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Query: UNIPROT:P06889 (Mol)
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We cloned two genes, KIN1 and KIN2, encoding kinesin-II homologues from the ciliate Tetrahymena thermophila and constructed strains lacking either KIN1 or KIN2 or both genes. Cells with a single disruption of either gene showed partly overlapping sets of defects in cell growth, motility, ciliary assembly, and thermoresistance. Deletion of both genes resulted in loss of cilia and arrests in cytokinesis. Mutant cells were unable to assemble new cilia or to maintain preexisting cilia. Double knockout cells were not viable on a standard medium but could be grown on a modified medium on which growth does not depend on phagocytosis. Double knockout cells could be rescued by transformation with a gene encoding an epitope-tagged Kin1p. In growing cells, epitope-tagged Kin1p preferentially accumulated in cilia undergoing active assembly. Kin1p was also detected in the cell body but did not show any association with the cleavage furrow. The cell division arrests observed in kinesin-II knockout cells appear to be induced by the loss of cilia and resulting cell paralysis.
Mol Biol Cell 1999 Oct
PMID:Kinesin-II is preferentially targeted to assembling cilia and is required for ciliogenesis and normal cytokinesis in Tetrahymena. 1051 52

Several novel members of the kinesin superfamily, until now identified only in plants, are unique in their ability to bind calmodulin in the presence of Ca(2+). Here, we identify the first such kinesin in an animal system. Sequence analysis of this new motor, called kinesin-C, predicts that it is a large carboxy-terminal kinesin, 1624 amino acid residues in length, with a predicted molecular mass of 181 kDa. Kinesin-C is predicted to contain a kinesin motor domain at its carboxy terminus, linked to a segment of alpha-helical coiled-coil 950 amino acid residues long, ending with an amino-terminal proline-rich tail domain. A putative calmodulin-binding domain resides at the extreme carboxy terminus of the motor polypeptide, and recombinant kinesin-C binds to a calmodulin-affinity column in a Ca(2+)-dependent fashion. The presence of this novel calmodulin-binding motor in sea urchin embryos suggests that it plays a critical role in Ca(2+)-dependent events during early sea urchin development.
J Mol Biol 1999 Nov 19
PMID:Identification of kinesin-C, a calmodulin-binding carboxy-terminal kinesin in animal (Strongylocentrotus purpuratus) cells. 1055 23

In axons, organelles move away from (anterograde) and toward (retrograde) the cell body along microtubules. Previous studies have provided compelling evidence that conventional kinesin is a major motor for anterograde fast axonal transport. It is reasonable to expect that cytoplasmic dynein is a fast retrograde motor, but relatively few tests of dynein function have been reported with neurons of intact organisms. In extruded axoplasm, antibody disruption of kinesin or the dynactin complex (a dynein activator) inhibits both retrograde and anterograde transport. We have tested the functions of the cytoplasmic dynein heavy chain (cDhc64C) and the p150(Glued) (Glued) component of the dynactin complex with the use of genetic techniques in Drosophila. cDhc64C and Glued mutations disrupt fast organelle transport in both directions. The mutant phenotypes, larval posterior paralysis and axonal swellings filled with retrograde and anterograde cargoes, were similar to those caused by kinesin mutations. Why do specific disruptions of unidirectional motor systems cause bidirectional defects? Direct protein interactions of kinesin with dynein heavy chain and p150(Glued) were not detected. However, strong dominant genetic interactions between kinesin, dynein, and dynactin complex mutations in axonal transport were observed. The genetic interactions between kinesin and either Glued or cDhc64C mutations were stronger than those between Glued and cDhc64C mutations themselves. The shared bidirectional disruption phenotypes and the dominant genetic interactions demonstrate that cytoplasmic dynein, the dynactin complex, and conventional kinesin are interdependent in fast axonal transport.
Mol Biol Cell 1999 Nov
PMID:Cytoplasmic dynein, the dynactin complex, and kinesin are interdependent and essential for fast axonal transport. 1056 67

The RHO1 gene encodes a yeast homolog of the mammalian RhoA protein. Rho1p is localized to the growth sites and is required for bud formation. We have recently shown that Bni1p is one of the potential downstream target molecules of Rho1p. The BNI1 gene is implicated in cytokinesis and the establishment of cell polarity in Saccharomyces cerevisiae but is not essential for cell viability. In this study, we screened for mutations that were synthetically lethal in combination with a bni1 mutation and isolated two genes. They were the previously identified PAC1 and NIP100 genes, both of which are implicated in nuclear migration in S. cerevisiae. Pac1p is a homolog of human LIS1, which is required for brain development, whereas Nip100p is a homolog of rat p150(Glued), a component of the dynein-activated dynactin complex. Disruption of BNI1 in either the pac1 or nip100 mutant resulted in an enhanced defect in nuclear migration, leading to the formation of binucleate mother cells. The arp1 bni1 mutant showed a synthetic lethal phenotype while the cin8 bni1 mutant did not, suggesting that Bni1p functions in a kinesin pathway but not in the dynein pathway. Cells of the pac1 bni1 and nip100 bni1 mutants exhibited a random distribution of cortical actin patches. Cells of the pac1 act1-4 mutant showed temperature-sensitive growth and a nuclear migration defect. These results indicate that Bni1p regulates microtubule-dependent nuclear migration through the actin cytoskeleton. Bni1p lacking the Rho-binding region did not suppress the pac1 bni1 growth defect, suggesting a requirement for the Rho1p-Bni1p interaction in microtubule function.
Mol Cell Biol 1999 Dec
PMID:Bni1p regulates microtubule-dependent nuclear migration through the actin cytoskeleton in Saccharomyces cerevisiae. 1056 27

We have compared the interaction of ncd (non-claret disjunctional), a kinesin related protein, with microtubules and tubulin heterodimer. Ultracentrifugation experiments revealed that the ncd motor domain, residues 335-700 (ncd335), does not induce tubulin polymerization but stabilizes pre-formed microtubules with a maximum effect at a 1:1 ncd335:tubulin ratio. Ncd335 binding to tubulin or microtubules was estimated by following the change in fluorescence polarization of an exogenous dye attached to Cys670 of ncd335. Ncd335 binding to tubulin (containing GTP or GDP-bound) is characterized by a 2:1 stoichiometry, a higher affinity and an increased sensitivity towards salt, ADP, ATP and AMPPNP, as compared with ncd335 binding to microtubules. Maximum ATPases were 0.06-0.08 sec(-1) and 1.8-2.0 sec(-1) for the ncd335-tubulin and ncd335-microtubules complexes, respectively. Only the polymerized complex is fully functional, suggesting the presence of additional contacts between adjacent protofilaments. Moreover, the data reveal that the oligomeric state of microtubules is a potent regulator for the activity of kinesin related proteins.
J Mol Biol 2000 Jan 14
PMID:Regulation of ncd by the oligomeric state of tubulin. 1062 29

In microtubule (MT) translocation assays, using colloidal gold particles coupled to monoclonal tubulin antibodies to mark positions along MTs, we found that relative motion is possible between the gold particle and an MT, gliding on dynein or kinesin. Such motion evidently occurred by an affinity release and rebinding mechanism that did not require motor activity on the particle. As the MTs moved, particles drifted to the trailing edge of the MT and then were released. Sometimes the particles transferred from one MT to another, moving orthogonally. Although motion of the particles was uniformly rearward, movement was toward the (-) or (+) end of the MT, depending on whether dynein or kinesin, respectively, was used in the assay. These results open possibilities for physiological mechanisms of organelle and other movement that, although dependent on motor-driven microtubule transport, do not require direct motor attachment between the organelle and the microtubule. Our observations on the direction of particle drift and time of release may also provide confirmation in a dynamic system for the conclusion that beta tubulin is exposed at the (+) end of the MT.
Mol Biol Cell 2000 Jan
PMID:Evidence for a novel affinity mechanism of motor-assisted transport along microtubules. 1063 99

Treatment of cultured cells with brefeldin A (BFA) induces the formation of extensive membrane tubules from the Golgi apparatus, trans-Golgi network, and early endosomes in a microtubule-dependent manner. We have reconstituted this transport process in vitro using Xenopus egg cytosol and a rat liver Golgi-enriched membrane fraction. The presence of BFA results in the formation of an intricate, interconnected tubular membrane network, a process that, as in vivo, is inhibited by nocodazole, the H1 anti-kinesin monoclonal antibody, and by membrane pretreatment with guanosine 5'-O-(3-thiotriphosphate). Surprisingly, membrane tubule formation is not due to the action of conventional kinesin or any of the other motors implicated in Golgi membrane dynamics. Two candidate motors of approximately 100 and approximately 130 kDa have been identified using the H1 antibody, both of which exhibit motor properties in a biochemical assay. Finally, BFA-induced membrane tubule formation does not occur in metaphase cytosol, and because membrane binding of both candidate motors is not altered after incubation in metaphase compared with interphase cytosol, these results suggest that either the ATPase or microtubule-binding activity of the relevant motor is cell cycle regulated.
Mol Biol Cell 2000 Mar
PMID:Brefeldin A-dependent membrane tubule formation reconstituted in vitro is driven by a cell cycle-regulated microtubule motor. 1071 11

Null mutations in the Drosophila Kinesin heavy chain gene (Khc), which are lethal during the second larval instar, have shown that conventional kinesin is critical for fast axonal transport in neurons, but its functions elsewhere are uncertain. To test other tissues, single imaginal cells in young larvae were rendered null for Khc by mitotic recombination. Surprisingly, the null cells produced large clones of adult tissue. The rates of cell proliferation were not reduced, indicating that conventional kinesin is not essential for cell growth or division. This suggests that in undifferentiated cells vesicle transport from the Golgi to either the endoplasmic reticulum or the plasma membrane can proceed at normal rates without conventional kinesin. In adult eye clones produced by null founder cells, there were some defects in differentiation that caused mild ultrastructural changes, but they were not consistent with serious problems in the positioning or transport of endoplasmic reticulum, mitochondria, or vesicles. In contrast, defective cuticle deposition by highly elongated Khc null bristle shafts suggests that conventional kinesin is critical for proper secretory vesicle transport in some cell types, particularly ones that must build and maintain long cytoplasmic extensions. The ubiquity and evolutionary conservation of kinesin heavy chain argue for functions in all cells. We suggest interphase organelle movements away from the cell center are driven by multilayered transport mechanisms; that is, individual organelles can use kinesin-related proteins and myosins, as well as conventional kinesin, to move toward the cell periphery. In this case, other motors can compensate for the loss of conventional kinesin except in cells that have extremely long transport tracks.
Mol Biol Cell 2000 Apr
PMID:Clonal tests of conventional kinesin function during cell proliferation and differentiation. 1074 33

The interactions of monomeric and dimeric kinesin and ncd constructs with microtubules have been investigated using cryo-electron microscopy (cryo-EM) and several biochemical methods. There is a good consensus on the structure of dimeric ncd when bound to a tubulin dimer showing one head attached directly to tubulin, and the second head tethered to the first. However, the 3D maps of dimeric kinesin motor domains are still quite controversial and leave room for different interpretations. Here we reinvestigated the microtubule binding patterns of dimeric kinesins by cryo-EM and digital 3D reconstruction under different nucleotide conditions and different motor:tubulin ratios, and determined the molecular mass of motor-tubulin complexes by STEM. Both methods revealed complementary results. We found that the ratio of bound kinesin motor-heads to alphabeta-tubulin dimers was never reaching above 1.5 irrespective of the initial mixing ratios. It appears that each kinesin dimer occupies two microtubule-binding sites, provided that there is a free one nearby. Thus the appearances of different image reconstructions can be explained by non-specific excess binding of motor heads. Consequently, the use of different apparent density distributions for docking the X-ray structures onto the microtubule surface leads to different and mutually exclusive models. We propose that in conditions of stoichiometric binding the two heads of a kinesin dimer separate and bind to different tubulin subunits. This is in contrast to ncd where the two heads remain tightly attached on the microtubule surface. Using dimeric kinesin molecules crosslinked in their neck domain we also found that they stabilize protofilaments axially, but not laterally, which is a strong indication that the two heads of the dimers bind along one protofilament, rather than laterally bridging two protofilaments. A molecular walking model based on these results summarizes our conclusions and illustrates the implications of symmetry for such models.
J Mol Biol 2000 Apr 14
PMID:A new look at the microtubule binding patterns of dimeric kinesins. 1076 75

HIKE is a highly conserved sequence motif identified as a candidate pleckstrin-homology (PH) domain binding site in Gbeta proteins, protein kinases, ankyrin and kinesin. HIKE motifs occur also in gelsolin, neurogranin, neuromodulin and in the PH domain of Bruton tyrosin kinase (BTK). Phosphatidylinositol-binding sequences more distantly related to HIKE are present in gelsolin, in the G protein-coupled receptor kinase 4 and in Trop-2. HIKE regions have been demonstrated to bind both proteins and lipids, and to regulate the interaction of Gbeta, neuromodulin and the BTK PH domain with downstream effectors and the cell membrane. Remarkably, mutations of the HIKE regions are common in diverse human genetic diseases. Several HIKE mutations in protein kinases lead to constitutive activation and cellular transformation, e.g. in MEN-2B, acute myeloid and mast cell leukemias, hereditary papillary renal carcinomas and multiple myeloma. Kinase-inactivating HIKE mutations cause Hirschsprung's disease, piebaldism, insulin resistance and developmental dysplasias. HIKE mutations in the PH domain of BTK lead to X-linked agammaglobulinemia, and different forms of amyloidosis are caused by mutations of HIKE-bearing molecules, for example gelsolin, Ret and Trop-2. Thus, quite diverse genetic diseases might share common molecular mechanisms. These include altered interactions of the mutated molecules with downstream effectors or the cell membrane, and defects in intracellular transport.
Hum Mol Genet 2000 Apr 12
PMID:Large and diverse numbers of human diseases with HIKE mutations. 1076 24


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