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)

Purified Golgi membranes were mixed with cytosol and microtubules (MTs) and observed by video enhanced light microscopy. Initially, the membranes appeared as vesicles that moved along MTs. As time progressed, vesicles formed aggregates from which membrane tubules emerged, traveled along MTs, and eventually generated extensive reticular networks. Membrane motility required ATP, occurred mainly toward MT plus ends, and was inhibited almost completely by the H1 monoclonal antibody to kinesin heavy chain, 5'-adenylylimidodiphosphate, and 100 microM but not 20 microM vanadate. Motility was also blocked by GTPgammaS or A1F4- but was insensitive to A1C13, NaF, staurosporin, or okadaic acid. The targets for GTPgammaS and A1F4- were evidently of cytosolic origin, did not include kinesin or MTs, and were insensitive to several probes for trimeric G proteins. Transport of Golgi membranes along MTs mediated by a kinesin has thus been reconstituted in vitro. The motility is regulated by one or more cytosolic GTPases but not by protein kinases or phosphatases that are inhibited by staurosporin or okadaic acid, respectively. The pertinent GTPases are likely to be small G proteins or possibly dynamin. The in vitro motility may correspond to Golgi-to-ER or Golgi-to-cell surface transport in vivo.
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PMID:In vitro reconstitution of microtubule plus end-directed, GTPgammaS-sensitive motility of Golgi membranes. 976 38

Caenorhabditis elegans dynamin is expressed at high levels in neurons and at lower levels in other cell types, consistent with the important role that dynamin plays in the recycling of synaptic vesicles. Indirect immunofluorescence showed that dynamin is concentrated along the dorsal and ventral nerve cords and in the synapse-rich nerve ring. Green fluorescent protein (GFP) fused to the N terminus of dynamin is localized to synapse-rich regions. Furthermore, this chimera was detected along the apical membrane of intestinal cells, in spermathecae, and in coelomocytes. Dynamin localization was not affected by disrupting axonal transport of synaptic vesicles in the unc-104 (kinesin) mutant. To investigate the alternative mechanisms that dynamin might use for translocation to the synapse, we systematically tested the localization of different protein domains by fusion to GFP. Localization of each chimera was measured in one specific neuron, the ALM. The GTPase, a middle domain, and the putative coiled coil each contribute to synaptic localization. Surprisingly, the pleckstrin homology domain and the proline-rich domain, which are known to bind to coated-pit constituents, did not contribute to synaptic localization. The GFP-GTPase chimera was most strongly localized, although the GTPase domain has no known interactions with proteins other than with dynamin itself. Our results suggest that different dynamin domains contribute to axonal transport and the sequestration of a pool of dynamin molecules in synaptic cytosol.
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PMID:Contribution of the GTPase domain to the subcellular localization of dynamin in the nematode Caenorhabditis elegans. 980 8

A novel protein with ATPase activity was purified from the cytoplasmic extracts of maize pollen by acetone precipitation, ammonium sulfate fractionation, followed by DEAE-Sephadex A(50) and Mono S ion-exchange chromatography. The molecular weight was about 28 kD as determined by SDS-PAGE and the isoelectric point was pH 8.3 by IEF-PAGE. Western blotting analysis showed the 28 kD protein had no specific immuno-reactions with the anti-kinesin monoclonal or the anti-dynamin polyclonal antibodies. The maximum ultraviolet absorbance was at 278 nm, CD spectrum analysis showed the that 28 kD protein with the feature of a globulin. Pharmacological studies indicated that the enzyme activity was strongly inhibited by Na(3)VO(4) but insensitive to NEM. It was inhibited about 50% by NaF. Oligomycin, KNO(3) and ouabain had no effects on its ATPase activity.
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PMID:Purification of the 28 kD Protein from Maize Pollen and Studies on Its Properties. 1223 28

The mechanism of cell wall formation after male meiosis was studied in microsporocytes of Arabidopsis thaliana (L.) Heynh. by means of thin-section and immuno-electron microscopy and dual-axis electron tomography of high-pressure-frozen/freeze-substituted cells. The cellularization of four-nucleate microsporocytes involves a novel type of cell plate, called a post-meiotic-type cell plate. As in the syncytial endosperm, the microsporocyte cell plates assemble in association with mini-phragmoplasts. However, in contrast to the endosperm cell plates, post-meiotic type cell plates arise simultaneously across the entire division plane. Vesicles are transported along mini-phragmoplast microtubules by putative kinesin proteins and, prior to fusion, they become connected together by 24-nm-long linkers that resemble exocyst complexes. These vesicles fuse with each other to form wide tubules and wide tubular networks. In contrast to endosperm cell plates, the wide tubular networks in microsporocytes completely lack callose and do not appear to be constricted by dynamin rings. The most peripheral wide tubular networks begin to fuse with the plasma membrane before the more central cell plate assembly sites become integrated into a coherent cell plate. Fusion with the parental plasma membrane triggers callose synthesis and the wide tubular domains are converted into convoluted sheets. As the peripheral convoluted sheets accumulate callose and arabinogalactan proteins, they are converted into stub-like projections, which grow centripetally, i.e. toward the interior of the syncytium, fusing with the wide tubular networks already assembled in the division plane. We also demonstrate that the ribosome-excluding cell plate assembly matrix is delivered to the mini-phragmoplast with the first vesicles, and encompasses all the linked vesicles and intermediate stages in cell plate formation.
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PMID:Electron tomographic analysis of post-meiotic cytokinesis during pollen development in Arabidopsis thaliana. 1461 Jun 76

Dynamin, a crucial factor in endocytosis, is a member of a family of GTPases that participates in membrane fission. It was initially proposed to act as a machine that constricts and cuts the neck of nascent vesicles in a GTP-hydrolysis-dependent reaction, but subsequent studies suggested alternative models. Here we monitored the effect of nucleotides on dynamin-coated lipid tubules in real time. Addition of GTP, but not of GDP or GTP-gammaS, resulted in twisting of the tubules and supercoiling, suggesting a rotatory movement of the helix turns relative to each other during GTP hydrolysis. Rotation was confirmed by the movement of beads attached to the tubules. Twisting activity produced a longitudinal tension that was released by tubule breakage when both ends of the tubule were anchored. Fission also occurred when dynamin and GTP were added to lipid tubules that had been generated from liposomes by the motor activity of kinesin on microtubules. No fission events were observed in the absence of longitudinal tension. These findings demonstrate a mechanoenzyme activity of dynamin in endocytosis, but also imply that constriction is not sufficient for fission. At the short necks of endocytic vesicles, other factors leading to tension may cooperate with the constricting activity of dynamin to induce fission.
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PMID:GTP-dependent twisting of dynamin implicates constriction and tension in membrane fission. 1664 39

Emerging evidence has suggested that glycogen synthase kinase 3 (GSK-3) is a key regulatory kinase involved in a plethora of processes in the nervous system, including neuronal development, mood stabilization, and neurodegeneration. However, the cellular mechanisms underlying the actions of GSK-3 remain to be identified. In this study, we examined the impact of GSK-3 on the N-methyl-D-aspartate (NMDA) receptor channel, a central player involved in cognitive and emotional processes. We found that application of various structurally different GSK-3 inhibitors caused a long-lasting reduction of NMDA receptor-mediated ionic and synaptic current in cortical pyramidal neurons. Cellular knockdown of GSK-3beta in neuronal cultures with a small interfering RNA led to smaller NMDA receptor current and loss of its regulation by GSK-3 inhibitors. The NR2B subunit-containing NMDA receptor was the primary target of GSK-3, but the GSK-3 modulation of NMDAR current did not involve the motor protein kinesin superfamily member 17-based transport of NR2B-containing vesicles along microtubules. Combined electrophysiological, immunocytochemical, and biochemical evidence indicated that GSK-3 inhibitors induced the down-regulation of NMDAR current through increasing the Rab5-mediated and PSD-95-regulated NMDAR internalization in a clathrin/dynamin-dependent manner.
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PMID:Glycogen synthase kinase 3 regulates N-methyl-D-aspartate receptor channel trafficking and function in cortical neurons. 1740 Jul 62

Mitochondria undergo dramatic rearrangement during Drosophila spermatogenesis. In wild type testes, the many small mitochondria present in pre-meiotic spermatocytes later aggregate, fuse, and interwrap in post-meiotic haploid spermatids to form the spherical Nebenkern, whose two giant mitochondrial compartments later unfurl and elongate beside the growing flagellar axoneme. Drp1 encodes a dynamin-related protein whose homologs in many organisms mediate mitochondrial fission and whose Drosophila homolog is known to govern mitochondrial morphology in neurons. The milton gene encodes an adaptor protein that links mitochondria with kinesin and that is required for mitochondrial transport in Drosophila neurons. To determine the roles of Drp1 and Milton in spermatogenesis, we used the FLP-FRT mitotic recombination system to generate spermatocytes homozygous for mutations in either gene in an otherwise heterozygous background. We found that absence of Drp1 leads to abnormal clustering of mitochondria in mature primary spermatocytes and aberrant unfurling of the mitochondrial derivatives in early Drp1 spermatids undergoing axonemal elongation. In milton spermatocytes, mitochondria are distributed normally; however, after meiosis, the Nebenkern is not strongly anchored to the nucleus, and the mitochondrial derivatives do not elongate properly. Our work defines specific functions for Drp1 and Milton in the anchoring, unfurling, and elongation of mitochondria during sperm formation.
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PMID:Roles for Drp1, a dynamin-related protein, and milton, a kinesin-associated protein, in mitochondrial segregation, unfurling and elongation during Drosophila spermatogenesis. 1869 63

Embryonic CNS neurons can migrate from the ventricular zone to their final destination by radial glial-guided locomotion. Another less appreciated mechanism is somal translocation, where the young neuron maintains its primitive ventricular and pial processes, through which the cell body moves. A major problem in studying translocation has been the identification of neuronal-specific markers that appear in primitive, radially shaped cells. We used enhanced yellow fluorescent protein under control of the Pet-1 enhancer/promoter region (ePet-EYFP), a specific marker of early differentiated serotonergic neurons, to study their migration via immunohistology and time-lapse imaging of living slice cultures. As early as E10.0, ePet-EYFP-expressing neurons were axonless, radially oriented, and spanned the entire neuroepithelium. The soma translocated within the pial process toward the pial surface and could also translocate through its neurites, which sprouted from the pial process. The dynamin inhibitor dynasore significantly reduced translocation velocity, while the nonmuscle myosin II inhibitor blebbistatin and the kinesin inhibitor AMP-PNP had no significant effect. Here we show for the first time that serotonergic neurons migrate by somal translocation mediated, in part, by dynamin.
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PMID:Serotonergic neurons migrate radially through the neuroepithelium by dynamin-mediated somal translocation. 2007 6

Ostreococcus tauri is a unicellular green alga and amongst the smallest and simplest free-living eukaryotes. The O. tauri genome sequence was determined in 2006. Molecular, physiological and taxonomic data that has been generated since then highlight its potential as a simple model species for algae and plants. However, its proteome remains largely unexplored. This paper describes the global proteomic study of O. tauri, using mass spectrometry-based approaches: phosphopeptide enrichment, cellular fractionation, label-free quantification and (15)N metabolic labeling. The O. tauri proteome was analyzed under the following conditions: sampling at different times during the circadian cycle, after 24h of illumination, after 24h of darkness and under various nitrogen source supply levels. Cell cycle related proteins such as dynamin and kinesin were significantly up-regulated during the daylight-to-darkness transition. This is reflected by their higher intensity at ZT13 and this transition phase coincides with the end of mitosis. Proteins involved in several metabolic mechanisms were found to be up-regulated under low nitrogen conditions, including carbon storage pathways, glycolysis, phosphate transport, and the synthesis of inorganic polyphosphates. Ostreococcus tauri responds to low nitrogen conditions by reducing its nitrogen assimilation machinery which suggests an atypical adaptation mechanism for coping with a nutrient-limited environment.
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PMID:Shotgun proteomic analysis of the unicellular alga Ostreococcus tauri. 2163 80

Unlike apoptosis, necrotic cell death is characterized by marked loss of plasma membrane integrity. Leakage of cytoplasmic material to the extracellular space contributes to cell demise, and is the cause of acute inflammatory responses, which typically accompany necrosis. The mechanisms underlying plasma membrane damage during necrotic cell death are not well understood. We report that endocytosis is critically required for the execution of necrosis. Depletion of the key endocytic machinery components dynamin, synaptotagmin and endophilin suppresses necrotic neurodegeneration induced by diverse genetic and environmental insults in C. elegans. We used genetically encoded fluorescent markers to monitor the formation and fate of specific types of endosomes during cell death in vivo. Strikingly, we find that the number of early and recycling endosomes increases sharply and transiently upon initiation of necrosis. Endosomes subsequently coalesce around the nucleus and disintegrate during the final stage of necrosis. Interfering with kinesin-mediated endosome trafficking impedes cell death. Endocytosis synergizes with autophagy and lysosomal proteolytic mechanisms to facilitate necrotic neurodegeneration. These findings demonstrate a prominent role for endocytosis in cellular destruction during neurodegeneration, which is likely conserved in metazoans.
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PMID:Endocytosis and intracellular trafficking contribute to necrotic neurodegeneration in C. elegans. 2215 48


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