Kinesin Motor Domain Research Articles

A Change in the Selective Translocation of the Kinesin-1 Motor Domain Marks the Initial Specification of the Axon

We used the accumulation of constitutively active kinesin motor domains as a measure of where kinesins translocate in developing neurons. Throughout development, truncated Kinesin-3 accumulates at the tips of all neurites. In contrast, Kinesin-1 selectively accumulates in only a subset of neurites. Before neurons become polarized, truncated Kinesin-1 accumulates transiently in a single neurite. Coincident with axon specification, truncated Kinesin-1 accumulates only in the emerging axon and no longer appears in any other neurite. The translocation of Kinesin-1 along a biochemically distinct track leading to the nascent axon could ensure the selective delivery of Kinesin-1 cargoes to the axon and hence contribute to its molecular specification. Imaging YFP-tagged truncated Kinesin-1 provides the most precise definition to date of when neuronal polarity first emerges and allows visualization of the molecular differentiation of the axon in real time.

Mutation in KIF5A can also cause adult-onset hereditary spastic paraplegia

Autosomal dominant hereditary spastic paraplegia (AD HSP) linked to chromosome 12q (SPG10) is caused by mutations in the neuronal kinesin heavy-chain KIF5A gene. This is a rare cause of AD HSP, and only two disease-causing mutations have been reported thus far. In both instances, affected individuals harboring mutations in the KIF5A gene displayed symptom onset at a very early age. Here we present the results of clinical and genetic analyses of a large kindred with uncomplicated AD HSP. We were able to establish a definitive linkage to the SPG10 locus, and sequencing of the KIF5A gene revealed a heterozygous missense mutation 1,035 A>G in exon 10, resulting in tyrosine-to-cysteine substitution. This mutation is located in a highly conserved kinesin motor domain of the neuronal kinesin heavy-chain protein, but in contrast to two previously reported missense mutations, the age of symptom onset in our family was much later, with an average age of 36.1+/-4 years. Our results demonstrate that mutations in the KIF5A gene can also be associated with an adult age of onset of AD HSP.

Interaction of kinesin motors, microtubules, and MAPs

Kinesins are a family of microtubule-dependent motor proteins that carry cargoes such as vesicles, organelles, or protein complexes along microtubules. Here we summarize structural studies of the "conventional" motor protein kinesin-1 and its interactions with microtubules, as determined by X-ray crystallography and cryo-electron microscopy. In particular, we consider the docking between the kinesin motor domain and tubulin subunits and summarize the evidence that kinesin binds mainly to beta tubulin with the switch-2 helix close to the intradimer interface between alpha and beta tubulin.

Structural analysis of the ZEN-4/CeMKLP1 motor domain and its interaction with microtubules

The centralspindlin complex is required for the assembly and maintenance of the central spindle during late anaphase and the completion of cytokinesis. It is composed of two copies each of the kinesin-like protein ZEN-4, a Caenorhabditis elegans MKLP-1 (Kinesin-6 family), and the RhoGAP CYK-4. By using cryo-electron microscopy and helical 3D reconstruction, we are investigating the structural features of the interactions between monomeric and dimeric motor domain constructs of ZEN-4 and microtubules. We have calculated helically averaged 3D maps of microtubules decorated with ZEN-4 motor domain in the presence of AMP-PNP, ADP, ADP-AlF 4 −, and nucleotide-free conditions. We used statistical difference mapping to compare these maps among each other and to related maps obtained from microtubules decorated with a well-characterized Kinesin-1 motor domain from Neurospora crassa. Thereby, we found distinct structural features in microtubule–ZEN-4 complexes that may directly relate to the functional properties of ZEN-4 and centralspindlin. Furthermore, we investigated the location, structure, and function of a highly conserved extension of ∼50 residues unique to the Kinesin-6 subfamily, located in the motor core loop6/β4 region.

Cik1 Targets the Minus-End Kinesin Depolymerase Kar3 to Microtubule Plus Ends

Kar3, a Saccharomyces cerevisiae Kinesin-14, is essential for karyogamy and meiosis I but also has specific functions during vegetative growth. For its various roles, Kar3 forms a heterodimer with either Cik1 or Vik1, both of which are noncatalytic polypeptides. Here, we present the first biochemical characterization of Kar3Cik1, the kinesin motor that is essential for karyogamy. Kar3Cik1 depolymerizes microtubules from the plus end and promotes robust minus-end-directed microtubule gliding. Immunolocalization studies show that Kar3Cik1 binds preferentially to one end of the microtubule, whereas the Kar3 motor domain, in the absence of Cik1, exhibits significantly higher microtubule lattice binding. Kar3Cik1-promoted microtubule depolymerization requires ATP turnover, and the kinetics fit a single exponential function. The disassembly mechanism is not microtubule catastrophe like that induced by the MCAK Kinesin-13s. Soluble tubulin does not activate the ATPase activity of Kar3Cik1, and there is no evidence of Kar3Cik1(.)tubulin complex formation as observed for MCAK. These results reveal a novel mechanism to regulate microtubule depolymerization. We propose that Cik1 targets Kar3 to the microtubule plus end. Kar3Cik1 then uses its minus-end-directed force to depolymerize microtubules from the plus end, with each tubulin-subunit release event tightly coupled to one ATP turnover.

Mitotic Kinesins: Prospects for Antimitotic Drug Discovery

Kinesins, mechanochemical enzymes that utilize the energy of ATP to translocate along or destabilize microtubules, are essential for accurate completion of cell division. Recently, small moleculer inhibitors of one kinesin, kinesin spindle protein (KSP/Eg5/kinesin5), have been shown to be efficacious in pre-clinical studies, with one quinazolinone-based inhibitor advancing to Phase II clinical trials as a potential anticancer chemotherapeutic agent. This highlights the potential of KSP and other mitotic kinesins as targets for chemotherapeutic intervention. Ten other kinesins have been shown to play essential roles in cell division and thus may provide additional therapeutic opportunities. In this review, the biological roles of these proteins are described with emphasis on their importance to cell proliferation. In addition, kinesin motor domain structure and mechanism are described with particular attention given to the conformational changes that offer opportunities for chemical inhibition. Finally, a current list of KSP inhibitor classes is described in the context of their potential as clinical leads.

KIF27 is one of orthologs for Drosophila Costal-2

Signals of Hedgehog family proteins (SHH, IHH and DHH) are transduced through Patched family receptors (PTCH1 and PTCH2) and Smoothened (SMO) to GLI family transcription factors (GLI1, GLI2 and GLI3). SHH plays a key role in development and progression of pancreatic cancer, gastric cancer, basal cell carcinoma, and brain tumors. Drosophila Costal-2 (Cos2) is implicated in the Hedgehog pathway through the interaction with Smoothened (Smo), Cubitus interruptus (Ci), Fused (Fu), and microtubule; however, mammalian ortholog of Drosophila Cos2 remained to be identified. Here we identified and characterized human ortholog of Drosophila Cos2 by using bioinformatics. Full-length Drosophila Cos2 was most homologous to human KIF27, followed by mouse Kif7, and other KIF family members. KIF27 gene at human chromosome 9q22.1 and KIF7 gene at human chromosome 15q26.1 were paralogs within the human genome. Phylogenetic analysis revealed that KIF27, Kif7, KIF4A, KIF4B and KIF21A constitute the KIF27 subfamily among mammalian Kinesin family. Drosophila Cos2 protein consists of Kinesin motor (KISc) domain, Ci-binding domain, and Smo-binding domain. KIF27 itself shared the common domain structure with Drosophila Cos2, while other members of KIF27 subfamily shared partial domain structure with Drosophila Cos2. These facts indicate that KIF27 is one of mammalian orthologs for Drosophila Cos2.

Cytoskeletal Motors in Arabidopsis. Sixty-One Kinesins and Seventeen Myosins

Cytoskeletal motor proteins are ATPases that use the energy released from ATP hydrolysis to move along the cytoskeletal elements of microtubules and actin microfilaments. Found among all eukaryotic organisms, kinesins are microtubule-based motor proteins with a conserved kinesin motor domain, and myosins are actin microfilament-based motor proteins with a conserved myosin motor domain. Cytoskeletal motor proteins directly contribute to the organization of various cytoskeletal arrays during cell division and cell growth in plant tissues. They are also responsible for the motility of molecules and organelles, and the segregation of genetic materials during mitosis and meiosis. In the genome of the model plant Arabidopsis (Arabidopsis thaliana), there are at least 61 genes encoding kinesins and 17 genes encoding myosins. Most Arabidopsis kinesins and all myosins are evolutionarily divergent from their counterparts in animals and fungi. Little is known about the functions of most plant kinesins and myosins. Arabidopsis kinesins form a number of subfamilies. The mitotic kinesins in the BIMC/Kinesin-5 and the NCD/Kinesin-14 subfamilies appear to be similar to those in fungi and animals. Others, however, are very divergent, as their nonmotor sequences are unique to plants. Some of Arabidopsis kinesins are associated with microtubules, mitochondria, Golgi stacks, and vesicles. They affect microtubule organization, organelle distribution, and vesicle transport, respectively. Ultimately, Arabidopsis kinesins contribute directly or indirectly to cell division and cell growth in various tissues. Arabidopsis myosins are classified into two subfamilies: class VIII and class XI. The class XI myosins are associated with various organelles/vesicles. Functions of Arabidopsis myosins are still elusive. Future efforts will be devoted to deciphering not only the functions of these motors by molecular genetics but also the molecular mechanisms underlying how these roles are played.

Cell cycle regulation of central spindle assembly.

The bipolar mitotic spindle is responsible for segregating sister chromatids at anaphase. Microtubule motor proteins generate spindle bipolarity and enable the spindle to perform mechanical work. A major change in spindle architecture occurs at anaphase onset when central spindle assembly begins. This structure regulates the initiation of cytokinesis and is essential for its completion. Central spindle assembly requires the centralspindlin complex composed of the Caenorhabditis elegans ZEN-4 (mammalian orthologue MKLP1) kinesin-like protein and the Rho family GAP CYK-4 (MgcRacGAP). Here we describe a regulatory mechanism that controls the timing of central spindle assembly. The mitotic kinase Cdk1/cyclin B phosphorylates the motor domain of ZEN-4 on a conserved site within a basic amino-terminal extension characteristic of the MKLP1 subfamily. Phosphorylation by Cdk1 diminishes the motor activity of ZEN-4 by reducing its affinity for microtubules. Preventing Cdk1 phosphorylation of ZEN-4/MKLP1 causes enhanced metaphase spindle localization and defects in chromosome segregation. Thus, phosphoregulation of the motor domain of MKLP1 kinesin ensures that central spindle assembly occurs at the appropriate time in the cell cycle and maintains genomic stability.

Kinesin heads fused to hinge-free myosin tails drive efficient motility

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 (∼0.6 μms−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.

Development of high-throughput screens for discovery of kinesin adenosine triphosphatase modulators

Kinesins are a group of related molecular motor proteins that have great potential as targets for antimitotic drug development. We have developed two novel assays, one end-point and one kinetic, that are useful for the discovery and optimization of kinesin modulators. Both assays measure inorganic phosphate (Pi) generated by microtubule-activated kinesin adenosine triphosphatase activity. The assays were validated using the mitotic Eg5 kinesin-specific inhibitor, monastrol. A panel of nine kinesin motor domain proteins, representing 8 of the 14 classes of kinesins, was screened. The coefficient of variation for both assays was determined to be 4–14% depending on the panel member. Using the Eg5 kinetic assay with monastrol the IC50 value was 12 μM, which agrees well with previously published results. Two other closely related mitotic kinesins (AnBimC and MKLP1) were found to have IC50 values in the millimolar range. The other panel members (kinesin heavy chain, chromokinesin KIF4A, KIF3C, CENP-E, MCAK, and KIFC3) were not significantly inhibited by millimolar levels of monastrol. It is anticipated that screening of the nine-member panel of kinesins in these assays will serve as a platform for the discovery and development of specific kinesin modulators.

Microtubule−Kinesin Interface Mutants Reveal a Site Critical for Communication

Strict coordination of the two motor domains of kinesin is required for driving the processive movement of organelles along microtubules. Glutamate 164 of the kinesin heavy chain was shown to be critical for kinesin function through in vivo genetics in Drosophila melanogaster. The mutant motor E164K exhibited reduced steady-state ATPase activity and higher affinity for both ATP and microtubules. Moreover, an alanine substitution at this position (E164A) caused similar defects. It became stalled on the microtubule and was unable to bind and hydrolyze ATP at the second motor domain. Glu(164), which has been conserved through evolution, is located at the motor-microtubule interface close to key residues on helix alpha12 of beta-tubulin. We explored further the contributions of Glu(164) to motor function using several site-directed mutant proteins: E164K, E164N, E164D, E164Q, and D165A. The results indicate that the microtubule-E164K complex can only bind and hydrolyze one ATP. ATP with increased salt was able to dissociate a population of E164K motors from the microtubule but could not dissociate E164A. We tested the basis of the stabilized microtubule interaction with E164K, E164N, and E164A. The results provide new insights about the motor-microtubule interface and the pathway of communication for processive motility.

3P183 特徴的ループL5に変異を与えたキネシンの性質(分子モーター)

3P183 特徴的ループL5に変異を与えたキネシンの性質(分子モーター)

The Kinesin Family Member BimC Contains a Second Microtubule Binding Region Attached to the N terminus of the Motor Domain

The kinesin family member BimC has a highly positively charged domain of approximately 70 amino acids at the N terminus of the motor domain. Motor domain constructs of BimC were prepared with and without this extra domain to determine its influence. The level of microtubules needed for half saturation of the ATPase of BimC motor domain constructs is reduced by approximately 7000-fold at low ionic strength upon addition of this extra N-terminal extension. Although the change in microtubule affinity is less at higher salt, addition of the N-terminal domain still produces a 20-fold increase in affinity for microtubules in 200 mm potassium acetate. A fusion protein of the N-terminal domain and thioredoxin binds tightly to MTs at low salt, consistent with the increased affinity of motor domain constructs (which contain the N-terminal domain) being due to the additional binding of the N-terminal domain to the microtubule. Hydrodynamic analysis indicates that the N-terminal extension is in a highly extended conformation, suggesting that it may be intrinsically disordered. Fusion of the N-terminal extension of BimC onto the motor domain of conventional kinesin produces a similar large increase in microtubule affinity without significant reduction in kcat or velocity in an in vitro motility assay, suggesting that the N-terminal extension can act in a modular manner to increase the microtubule affinity of kinesin motor domains without a decrease in velocity.

Complex Formation with Kinesin Motor Domains Affects the Structure of Microtubules

Microtubules are highly dynamic components of the cytoskeleton. They are important for cell movement and they are involved in a variety of transport processes together with motor proteins, such as kinesin. The exact mechanism of these transport processes is not known and so far the focus has been on structural changes within the motor domains, but not within the underlying microtubule structure. Here we investigated the interaction between kinesin and tubulin and our experimental data show that microtubules themselves are changing structure during that process. We studied unstained, vitrified samples of microtubules composed of 15 protofilaments using cryo electron microscopy and helical image analysis. 3D maps of plain microtubules and microtubules decorated with kinesin have been reconstructed to ∼17 Å resolution. The αβ-tubulin dimer could be identified and, according to our data, α- and β-tubulin adopt different conformations in plain microtubules. Significant differences were detected between maps of plain microtubules and microtubule–kinesin complexes. Most pronounced is the continuous axial inter-dimer contact in the microtubule–kinesin complex, suggesting stabilized protofilaments along the microtubule axis. It seems, that mainly structural changes within α-tubulin are responsible for this observation. Lateral effects are less pronounced. Following our data, we believe, that microtubules play an active role in intracellular transport processes through modulations of their core structure.

Identification and phylogenetic analysis of Dictyostelium discoideum kinesin proteins.

BackgroundKinesins constitute a large superfamily of motor proteins in eukaryotic cells. They perform diverse tasks such as vesicle and organelle transport and chromosomal segregation in a microtubule- and ATP-dependent manner. In recent years, the genomes of a number of eukaryotic organisms have been completely sequenced. Subsequent studies revealed and classified the full set of members of the kinesin superfamily expressed by these organisms. For Dictyostelium discoideum, only five kinesin superfamily proteins (Kif's) have already been reported.ResultsHere, we report the identification of thirteen kinesin genes exploiting the information from the raw shotgun reads of the Dictyostelium discoideum genome project. A phylogenetic tree of 390 kinesin motor domain sequences was built, grouping the Dictyostelium kinesins into nine subfamilies. According to known cellular functions or strong homologies to kinesins of other organisms, four of the Dictyostelium kinesins are involved in organelle transport, six are implicated in cell division processes, two are predicted to perform multiple functions, and one kinesin may be the founder of a new subclass.ConclusionThis analysis of the Dictyostelium genome led to the identification of eight new kinesin motor proteins. According to an exhaustive phylogenetic comparison, Dictyostelium contains the same subset of kinesins that higher eukaryotes need to perform mitosis. Some of the kinesins are implicated in intracellular traffic and a small number have unpredictable functions.

Configuration of the two kinesin motor domains during ATP hydrolysis.

To understand the mechanism of kinesin movement we have investigated the relative configuration of the two kinesin motor domains during ATP hydrolysis using fluorescence polarization microscopy of ensemble and single molecules. We found that: (i) in nucleotide states that induce strong microtubule binding, both motor domains are bound to the microtubule with similar orientations; (ii) this orientation is maintained during processive motion in the presence of ATP; (iii) the neck-linker region of the motor domain has distinct configurations for each nucleotide condition tested. Our results fit well with a hand-over-hand type movement mechanism and suggest how the ATPase cycle in the two motor domains is coordinated. We propose that the motor neck-linker domain configuration controls ADP release.

Stepping and Stretching: HOW KINESIN USES INTERNAL STRAIN TO WALK PROCESSIVELY

The ability of kinesin to travel long distances on its microtubule track without dissociating has led to a variety of models to explain how this remarkable degree of processivity is maintained. All of these require that the two motor domains remain enzymatically "out of phase," a behavior that would ensure that, at any given time, one motor is strongly attached to the microtubule. The maintenance of this coordination over many mechanochemical cycles has never been explained, because key steps in the cycle could not be directly observed. We have addressed this issue by applying several novel spectroscopic approaches to monitor motor dissociation, phosphate release, and nucleotide binding during processive movement by a dimeric kinesin construct. Our data argue that the major effect of the internal strain generated when both motor domains of kinesin bind the microtubule is to block ATP from binding to the leading motor. This effect guarantees the two motor domains remain out of phase for many mechanochemical cycles and provides an efficient and adaptable mechanism for the maintenance of processive movement.

Repeat motifs of tau bind to the insides of microtubules in the absence of taxol.

The tau family of microtubule-associated proteins has a microtubule-binding domain which includes three or four conserved sequence repeats. Pelleting assays show that when tubulin and tau are co- assembled into microtubules, the presence of taxol reduces the amount of tau incorporated. In the absence of taxol, strong binding sites for tau are filled by one repeat motif per tubulin dimer; additional tau molecules bind more weakly. We have labelled a repeat motif with nanogold and used three-dimensional electron cryomicroscopy to compare images of microtubules assembled with labelled or unlabelled tau. With kinesin motor domains bound to the microtubule outer surface to distinguish between alpha- and beta-tubulin, we show that the gold label lies on the inner surface close to the taxol binding site on beta-tubulin. Loops within the repeat motifs of tau have sequence similarity to an extended loop which occupies a site in alpha-tubulin equivalent to the taxol-binding pocket in beta-tubulin. We propose that loops in bound tau stabilize microtubules in a similar way to taxol, although with lower affinity so that assembly is reversible.

分子モーターキネシンの特徴的ループL5の役割

分子モーターキネシンの特徴的ループL5の役割