microtubule-independent. Finally, this stimulation by microtubules depends on microtubule-associated proteins and requires the presence of the minus-end directed motor cytoplasmic dynein, but not the plus-end directed motor kinesin, in agreement with the microtubule organization in vivo. Our data strongly suggest that early and late endosomes are separate, highly dynamic organelles, which are connected by a microtubule-dependent vesicular transport step.
J Cell Biol 1993 Dec
PMID:Cytoplasmic dynein-dependent vesicular transport from early to late endosomes. 825 38

Kinesin is a motor protein that uses the energy derived from ATP hydrolysis to transport organelles along microtubules. By analyzing the thermal fluctuation of microtubules tethered to glass surfaces by single molecules of kinesin, we have measured the torsional flexibility of the motor protein. The torsional stiffness of kinesin, (117 +/- 19) x 10(-24) N.m.rad-1 (mean +/- SEM), is so low that one kT of energy (approximately 4.1 x 10(-21) J at room temperature) is sufficient to twist a kinesin molecule through more than 360 degrees from its resting orientation. Consistent with this flexibility, motility assays show that one or more kinesin molecules can move a microtubule equally well in any direction. These results explain how a motor on the surface of an organelle can rapidly bind to and capture a microtubule irrespective of the organelle's orientation. Furthermore, the flexibility ensures that several motors can efficiently work together even though they are randomly oriented on the surface of an organelle rather than being in precise arrays like the motors of muscle and cilia.
Proc Natl Acad Sci U S A 1993 Dec 15
PMID:Kinesin swivels to permit microtubule movement in any direction. 826 3

We report the isolation, sequence, and identification of a cDNA encoding the human kinesin light-chain (KLC) protein. The cDNA molecule consisted of 276 nucleotides of 5' untranslated region, the complete coding sequence of 1,710 nucleotides, and 322 nucleotides of 3' untranslated region. It encoded a polypeptide of 569 amino acids and a deduced molecular mass of 64,789 daltons. The predicted secondary internal structure of the KLC molecule consisted of about 27 contiguous repeats, each of approximately 21 amino acid residues, and could be divided into three domains. The amino-terminal domain consisted of heptad repeats typical of the rod domain of several cytoskeletal proteins. The central and carboxy-terminal domains consist of 21-mer repeats. KLC mRNA was expressed in most tissues analyzed. The gene, which was expressed in bacteria and Chinese hamster ovary cells, was provisionally assigned to the long arm of human chromosome 14.
DNA Cell Biol 1993 Dec
PMID:Cloning and genetic characterization of the human kinesin light-chain (KLC) gene. 827 21

Multiple transcripts coding for kinesin light chain isoforms are present in the tissues of the squid Loligo pealii. Isoform diversity arises through alternative RNA splicing in the amino and carboxyl termini of the putative proteins. Comparison to rat and Drosophila proteins demonstrates a remarkable conservation of structural domains and regulatory motifs. We have identified a PEST domain that may be the site of degradative uncoupling of kinesin functions. Selective transcript distribution occurs in disparate tissues, suggesting an adaptation toward specialized functions. Expression is highest in the nervous system and some evidence for neural-specific transcripts is provided. In neurons, this may relate to the differential targeting of specific membrane-bound organelles such as synaptic vesicles.
DNA Cell Biol 1993 Dec
PMID:Kinesin light chains: identification and characterization of a family of proteins from the optic lobe of the squid Loligo pealii. 827 23

One of the major functions of cytoplasmic microtubules is their involvement in maintenance of asymmetric cell shape. Microtubules were considered to perform this function working as rigid structural elements. At the same time, microtubules play a critical role in intracellular organelle transport, and this fact raises the possibility that the involvement of microtubules in maintenance of cell shape may be mediated by directed transport of certain cellular components to a limited area of the cell surface (e.g., to the leading edge) rather than by their functioning as a mechanical support. To test this hypothesis we microinjected cultured human fibroblasts with the antibody (called HD antibody) raised against kinesin motor domain highly conserved among the different members of kinesin superfamily. As was shown before this antibody inhibits kinesin-dependent microtubule gliding in vitro and interferes with a number of microtubule-dependent transport processes in living cells. Preimmune IgG fraction was used for control experiments. Injections of fibroblasts with HD antibody but not with preimmune IgG significantly reduced their asymmetry, resulting in loss of long processes and elongated cell shape. In addition, antibody injection suppressed pseudopodial activity at the leading edge of fibroblasts moving into an experimentally made wound. Analysis of membrane organelle distribution showed that kinesin antibody induced clustering of mitochondria in perinuclear region and their withdrawal from peripheral parts of the cytoplasm. HD antibody does not affect either density or distribution of cytoplasmic microtubules. The results of our experiments show that many changes of phenotype induced in cells by microtubule-depolymerizing agents can be mimicked by the inhibition of motor proteins, and therefore microtubule functions in maintaining of the cell shape and polarity are mediated by motor proteins rather than by being provided by rigidity of tubulin polymer itself.
J Cell Biol 1993 Dec
PMID:Microtubule-dependent control of cell shape and pseudopodial activity is inhibited by the antibody to kinesin motor domain. 827 99


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