Kinesin-related cut7 protein associates with mitotic and meiotic spindles in fission yeast.

Several mitotic and meiotic gene products are related to the microtubule motor kinesin, providing insight into the molecular basis of the complex motile events responsible for spindle formation and function. Of these genes, three have been shown to affect spindle structure when mutated. The most severe phenotype is seen in Aspergillus nidulans bimC and Schizosaccharomyces pombe cut7 mutants. In both fungi the intranuclear spindle is bipolar, with microtubules that emanate from spindle pole bodies at either pole, interdigitating in a central overlap zone. In bimC and cut7 mutants, microtubule interdigitation does not appear to take place, instead two unconnected half spindles form and chromosome separation fails. Here we report that cut7 protein concentrates on or near the spindle pole bodies throughout mitotic and meiotic nuclear division and associates with mitotic spindle microtubules in a stage-specific manner, associating with the mid-anaphase B midzone. In cut7ts mutants, spindle pole bodies stain but mitotic microtubules do not.     

A novel genome-wide fulllength kinesin prediction analysis reveals additional mammalian kinesins

Kinesins are microtubule-based motor proteins that perform diverse functions[1―6], including the transloca- tion of vesicles, organelles, chromosomes, proteincomplexes, RNA-binding proteins (RNPs), etc. They also help to orchestrate microtubule dynamics …     

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.     

Mediation of meiotic and early mitotic chromosome segregation in Drosophila by a protein related to kinesin.

Contrary to the traditional view that microtubules pull chromosomes polewards during the anaphase stage of meiotic and mitotic cell divisions, new evidence suggests that the chromosome movements are driven by a motor located at the kinetochore. The process of chromosome segregation involves proper arrangement of kinetochores for spindle attachment, followed by spindle attachment and chromosome movement. Mechanisms in Drosophila for chromosome segregation in meiosis differ in males and females, implying the action of different gene products in the two sexes. A product encoded at the claret locus in Drosophila is required for normal chromosome segregation in meiosis in females and in early mitotic divisions of the embryo. Here we show that the predicted amino-acid sequence of this product is related to the heavy chain of kinesin. The conserved region corresponds to the kinesin motor domain and includes the ATP-binding site and a region that can bind microtubules. A second region contains a leucine repeat motif which may mediate protein-subunit interactions necessary for attachment of chromosomes to the spindle. The mutant phenotype of chromosome nondisjunction and loss, and its similarity to the kinesin ATP-binding domain, suggest that the product encoded at claret not only stabilizes chromosome attachments to the spindle, but may also be a motor that drives chromosome segregation in female meiosis.     

A plus-end-directed motor enzyme that moves antiparallel microtubules in vitro localizes to the interzone of mitotic spindles.

Mitosis comprises a complex set of overlapping motile events, many of which involve microtubule-dependent motor enzymes. Here we describe a new member of the kinesin superfamily. The protein was originally identified as a spindle antigen by the CHO1 monoclonal antibody and shown to be required for mitotic progression. We have cloned the gene that encodes this antigen and found that its sequence contains a domain with strong sequence similarity to the motor domain of kinesin-like proteins. The product of this gene, expressed in bacteria, can cross-bridge antiparallel microtubules in vitro, and in the presence of Mg-ATP, microtubules slide over one another in a fashion reminiscent of microtubule movements during spindle elongation.     

Polarity controls forces governing asymmetric spindle positioning in the Caenorhabditis elegans embryo

Cell divisions that create daughter cells of different sizes are crucial for the generation of cell diversity during animal development. In such asymmetric divisions, the mitotic spindle must be asymmetrically positioned at the end of anaphase. The mechanisms by which cell polarity translates to asymmetric spindle positioning remain unclear. Here we examine the nature of the forces governing asymmetric spindle positioning in the single-cell-stage Caenorhabditis elegans embryo. To reveal the forces that act on each spindle pole, we removed the central spindle in living embryos either physically with an ultraviolet laser microbeam, or genetically by RNA-mediated interference of a kinesin. We show that pulling forces external to the spindle act on the two spindle poles. A stronger net force acts on the posterior pole, thereby explaining the overall posterior displacement seen in wild-type embryos. We also show that the net force acting on each spindle pole is under control of the par genes that are required for cell polarity along the anterior-posterior embryonic axis. Finally, we discuss simple mathematical models that describe the main features of spindle pole behaviour. Our work suggests a mechanism for generating asymmetry in spindle positioning by varying the net pulling force that acts on each spindle pole, thus allowing for the generation of daughter cells with different sizes.     

Molecular cloning, expression and biochemical property analysis of AtKP1, a kinesin gene from Arabidopsis thaliana

Kinesins are common in a variety of eukaryotic cells with diverse functions. A cDNA encoding a member of the Kinesin-14B subfamily is obtained using 3＇-RACE technology and named AtKP1 （for Arabidopsis kinesin protein 1）. This cDNA has a maximum open reading frame of 3.3 kb encoding a polypeptide of 1087 aa. Protein domain analysis shows that AtKP1 contains the motor domain and the calponin homology domain in the central and amino-terminal regions, respectively. The carboxyl-terminal region with 202 aa residues is diverse from other known kinesins. Northern blot analysis shows that AtKP1 is widely expressed at a higher level in seedlings than in mature plants. 2808 bp of the AtKP1 promoter region is cloned and fused to GUS. GUS expression driven by the AtKP1 promoter region shows that AtKP1 is mainly expressed in vasculature of young organs and young leaf trichomes, indicating that AtKP1 may participate in the differentiation or development of Arabidopsis thaliana vascular bundles and trichomes. A truncated AtKP1 protein containing the putative motor domain is expressed in E. coil and affinity-purified. In vitro characterizations indicate that the polypeptide has nucleotide-dependent microtubule-binding ability and microtubule-stimulated ATPase activity.     

Suppression of a myosin defect by a kinesin-related gene.

Motor proteins in cells include myosin, which is actin-based, and kinesin, dynein and dynamin, which are microtubule-based. Several proteins have recently been identified that have amino-acid sequences with similarity to the motor domains of either myosin or kinesin, but are otherwise dissimilar. This has led to the suggestion that these may all be motor proteins, but that they are specialized for moving different cargos. Genetic analysis can address the question of the different functions of these new proteins. Studies of a temperature-sensitive mutation (myo2-66) in a gene of the myosin superfamily (MYO2) have implicated the Myo2 protein (Myo2p) in the process of polarized secretion in yeast (Saccharomyces cerevisiae). To understand more about the role of Myo2p, we have looked for 'multicopy suppressors' (heterologous genes that, when overexpressed, can correct the temperature sensitivity of the myo2-66 mutant). Here we report the identification of such a suppressor (SMY1) that (surprisingly) encodes a predicted polypeptide sharing sequence similarity with the motor portion of proteins in the kinesin superfamily.     

Switch-based mechanism of kinesin motors

Kinesin motors are specialized enzymes that use hydrolysis of ATP to generate force and movement along their cellular tracks, the microtubules. Although numerous biochemical and biophysical studies have accumulated much data that link microtubule-assisted ATP hydrolysis to kinesin motion, the structural view of kinesin movement remains unclear. This study of the monomeric kinesin motor KIF1A combines X-ray crystallography and cryo-electron microscopy, and allows analysis of force-generating conformational changes at atomic resolution. The motor is revealed in its two functionally critical states-complexed with ADP and with a non-hydrolysable analogue of ATP. The conformational change observed between the ADP-bound and the ATP-like structures of the KIF1A catalytic core is modular, extends to all kinesins and is similar to the conformational change used by myosin motors and G proteins. Docking of the ADP-bound and ATP-like crystallographic models of KIF1A into the corresponding cryo-electron microscopy maps suggests a rationale for the plus-end directional bias associated with the kinesin catalytic core.     

Two mitotic kinesins cooperate to drive sister chromatid separation during anaphase

During anaphase identical sister chromatids separate and move towards opposite poles of the mitotic spindle. In the spindle, kinetochore microtubules have their plus ends embedded in the kinetochore and their minus ends at the spindle pole. Two models have been proposed to account for the movement of chromatids during anaphase. In the 'Pac-Man' model, kinetochores induce the depolymerization of kinetochore microtubules at their plus ends, which allows chromatids to move towards the pole by 'chewing up' microtubule tracks. In the 'poleward flux' model, kinetochores anchor kinetochore microtubules and chromatids are pulled towards the poles through the depolymerization of kinetochore microtubules at the minus ends. Here, we show that two functionally distinct microtubule-destabilizing KinI kinesin enzymes (so named because they possess a kinesin-like ATPase domain positioned internally within the polypeptide) are responsible for normal chromatid-to-pole motion in Drosophila. One of them, KLP59C, is required to depolymerize kinetochore microtubules at their kinetochore-associated plus ends, thereby contributing to chromatid motility through a Pac-Man-based mechanism. The other, KLP10A, is required to depolymerize microtubules at their pole-associated minus ends, thereby moving chromatids by means of poleward flux.     

Alp7/TACC is a crucial target in Ran-GTPase-dependent spindle formation in fission yeast

Microtubules are essential intracellular structures involved in several cellular phenomena, including polarity establishment and chromosome segregation. Because the nuclear envelope persists during mitosis (closed mitosis) in fission yeast (Schizosaccharomyces pombe), cytoplasmic microtubules must be reorganized into the spindle in the compartmentalized nucleus on mitotic entry. An ideal mechanism might be to take advantage of an evolutionarily conserved microtubule formation system that uses the Ran-GTPase nuclear transport machinery, but no targets of Ran for spindle formation have been identified in yeast. Here we show that a microtubule-associated protein, Alp7, which forms a complex with Alp14, is a target of Ran in yeast for spindle formation. The Ran-deficient pim1 mutant (pim1-F201S) failed to show mitosis-specific nuclear accumulation of Alp7. Moreover, this mutant exhibited compromised spindle formation and early mitotic delay. Importantly, these defects were suppressed by Alp7 that was artificially targeted to the nucleus by a Ran-independent and importin-alpha-mediated system. Thus, Ran targets Alp7-Alp14 to achieve nuclear spindle formation, and might differentiate its targets depending on whether the organism undergoes closed or open mitosis.     

Myosin VI is an actin-based motor that moves backwards.

Myosins and kinesins are molecular motors that hydrolyse ATP to track along actin filaments and microtubules, respectively. Although the kinesin family includes motors that move towards either the plus or minus ends of microtubules, all characterized myosin motors move towards the barbed (+) end of actin filaments. Crystal structures of myosin II (refs 3-6) have shown that small movements within the myosin motor core are transmitted through the 'converter domain' to a 'lever arm' consisting of a light-chain-binding helix and associated light chains. The lever arm further amplifies the motions of the converter domain into large directed movements. Here we report that myosin VI, an unconventional myosin, moves towards the pointed (-) end of actin. We visualized the myosin VI construct bound to actin using cryo-electron microscopy and image analysis, and found that an ADP-mediated conformational change in the domain distal to the motor, a structure likely to be the effective lever arm, is in the opposite direction to that observed for other myosins. Thus, it appears that myosin VI achieves reverse-direction movement by rotating its lever arm in the opposite direction to conventional myosin lever arm movement.     

The Drosophila claret segregation protein is a minus-end directed motor molecule

A product encoded at the claret locus in Drosophila is needed for normal chromosome segregation in meiosis in females and in early mitotic divisions of the embryo. The predicted amino-acid sequence of the segregation protein was shown recently to be strikingly similar to Drosophila kinesin heavy chain. We have expressed the claret segregation protein in bacteria and have found that the bacterially expressed protein has motor activity in vitro with several novel features. The claret motor is slow (4 microns min-1), unlike either kinesin or dyneins. It has the directionality, the ability to generate torque and the sensitivity to inhibitors reported previously for dyneins. The finding of minus-end directed motor activity for a protein with sequence similarity to kinesin suggests that the dynein and kinesin motor domains are ancestrally related. The minus-end directed motor activity of the claret motor is consistent with a role for this protein in producing chromosome movement along spindle microtubules during prometaphase and/or anaphase.     

ESCRT-III recognition by VPS4 ATPases

The ESCRT (endosomal sorting complex required for transport) pathway is required for terminal membrane fission events in several important biological processes, including endosomal intraluminal vesicle formation, HIV budding and cytokinesis. VPS4 ATPases perform a key function in this pathway by recognizing membrane-associated ESCRT-III assemblies and catalysing their disassembly, possibly in conjunction with membrane fission. Here we show that the microtubule interacting and transport (MIT) domains of human VPS4A and VPS4B bind conserved sequence motifs located at the carboxy termini of the CHMP1-3 class of ESCRT-III proteins. Structures of VPS4A MIT-CHMP1A and VPS4B MIT-CHMP2B complexes reveal that the C-terminal CHMP motif forms an amphipathic helix that binds in a groove between the last two helices of the tetratricopeptide-like repeat (TPR) of the VPS4 MIT domain, but in the opposite orientation to that of a canonical TPR interaction. Distinct pockets in the MIT domain bind three conserved leucine residues of the CHMP motif, and mutations that inhibit these interactions block VPS4 recruitment, impair endosomal protein sorting and relieve dominant-negative VPS4 inhibition of HIV budding. Thus, our studies reveal how the VPS4 ATPases recognize their CHMP substrates to facilitate the membrane fission events required for the release of viruses, endosomal vesicles and daughter cells.     

The orthodenticle gene is regulated by bicoid and torso and specifies Drosophila head development

In the Drosophila embryo, cell fate along the anterior-posterior axis is determined by maternally expressed genes. The activity of the bicoid (bcd) gene is required for the development of larval head and thoracic structures, and that of maternal torso (tor) for the development of the unsegmented region of the head (acron). In contrast to the case of thoracic and abdominal segmentation, the hierarchy of zygotically expressed genes controlling head development has not been clearly defined. The bcd protein, which is expressed in a gradient, activates zygotic expression of the gap gene hunchback (hb), but hb alone is not sufficient to specify head development. Driever et al. proposed that at least one other bcd-activated gene controls the development of head regions anterior to the hb domain. We report here that the homeobox gene orthodenticle (otd), which is involved in head development, could be such a gene. We also show that otd expression responds to the activity of the maternal tor gene at the anterior pole of the embryo.