Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.6.4.4 (kinesin)
 5,033 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The formation and function of axons depends on the microtubule-based transport of cellular components from their sites of synthesis in the neuronal cell body to their sites of utilization at the axon terminus. To directly visualize this axonal transport in a living organism, we constructed transgenic lines of Caenorhabditis elegans that express green fluorescent protein fused to the monomeric synaptic vesicle transport motor, UNC-104. This UNC-104:: GFP construct rescued the Unc-104 mutant phenotype and was expressed throughout the nervous system. Using time-lapse confocal fluorescence microscopy, we were able to visualize fluorescent motor proteins moving in both directions along neuronal processes, some of which were identified definitely as axons and others as dendrites. Using kymograph analysis, we followed the movement of >900 particles. Most of them moved in one direction, but not necessarily at the same velocity. Ten percent of the observed particles reversed direction of movement during the period of observation, and 10% exhibited periods of movement interspersed with pauses. During episodes of persistent movement, particles moved at an average velocity of 1.02 microm/sec, which is close to the in vitro velocity of microtubule gliding driven by purified monomeric kinesin at high motor density. To our knowledge, this is the first direct visualization and analysis of the movement of specifically labeled microtubule motor proteins along axons in vivo.
 ...
PMID:Direct visualization of the movement of the monomeric axonal transport motor UNC-104 along neuronal processes in living Caenorhabditis elegans. 1135 62

The motility of single one-headed kinesin molecules (K351 and K340), which were truncated fragments of Drosophila two-headed kinesin, has been tested using total internal reflection fluorescence microscopy. One-headed kinesin fragments moved continuously along the microtubules. The maximum distance traveled until the fragments dissociated from the microtubules for both K351 and K340 was approximately 600 nm. This value is considerably larger than the space resolution of the measurement system (SD approximately 30 nm). Although the movements of the fragments fluctuated in forward and backward directions, statistical analysis showed that the average movements for both K340 and K351 were toward the plus end of the microtubules, i.e., forward direction. When BDTC (a 1.3-S subunit of Propionibacterium shermanii transcarboxylase, which binds weakly to a microtubule), was fused to the tail (C-terminus) of K351, its movement was enhanced, smooth, and unidirectional, similar to that of the two-headed kinesin fragment, K411. However, the travel distance and velocity of K351BDTC molecules were approximately 3-fold smaller than that of K411. These observations suggest that a single kinesin head has basal motility, but coordination between the two heads is necessary for stabilizing the basal motility for the normal level of kinesin processivity.
 ...
PMID:Motility of single one-headed kinesin molecules along microtubules. 1160 95

The hand-over-hand stepping mechanism of kinesin at low loads is inadequately understood because the number of molecular steps taken per encounter with the microtubule is difficult to measure: optical traps do not register steps at zero load, while evanescent wave microscopy of single molecules of GFP-kinesin suffers from premature photobleaching. Obtaining low-load data is important because it can efficiently distinguish between alternative proposed mechanisms for molecular walking. We report a novel experiment that records the missing data. We fused kinesin to gelsolin, creating a construct that severs and caps rhodamine-phalloidin actin filaments, setting exactly one kinesin molecule on one end of each fluorescent actin filament. Single kinesin molecules labeled in this way can be tracked easily and definitively using a standard epifluorescence microscope. We use the new system to show that, contrary to a recent report, kinesin run length at low load is independent of ATP concentration in the muM to mM range of ATP concentration. Adding competitor ADP in the presence of saturating ATP decreases both velocity and run length. Based on these data, we propose a simplified model for the mechanism of processive stepping.
 ...
PMID:Direct long-term observation of kinesin processivity at low load. 1186 70

Plant kinesin-like calmodulin-binding protein (KCBP) is a novel member of the kinesin superfamily that interacts with calmodulin (CaM) via its CaM-binding domain (CBD). Activated CaM (Ca(2+)-CaM) has been shown to inhibit KCBP interaction with microtubules (MTs) thereby abolishing its motor- and MT-dependent ATPase activities. To test whether the fusion of CBD to non-CaM-binding kinesins confers Ca(2+)-CaM regulation, we fused the CBD of KCBP to the N or C terminus of a minus-end (non-claret disjunction) or C terminus of a plus-end (Drosophila kinesin) motor. Purified chimeric kinesins bound CaM in a Ca(2+)-dependent manner whereas non-claret disjunction, Drosophila kinesin, and KCBP that lack a CBD did not. As in the case of KCBP with CBD, the interaction of chimeric motors with MTs, as well as their MT-stimulated ATPase activity, was inhibited by Ca(2+)-CaM. The presence of a spacer between the motor and CBD did not alter Ca(2+)-CaM regulation. However, KCBP interaction with MTs and its MT-stimulated ATPase activity were not inhibited when the motor domain and CBD were added separately, suggesting that Ca(2+)-CaM regulation of CaM-binding motors occurs only when the CBD is attached to the motor domain. These results show that the fusion of the CBD to animal motors confers Ca(2+)-CaM regulation and suggest that the CBD functions as a modular domain in disrupting motor-MT interaction. Our data also support the hypothesis that CaM-binding kinesins may have evolved by addition of a CBD to a kinesin motor domain.
 ...
PMID:The calmodulin-binding domain from a plant kinesin functions as a modular domain in conferring Ca2+-calmodulin regulation to animal plus- and minus-end kinesins. 1237 58

Membrane-associated guanylate kinase homologues (MAGUKs) are generally found under the plasma membrane of cell-cell contact sites and function as scaffolding proteins by linking cytoskeletal and signaling molecules to transmembrane receptors. The correct targeting of MAGUKs is essential for their receptor-clustering function; however, the molecular mechanism of their intracellular transport is unknown. Here, we show that the guanylate kinase-like domain of human discs large protein binds directly within the amino acids 607-831 of the stalk domain of GAKIN, a kinesin-like protein of broad distribution. The primary structure of the binding segment, termed MAGUK binding stalk domain, is conserved in Drosophila kinesin-73 and some other motor and non-motor proteins. This stalk segment is not found in GKAP, a synaptic protein that interacts with the guanylate kinase-like domain, and unlike GKAP, the binding of GAKIN is not regulated by the intramolecular interactions within the discs large protein. The recombinant motor domain of GAKIN is an active microtubule-stimulated ATPase with k(cat) = 45 s(-1), K(0.5 (MT)) = 0.1 microm. Overexpression of green fluorescent protein-fused GAKIN in Madin-Darby canine kidney epithelial cells induced long projections with both GAKIN and endogenous discs large accumulating at the tip of these projections. Importantly, the accumulation of endogenous discs large was eliminated when a mutant GAKIN lacking its motor domain was overexpressed under similar conditions. Taken together, our results indicate that discs large is a cargo molecule of GAKIN and suggest a mechanism for intracellular trafficking of MAGUK-laden vesicles to specialized membrane sites in mammalian cells.
 ...
PMID:Direct interaction with a kinesin-related motor mediates transport of mammalian discs large tumor suppressor homologue in epithelial cells. 1249 41

CHO1 is a kinesin-like motor protein essential for cytokinesis in mammalian cells. To analyze how CHO1 functions, we established RNAi and genetic rescue assays. CHO1-depleted cells reached a late stage of cytokinesis but fused back to form binucleate cells because of the absence of the midbody matrix in the middle of the intercellular bridge. Expression of exogenous CHO1 restored the formation of the midbody matrix and rescued cytokinesis in siRNA-treated cells. By analyzing phenotypes rescued with different constructs, it was shown that both motor and stalk domains function in midbody formation, whereas the tail is essential for completion of cytokinesis after the midbody matrix has formed. During the terminal stage of cytokinesis, different subregions of the tail play distinctive roles in stabilizing the midbody matrix and maintaining an association between the midbody and cell cortex. These results demonstrate that CHO1 consists of functionally differentiated subregions that act in concert to ensure complete cell separation.
 ...
PMID:Role of the midbody matrix in cytokinesis: RNAi and genetic rescue analysis of the mammalian motor protein CHO1. 1507 67

Functional studies with ZWICHEL ( ZWI ), which encodes a Ca(2+)-calmodulin-regulated kinesin, have shown its involvement in trichome morphogenesis and cell division. To identify regulatory regions that control the ZWI expression pattern, we generated transgenic Arabidopsis plants with a GUS reporter driven by different lengths of the ZWI gene 5' region alone or 5' and 3' regions together. The 5' fusions contain varying lengths of the coding and non-coding regions of beta - HYDROXYISOBUTYRYL-CoA HYDROLASE 1 ( CHY1 ), which is upstream of ZWI, and a 162 bp intergenic region. In transgenic plants with 5' 460::GUS, GUS activity was observed primarily in the root hairs whereas transgenic plants with an additional 5' 266 bp region from the CHY1 gene (5' 726::GUS) showed strong GUS accumulation in the entire root including root hairs and root tip, calli and at various developmental stages in trichomes and pollen. However, very little GUS accumulation was detected in roots of dark-grown or root tips of cold-treated seedlings with 5' ZWI constructs. These results were further confirmed by quantifying GUS enzyme activity and transcripts in these seedlings. Calli and pollen transformed with the 5' distal 268 bp fused in antisense orientation to the proximal 460 bp did not show GUS expression. Further, IAA-treated dark-grown seedlings with 726::GUS, but not with 460::GUS, showed high GUS expression in specific regions (outer layer 2a cells) at the base of the lateral roots. The ZWI 3' region (3 kb) did not influence the GUS expression pattern driven by the 5' 726 bp. The absence of CHY1 transcripts in the chy1-2 mutant did not alter either ZWI expression or ZWI-mediated trichome morphogenesis. Thus, our data suggest that the 3' part of the CHY1 gene contains regulatory elements that control ZWI gene expression in dividing cells and other cells that exhibit polarized growth such as root hairs, pollen and trichomes. This is the first evidence that the regulatory regions conferring developmental and cell-specific expression of a gene reside in the introns and exons of its upstream protein-coding gene.
 ...
PMID:Developmental and cell-specific expression of ZWICHEL is regulated by the intron and exon sequences of its gene. 1515 28

The rat kinesin motor domain was fused at residues 433, 411, 376 or 367, respectively, to the C-terminal 1185, 1187, 1197 or 1185 residues of the brush border myosin tail. In motility assays, K433myt and K411myt, which preserve the head-proximal kinesin hinge, and K367myt, which deletes it, drove rapid microtubule sliding ( approximately 0.6 microms(-1)) that was optimal when the head-pairs were spaced apart by adding 1:1 headless myosin tails. K376myt, which partially deletes the head-proximal hinge, showed poor motility in sliding assays but wild type processivity, velocity and stall force in single molecule optical trapping. Accordingly, the head-proximal kinesin hinge is functionally dispensable.
 ...
PMID:Kinesin heads fused to hinge-free myosin tails drive efficient motility. 1522 8

Cellular motors (kinesin, dynein and myosin) are ubiquitous. A major task in cell biology is to determine how they function in cells. Here we focus on myosin 10, an intrafilopodial motor, and show how imaging green fluorescent protein fused to myosin 10 or its tail domains can help us understand the function of this myosin.
 ...
PMID:Imaging myosin 10 in cells. 1549 89

The LmjF01.0030 gene of Leishmania major Friedlin, annotated as 'MCAK-like', was confirmed as a kinesin with an internally located motor domain and termed LmjKIN13-1. Both the native form of the protein and a green fluorescent protein (GFP)-fused recombinant version were shown to be exclusively intranuclear, and, more specifically, to localize to the spindle and spindle poles. Cell cycle-dependent regulation of the protein levels was demonstrated using synchronized Leishmania cells: LmjKIN13-1 was highly abundant in the G2+M phase and present at very low levels after mitosis. Altogether, these features suggest that this protein participates in mitosis. The construction of systematic deletion mutants allowed the localization of the primary sequence regions responsible for nuclear targeting on the one hand, and for cell cycle-dependent variations on the other hand. A 42-amino-acid region of the carboxy(C)-terminal domain mediates nuclear import and could be defined as an atypical nuclear localization signal. Protein level regulation during the cell cycle was shown to also depend upon the C-terminal domain, where apparently redundant degradation signals are present. Putative degradation signals appear to be present on both sides and inside the nuclear localization signal. Further experiments strongly suggest a role for the ubiquitin/proteasome pathway in this cell cycle-dependent regulation. These data underline the importance of post-translational regulation of protein abundance in this ancestral eukaryote where transcriptional regulation seems to be rare or near absent.
 ...
PMID:Cell cycle-dependent expression regulation by the proteasome pathway and characterization of the nuclear targeting signal of a Leishmania major Kin-13 kinesin. 1643 Jun 91


<< Previous 1 2 3 4 5 6 Next >>