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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)

Dictyostelium discoideum, a unicellular eukaryote amenable to both biochemical and genetic dissection, provides an attractive system for studying microtubule-based transport. In this work, we have identified microtubule-based motor activities in Dictyostelium cell extracts and have partially purified a protein that induces microtubule translocation along glass surfaces. This protein, which sediments at approximately 9S in sucrose density gradients and is composed of a 105 kd polypeptide, generates anterograde movement along microtubules that is insensitive to 5 mM NEM (N-ethyl-maleimide) but sensitive to 200 microM vanadate, and has similar nucleotide-dependent microtubule binding properties to those of kinesins purified from mammals, sea urchin and Drosophila. This kinesin-like molecule from Dictyostelium, however, is immunologically distinct from bovine and squid neuronal kinesins and supports microtubule movement on glass at four-fold greater velocities (2.0 versus 0.5 microns/sec). Furthermore, AMP-PNP (adenylyl imidodiphosphate), which promotes attachment of previously characterized kinesins to microtubules, decreases the affinity of the Dictyostelium kinesin homolog for microtubules. Thus, an AMP-PNP-induced rigor binding may not be a characteristic of kinesins from lower eukaryotes.
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PMID:Identification of a kinesin-like microtubule-based motor protein in Dictyostelium discoideum. 253 Oct 77

Cytoplasmic dynein is a microtubule-based mechanochemical protein that plays an essential role in cell division, vesicle transport, and cytoplasmic membrane organization. As a molecular motor, dynein utilizes an ATP hydrolysis mechanism to bind and release microtubules and to undergo conformational changes that result in a net displacement towards the microtubule's minus end. To visualize structural features of this motor protein, we have begun to characterize the dynein head domain by electron microscopy and image processing. Transmission electron microscopy of negatively stained native dynein from Dictyostelium has been performed and images of the head domain have been aligned and analyzed with the software SPIDER. The resulting 2D averages show an oblong round shape composed of seven to eight globular domains or lobes that encircle a stain-filled area. A recombinant 380 kDa fragment of the dynein heavy chain encodes just the globular head domain; analysis of these particles reveals a high structural similarity with the native head domain. A prominent stalk can be seen in several projections of this fragment, suggesting a structure analogous to the B-link described for some axonemal dyneins. Single tilt pair images were used to compute low resolution 3D reconstructions of the dynein head domain. These show a flattened spheroidal shape of 13.5 nm in length with seven similar domains arranged in a ring. Slices through the reconstructions reveal a large central cavity. This is the first detailed description of the head domain structure for a dynein molecule. The presence of a central cavity and the outer globular features, along with its large size make dynein structurally distinct from either myosin or kinesin.
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PMID:Structural characterization of a dynein motor domain. 956 97

The cellular slime mold Dictyostelium discoideum is an attractive system for studying the roles of microtubule-based motility in cell development and differentiation. In this work, we report the first molecular characterization of kinesin-related proteins (KRPs) in Dictyostelium. A PCR-based strategy was used to isolate DNA fragments encoding six KRPs, several of which are induced during the developmental program that is initiated by starvation. The complete sequence of one such developmentally regulated KRP (designated K7) was determined and found to be a novel member of the kinesin superfamily. The motor domain of K7 is most similar to that of conventional kinesin, but unlike conventional kinesin, K7 is not predicted to have an extensive alpha-helical coiled-coil domain. The nonmotor domain is unusual and is rich in Asn, Gln, and Thr residues; similar sequences are found in other developmentally regulated genes in Dictyostelium. K7, expressed in Escherichia coli, supports plus end-directed microtubule motility in vitro at a speed of 0.14 micron/s, indicating that it is a bona fide motor protein. The K7 motor is found only in developing cells and reaches a peak level of expression between 12 and 16 h after starvation. By immunofluorescence microscopy, K7 localizes to a membranous perinuclear structure. To examine K7 function, we prepared a null cell line but found that these cells show no gross developmental abnormalities. However, when cultivated in the presence of wild-type cells, the K7-null cells are mostly absent from the prestalk zone of the slug. This result suggests that in a population composed largely of wild-type cells, the absence of the K7 motor protein interferes either with the ability of the cells to localize to the prestalk zone or to differentiate into prestalk cells.
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PMID:A developmentally regulated kinesin-related motor protein from Dictyostelium discoideum. 969 69

Three loop structures called the P-loop, switch I loop and switch II loop of myosin are major components of its ATPase site, and share structural and functional homology with the loop structures in other ATPases and GTPases such as kinesin and G-protein. Using the alanine scanning mutagenesis, structure-function relationship of the switch I and switch II loops in Dictyostelium myosin II was examined. Based on crystal structures of Dictyostelium myosin motor domain, functions of each residue in those loops are discussed.
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PMID:Structure-mutation analysis of the ATPase site of Dictyostelium discoideum myosin II. 994 64

Motor-powered movement along microtubule tracks is important for membrane organization and trafficking. However, the molecular basis for membrane transport is poorly understood, in part because of the difficulty in reconstituting this process from purified components. Using video microscopic observation of organelle transport in vitro as an assay, we have purified two polypeptides (245 and 170 kD) from Dictyostelium extracts that independently reconstitute plus-end-directed membrane movement at in vivo velocities. Both polypeptides were found to be kinesin motors, and the 245-kD protein (DdUnc104) is a close relative of Caenorhabditis elegans Unc104 and mouse KIF1A, neuron-specific motors that deliver synaptic vesicle precursors to nerve terminals. A knockout of the DdUnc104 gene produces a pronounced defect in organelle transport in vivo and in the reconstituted assay. Interestingly, DdUnc104 functions as a dimeric motor, in contrast to other members of this kinesin subfamily, which are monomeric.
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PMID:Reconstitution of membrane transport powered by a novel dimeric kinesin motor of the Unc104/KIF1A family purified from Dictyostelium. 1054 95

Phagocytosis and membrane traffic in general are largely dependent on the cytoskeleton and their associated molecular motors. The myosin family of motors, especially the unconventional myosins, interact with the actin cortex to facilitate the internalization of external materials during the early steps of phagocytosis. Members of the kinesin and dynein motor families, which mediate transport along microtubules (MTs), facilitate the intracellular processing of the internalized materials and the movement of membrane. Recent studies indicate that some unconventional myosins are also involved in membrane transport, and that the MT- and actin-dependent transport systems might interact with each other. Studies in Dictyostelium have led to the discovery of many motors involved in critical steps of phagocytosis and membrane transport. With the ease of genetic and biochemical approaches, the established functional analysis to test phagocytosis and vesicle transport, and the effort of the Dictyostelium cDNA and Genome Projects, Dictyostelium will continue to be a superb model system to study phagocytosis in particular and cytoskeleton and motors in general.
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PMID:Molecular motors and membrane traffic in Dictyostelium. 1125 37

Intracellular organelle transport is driven by motors that act upon microtubules or microfilaments. The microtubulebased motors, cytoplasmic dynein and kinesin, are believed to be responsible for retrograde and anterograde transport of intracellular cargo along microtubules. Many vesicles display bidirectional movement; however, the mechanism regulating directionality is unresolved. Directional movement might be accomplished by alternative binding of different motility factors to the cargo. Alternatively, different motors could associate with the same cargo and have their motor activity regulated. Although several studies have focused on the behavior of specific types of cargoes, little is known about the traffic of the motors themselves and how it correlates with cargo movement. To address this question, we studied cytoplasmic dynein dynamics in living Dictyostelium cells expressing dynein intermediate chain-green fluorescent protein (IC-GFP) fusion in an IC-null background. Dynein-associated structures display fast linear movement along microtubules in both minus-end and plus-end directions, with velocities similar to that of dynein and kinesin-like motors. In addition, dynein puncta often rapidly reverse their direction. Dynein stably associates with cargo moving in both directions as well as with those that rapidly reverse their direction of movement, suggesting that directional movement is not regulated by altering motor-cargo association but rather by switching activity of motors associated with the cargo. These observations suggest that both plus- and minus-end-directed motors associate with a given cargo and that coordinated regulation of motor activities controls vesicle directionality.
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PMID:Cytoplasmic dynein-associated structures move bidirectionally in vivo. 1189 93

Movement of membrane cargoes and chromosomes is driven by kinesin and dynein motors in most eukaryotic cells. In this review, we describe the known kinesin and dynein genes in Dictyostelium. Dictyostelium primarily utilizes two conventional kinesins, an Unc104/KIF1 kinesin, and cytoplasmic dynein to transport membrane organelles within its cytoplasm. We describe how the biological functions of these motors has been dissected through a combination of biochemical to genetic approaches.
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PMID:Kinesin motors and microtubule-based organelle transport in Dictyostelium discoideum. 1295 62

Actin filaments and microtubules are two major cytoskeletal systems involved in wide cellular processes, and the organizations of their filamentous networks are regulated by a large number of associated proteins. Recently, evidence has accumulated for the functional cooperation between the two filament systems via associated proteins. However, little is known about the interactions of the kinesin superfamily proteins, a class of microtubule-based motor proteins, with actin filaments. Here, we describe the identification and characterization of a novel kinesin-related protein named DdKin5 from Dictyostelium. DdKin5 consists of an N-terminal conserved motor domain, a central stalk region, and a C-terminal tail domain. The motor domain showed binding to microtubules in an ATP-dependent manner that is characteristic of kinesin-related proteins. We found that the C-terminal tail domain directly interacts with actin filaments and bundles them in vitro. Immunofluorescence studies showed that DdKin5 is specifically enriched at the actin-rich surface protrusions in cells. Overexpression of the DdKin5 protein affected the organization of actin filaments in cells. We propose that a kinesin-related protein, DdKin5, is a novel actin-bundling protein and a potential cross-linker of actin filaments and microtubules associated with specific actin-based structures in Dictyostelium.
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PMID:A novel actin-bundling kinesin-related protein from Dictyostelium discoideum. 1462 97

UNC-104 (KIF1A) is a kinesin motor that transports synaptic vesicles from the neuronal cell body to the terminal. Previous in vitro studies have shown that a Dictyostelium relative of UNC-104 transports liposomes containing acidic phospholipids, but whether this interaction is needed for the recognition and transport of synaptic vesicles in metazoans remains unexplored. Here, we have introduced mutations in the nonmotor domain of UNC-104 and examined whether these mutant motors can rescue an unc-104 Caenorhabditis elegans strain. We show that a pleckstrin homology (PH) domain in UNC-104 is essential for membrane transport in living C. elegans, that this PH domain binds specifically to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), and that point mutants in the PH domain that interfere with PI(4,5)P(2) binding in vitro also interfere with UNC-104 function in vivo. Several other lipid-binding modules could not effectively substitute for the UNC-104 PH domain in this in vivo assay. Real time imaging also revealed that a lipid-binding point mutation in the PH domain reduced movement velocity and processivity of individual UNC-104::GFP punctae in neurites. These results reveal a critical role for PI(4,5)P(2) binding in UNC-104-mediated axonal transport and shows that the cargo-binding properties of the distal PH domain can affect motor output.
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PMID:The lipid binding pleckstrin homology domain in UNC-104 kinesin is necessary for synaptic vesicle transport in Caenorhabditis elegans. 1515 10


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