Motor activity and mitotic spindle localization of the Drosophila kinesin-like protein KLP61F.

The KLP61F gene product is essential for Drosophila development. Mutations in KLP61F display a mitotic arrest phenotype caused by a failure in the proper separation of duplicated centrosomes (Heck et al., 1993). Sequence analysis of KLP61F identified it as a member of the bimC family of kinesin-like microtubule motor proteins. Here we report that KLP61F is distinct from KRP130, a kinesin-like protein recently purified from Drosophila embryos and suggested to be the product of the KLP61F gene (Cole et al., 1994). We also characterized recombinant KLP61F and found that it possesses microtubule-stimulated ATPase and microtubule translocation activities in vitro. In addition, we have used an affinity-purified, KLP61F-specific antiserum to localize native KLP61F and an epitope-tagged KLP61F fusion protein during various stages of mitosis in Drosophila syncytial blastoderm embryos. From early prophase through anaphase, KLP61F is coincident with the distribution of tubulin. Together these results confirm the existence of multiple bimC-like kinesins in Drosophila and suggest that KLP61F function is intrinsic to the mitotic spindle.     

Mutation of a gene that encodes a kinesin-like protein blocks nuclear division in A. nidulans

In A. nidulans, the temperature-sensitive cell cycle mutation bimC4 causes an elevated mitotic index at restrictive temperature. Under restrictive conditions the mutation interferes with separation of the spindle pole bodies, causes abnormal spindle morphology, and prevents nuclear division. We have cloned and sequenced the wild-type bimC gene. The predicted protein product has homology to Drosophila kinesin heavy chain. We conclude that this kinesin-like protein has an important role in nuclear division in Aspergillus.     

Functional coordination of three mitotic motors in Drosophila embryos

It is well established that multiple microtubule-based motors contribute to the formation and function of the mitotic spindle, but how the activities of these motors interrelate remains unclear. Here we visualize spindle formation in living Drosophila embryos to show that spindle pole movements are directed by a temporally coordinated balance of forces generated by three mitotic motors, cytoplasmic dynein, KLP61F, and Ncd. Specifically, our findings suggest that dynein acts to move the poles apart throughout mitosis and that this activity is augmented by KLP61F after the fenestration of the nuclear envelope, a process analogous to nuclear envelope breakdown, which occurs at the onset of prometaphase. Conversely, we find that Ncd generates forces that pull the poles together between interphase and metaphase, antagonizing the activity of both dynein and KLP61F and serving as a brake for spindle assembly. During anaphase, however, Ncd appears to have no effect on spindle pole movements, suggesting that its activity is down-regulated at this time, allowing dynein and KLP61F to drive spindle elongation during anaphase B.     

Tyrosines in the Kinesin-5 Head Domain Are Necessary for Phosphorylation by Wee1 and for Mitotic Spindle Integrity

Mitotic spindle assembly and maintenance relies on kinesin-5 motors that act as bipolar homotetramers to crosslink microtubules [1], [2], [3], [4] and [5]. Kinesin-5 motors have been the subject of extensive structure-function analysis [5], but the regulation of their activity in the context of mitotic progression remains less well understood [2]. We report here that Drosophila kinesin-5 (KLP61F) is regulated by Drosophila Wee1 (dWee1). Wee1 tyrosine kinases are known to regulate mitotic entry via inhibitory phosphorylation of Cdk1 [6], [7], [8], [9] and [10]. Recently, we showed that dWee1 also plays a role in mitotic spindle positioning through γ-tubulin and spindle fidelity through an unknown mechanism [11]. Here, we investigated whether a KLP61F-dWee1 interaction could explain the latter role of dWee1. We found that dWee1 phosphorylates KLP61F in vitro on three tyrosines within the head domain, the catalytic region that mediates movement along microtubules. In vivo, KLP61F with tyrosine→phenylalanine mutations fails to complement a klp61f mutant and dominantly induces spindle defects similar to ones seen in dwee1 mutants. We propose that phosphorylation of the KLP61F catalytic domain by dWee1 is important for the motor's function. This study identifies a second substrate for a Wee1 kinase and provides evidence for phosphoregulation of a kinesin in the head domain.     

Dynamic partitioning of mitotic kinesin-5 cross-linkers between microtubule-bound and freely diffusing states

The dynamic behavior of homotetrameric kinesin-5 during mitosis is poorly understood. Kinesin-5 may function only by binding, cross-linking, and sliding adjacent spindle microtubules (MTs), or, alternatively, it may bind to a stable "spindle matrix" to generate mitotic movements. We created transgenic Drosophila melanogaster expressing fluorescent kinesin-5, KLP61F-GFP, in a klp61f mutant background, where it rescues mitosis and viability. KLP61F-GFP localizes to interpolar MT bundles, half spindles, and asters, and is enriched around spindle poles. In fluorescence recovery after photobleaching experiments, KLP61F-GFP displays dynamic mobility similar to tubulin, which is inconsistent with a substantial static pool of kinesin-5. The data conform to a reaction-diffusion model in which most KLP61F is bound to spindle MTs, with the remainder diffusing freely. KLP61F appears to transiently bind MTs, moving short distances along them before detaching. Thus, kinesin-5 motors can function by cross-linking and sliding adjacent spindle MTs without the need for a static spindle matrix.     

Suppression of the bimC4 mitotic spindle defect by deletion of klpA, a gene encoding a KAR3-related kinesin-like protein in Aspergillus nidulans

To investigate the relationship between structure and function of kinesin-like proteins, we have identified by polymerase chain reaction (PCR) a new kinesin-like protein in the filamentous fungus Aspergillus nidulans, which we have designated KLPA. DNA sequence analysis showed that the predicted KLPA protein contains a COOH terminal kinesin-like motor domain. Despite the structural similarity of KLPA to the KAR3 and NCD kinesin-like proteins of Saccharomyces cerevisiae and Drosophila melanogaster, which also posses COOH-terminal kinesin-like motor domains, there are no significant sequence similarities between the nonmotor or tail portions of these proteins. Nevertheless, expression studies in S. cerevisiae showed that klpA can complement a null mutation in KAR3, indicating that primary amino acid sequence conservation between the tail domains of kinesin-like proteins is not necessarily required for conserved function. Chromosomal deletion of the klpA gene exerted no observable mutant phenotype, suggesting that in A. nidulans there are likely to be other proteins functionally redundant with KLPA. Interestingly, the temperature sensitive phenotype of a mutation in another gene, bimC, which encodes a kinesin-like protein involved in mitotic spindle function in A. nidulans, was suppressed by deletion of klpA. We hypothesize that the loss of KLPA function redresses unbalanced forces within the spindle caused by mutation in bimC, and that the KLPA and BIMC kinesin-like proteins may play opposing roles in spindle function.     

pkl1+and klp2+: Two Kinesins of the Kar3 Subfamily in Fission Yeast Perform Different Functions in Both Mitosis and Meiosis

We have identified Klp2p, a new kinesin-like protein (KLP) of the KAR3 subfamily in fission yeast. The motor domain of this protein is 61% identical and 71% similar to Pkl1p, another fission yeast KAR3 protein, yet the two enzymes are different in behavior and function. Pkl1p is nuclear throughout the cell cycle, whereas Klp2p is cytoplasmic during interphase. During mitosis Klp2p enters the nucleus where it forms about six chromatin-associated dots. In metaphase-arrested cells these migrate back and forth across the nucleus. During early anaphase they segregate with the chromosomes into two sets of about three, fade, and are replaced by other dots that form on the spindle interzone. Neither klp2(+) nor pkl1(+) is essential, and the double deletion is also wild type for both vegetative and sexual reproduction. Each deletion rescues different alleles of cut7(ts), a KLP that contributes to spindle formation and elongation. When either or both deletions are combined with a dynein deletion, vegetative growth is normal, but sexual reproduction fails: klp2 Delta,dhc1-d1 in karyogamy, pkl1 Delta,dhc1-d1 in multiple phases of meiosis, and the triple deletion in both. Deletion of Klp2p elongates a metaphase-arrested spindle, but pkl1 Delta shortens it. The anaphase spindle of klp2 Delta becomes longer than the cell, leading it to curl around the cell's ends. Apparently, Klp2p promotes spindle disassembly and contributes to the behavior of mitotic chromosomes.     

BimC motor protein KLP61F cycles between mitotic spindles and fusomes in Drosophila germ cells.

KLP61F in Drosophila is a member of the BimC family of kinesins and, as for other family members [1], is required for spindle assembly [2] [3]. KLP61F is a bipolar homotetramer that cross-links spindle microtubules [4]. It is not known, however, whether the function of KLP61F is dedicated to mitosis or whether KLP61F interacts exclusively with microtubules. Previous work suggested that KLP61F functions during interphase in proliferating germ cells [3]. Cytokinesis is incomplete in germ cells and a branched cortical structure known as a fusome extrudes through intercellular bridges called ring canals. Here I show that, in germ cells, KLP61F cycles between spindles during mitosis and fusomes during interphase. Inspection of fusome-deficient hu-li tai shao (hts) mutants indicated that KLP61F gains fusome-dependent interactions near telophase that mediate its incorporation into these structures. KLP61F proved to be maintained in fusomes by microtubule-independent, detergent-resistant interactions. Inspection of KLP61F mutants indicated that KLP61F is required to recruit fusome material to spindle midbodies near telophase and for normal fusome organization. These observations suggest that KLP61F is bifunctional in germ cells, with microtubule-dependent functions in spindle assembly and microtubule-independent functions in fusome organization. Cytological analyses with antibodies against phosphorylated Eg5 peptide [4] suggest that cycling of KLP61F might reflect phosphorylation.     

The chromokinesin, KLP3A, dives mitotic spindle pole separation during prometaphase and anaphase and facilitates chromatid motility

Mitosis requires the concerted activities of multiple microtubule (MT)-based motor proteins. Here we examined the contribution of the chromokinesin, KLP3A, to mitotic spindle morphogenesis and chromosome movements in Drosophila embryos and cultured S2 cells. By immunofluorescence, KLP3A associates with nonfibrous punctae that concentrate in nuclei and display MT-dependent associations with spindles. These punctae concentrate in indistinct domains associated with chromosomes and central spindles and form distinct bands associated with telophase midbodies. The functional disruption of KLP3A by antibodies or dominant negative proteins in embryos, or by RNA interference (RNAi) in S2 cells, does not block mitosis but produces defects in mitotic spindles. Time-lapse confocal observations of mitosis in living embryos reveal that KLP3A inhibition disrupts the organization of interpolar (ip) MTs and produces short spindles. Kinetic analysis suggests that KLP3A contributes to spindle pole separation during the prometaphase-to-metaphase transition (when it antagonizes Ncd) and anaphase B, to normal rates of chromatid motility during anaphase A, and to the proper spacing of daughter nuclei during telophase. We propose that KLP3A acts on MTs associated with chromosome arms and the central spindle to organize ipMT bundles, to drive spindle pole separation and to facilitate chromatid motility.     

A Chlamydomonas Homologue of the Putative Murine t Complex Distorter Tctex-2 Is an Outer Arm Dynein Light Chain

The kinesin superfamily is a large group of proteins (kinesin-like proteins [KLPs]) that share sequence similarity with the microtubule (MT) motor kinesin. Several members of this superfamily have been implicated in various stages of mitosis and meiosis. Here we report our studies on KLP67A of Drosophila. DNA sequence analysis of KLP67A predicts an MT motor protein with an amino-terminal motor domain. To prove this directly, KLP67A expressed in Escherichia coli was shown in an in vitro motility assay to move MTs in the plus direction. We also report expression analyses at both the mRNA and protein level, which implicate KLP67A in the localization of mitochondria in undifferentiated cell types. In situ hybridization studies of the KLP67A mRNA during embryogenesis and larval central nervous system development indicate a proliferation-specific expression pattern. Furthermore, when affinity-purified anti-KLP67A antisera are used to stain blastoderm embryos, mitochondria in the region of the spindle asters are labeled. These data suggest that KLP67A is a mitotic motor of Drosophila that may have the unique role of positioning mitochondria near the spindle.     

Spindle pole organization in Drosophila S2 cells by dynein, abnormal spindle protein (Asp), and KLP10A

Dynein is a critical mitotic motor whose inhibition causes defects in spindle pole organization and separation, chromosome congression or segregation, and anaphase spindle elongation, but results differ in different systems. We evaluated the functions of the dynein-dynactin complex by using RNA interference (RNAi)-mediated depletion of distinct subunits in Drosophila S2 cells. We observed a striking detachment of centrosomes from spindles, an increase in spindle length, and a loss of spindle pole focus. RNAi depletion of Ncd, another minus-end motor, produced disorganized spindles consisting of multiple disconnected mini-spindles, a different phenotype consistent with distinct pathways of spindle pole organization. Two candidate dynein-dependent spindle pole organizers also were investigated. RNAi depletion of the abnormal spindle protein, Asp, which localizes to focused poles of control spindles, produced a severe loss of spindle pole focus, whereas depletion of the pole-associated microtubule depolymerase KLP10A increased spindle microtubule density. Depletion of either protein produced long spindles. After RNAi depletion of dynein-dynactin, we observed subtle but significant mislocalization of KLP10A and Asp, suggesting that dynein-dynactin, Asp, and KLP10A have complex interdependent functions in spindle pole focusing and centrosome attachment. These results extend recent findings from Xenopus extracts to Drosophila cultured cells and suggest that common pathways contribute to spindle pole organization and length determination.     

A homotetrameric kinesin-5, KLP61F, bundles microtubules and antagonizes Ncd in motility assays

BACKGROUND: Mitosis depends upon the cooperative action of multiple microtubule (MT)-based motors. Among these, a kinesin-5, KLP61F, and the kinesin-14, Ncd, are proposed to generate antagonistic-sliding forces that control the spacing of the spindle poles. We tested whether purified KLP61F homotetramers and Ncd homodimers can generate a force balance capable of maintaining a constant spindle length in Drosophila embryos. RESULTS: Using fluorescence microscopy and cryo-EM, we observed that purified full-length, motorless, and tailless KLP61F tetramers (containing a tetramerization domain) and Ncd dimers can all cross-link MTs into bundles in MgATP. In multiple-motor motility assays, KLP61F and Ncd drive plus-end and minus-end MT sliding at 0.04 and 0.1 microm/s, respectively, but the motility of either motor is decreased by increasing the mole fraction of the other. At the "balance point," the mean velocity was zero and MTs paused briefly and then oscillated, taking approximately 0.3 microm excursions at approximately 0.02 microm/s toward the MT plus end and then the minus end. CONCLUSIONS: The results, combined with quantitative analysis, suggest that these motors could act as mutual brakes to modulate the rate of pole-pole separation and could maintain a prometaphase spindle displaying small fluctuations in its steady-state length.     

The Drosophila kinesin-like protein KLP67A is essential for mitotic and male meiotic spindle assembly

We have performed a mutational analysis together with RNA interference to determine the role of the kinesin-like protein KLP67A in Drosophila cell division. During both mitosis and male meiosis, Klp67A mutations cause an increase in MT length and disrupt discrete aspects of spindle assembly, as well as cytokinesis. Mutant cells exhibit greatly enlarged metaphase spindle as a result of excessive MT polymerization. The analysis of both living and fixed cells also shows perturbations in centrosome separation, chromosome segregation, and central spindle assembly. These data demonstrate that the MT plus end-directed motor KLP67A is essential for spindle assembly during mitosis and male meiosis and suggest that the regulation of MT plus-end polymerization is a key determinant of spindle architecture throughout cell division.     

A microtubule-destabilizing kinesin motor regulates spindle length and anchoring in oocytes

The kinesin-13 motor, KLP10A, destabilizes microtubules at their minus ends in mitosis and binds to polymerizing plus ends in interphase, regulating spindle and microtubule dynamics. Little is known about kinesin-13 motors in meiosis. In this study, we report that KLP10A localizes to the unusual pole bodies of anastral Drosophila melanogaster oocyte meiosis I spindles as well as spindle fibers, centromeres, and cortical microtubules. We frequently observe the pole bodies attached to cortical microtubules, indicating that KLP10A could mediate spindle anchoring to the cortex via cortical microtubules. Oocytes treated with drugs that suppress microtubule dynamics exhibit spindles that are reoriented more vertically to the cortex than untreated controls. A dominant-negative klp10A mutant shows both reoriented and shorter oocyte spindles, implying that, unexpectedly, KLP10A may stabilize rather than destabilize microtubules, regulating spindle length and positioning the oocyte spindle. By altering microtubule dynamics, KLP10A could promote spindle reorientation upon oocyte activation.     

Ran is required before metaphase for spindle assembly and chromosome alignment and after metaphase for chromosome segregation and spindle midbody organization

The Ran pathway has been shown to have a role in spindle assembly. However, the extent of the role of the Ran pathway in mitosis in vivo is unclear. We report that perturbation of the Ran pathway disrupted multiple steps of mitosis in syncytial Drosophila embryos and uncovered new mitotic processes that are regulated by Ran. During the onset of mitosis, the Ran pathway is required for the production, organization, and targeting of centrosomally nucleated microtubules to chromosomes. However, the role of Ran is not restricted to microtubule organization, because Ran is also required for the alignment of chromosomes at the metaphase plate. In addition, the Ran pathway is required for postmetaphase events, including chromosome segregation and the assembly of the microtubule midbody. The Ran pathway mediates these mitotic events, in part, by facilitating the correct targeting of the kinase Aurora A and the kinesins KLP61F and KLP3A to spindles.     

Antagonistic microtubule-sliding motors position mitotic centrosomes in Drosophila early embryos

The positioning of centrosomes, or microtubule-organizing centres, within cells plays a critical part in animal development. Here we show that, in Drosophila embryos undergoing mitosis, the positioning of centrosomes within bipolar spindles and between daughter nuclei is determined by a balance of opposing forces generated by a bipolar kinesin motor, KLP61F, that is directed to microtubule plus ends, and a carboxy-terminal kinesin motor, Ncd, that is directed towards microtubule minus ends. This activity maintains the spacing between separated centrosomes during prometaphase and metaphase, and repositions centrosomes and daughter nuclei during late anaphase and telophase. Surprisingly, we do not observe a function for KLP61F in the initial separation of centrosomes during prophase. Our data indicate that KLP61F and Ncd may function by crosslinking and sliding antiparallel spindle microtubules in relation to one another, allowing KLP61F to push centrosomes apart and Ncd to pull them together.     

Localization of a kinesin-like protein in generative cells of tobacco

Summary We have obtained immunofluorescence and immunoblot evidence for the presence of kinesin-like protein (KLP) in pollen tubes of tobacco using an antibody generated against peptides encoded by theKATA gene ofArabidopsis. This antibody recognizes an M_r 140,000 polypeptide inArabidopsis seedlings, and stains the mitotic apparatus in this species as well as in tobacco suspension cells. In tobacco pollen tubes prepared for dual immunofluorescence localizations of KLP and -tubulin, the antibody binds transiently to microtubule (Mt) bundles and the nucleus in premitotic generative cells; it then stains the developing mitotic apparatus as the nuclear envelope breaks down. By metaphase, fluorescence is located over kinetochore fibers and associated Mts. Localization of KLP is concentrated in the midzone during anaphase, and by early cytokinesis, it closely brackets the cell plate. Phragmoplast fluorescence then spreads along the phragmoplast distal to the cell plate. Punctate staining is also detected along vegetative Mts. No KLP localization is seen in pollen tubes treated with antibody after it had been preadsorbed to the antigenic peptides. The antibody recognizes an M_r 110,000 polypeptide in extracts of tobacco pollen tubes, and a polypeptide of somewhat lower M_r inTradescantia pollen tubes. Our results show that KLP(s) related to KatAp are present in tobacco generative cells and may play roles in the organization and/or operation of the mitotic apparatus and phragmoplast.Abbreviations KLP kinesin-like protein - Mt microtubule - MA mitotic apparatus Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Human Nek7-interactor RGS2 is required for mitotic spindle organization

The mitotic spindle apparatus is composed of microtubule (MT) networks attached to kinetochores organized from 2 centrosomes (a.k.a. spindle poles). In addition to this central spindle apparatus, astral MTs assemble at the mitotic spindle pole and attach to the cell cortex to ensure appropriate spindle orientation. We propose that cell cycle-related kinase, Nek7, and its novel interacting protein RGS2, are involved in mitosis regulation and spindle formation. We found that RGS2 localizes to the mitotic spindle in a Nek7-dependent manner, and along with Nek7 contributes to spindle morphology and mitotic spindle pole integrity. RGS2-depletion leads to a mitotic-delay and severe defects in the chromosomes alignment and congression. Importantly, RGS2 or Nek7 depletion or even overexpression of wild-type or kinase-dead Nek7, reduced γ-tubulin from the mitotic spindle poles. In addition to causing a mitotic delay, RGS2 depletion induced mitotic spindle misorientation coinciding with astral MT-reduction. We propose that these phenotypes directly contribute to a failure in mitotic spindle alignment to the substratum. In conclusion, we suggest a molecular mechanism whereupon Nek7 and RGS2 may act cooperatively to ensure proper mitotic spindle organization.     

Human Nek7-interactor RGS2 is required for mitotic spindle organization

The mitotic spindle apparatus is composed of microtubule (MT) networks attached to kinetochores organized from 2 centrosomes (a.k.a. spindle poles). In addition to this central spindle apparatus, astral MTs assemble at the mitotic spindle pole and attach to the cell cortex to ensure appropriate spindle orientation. We propose that cell cycle-related kinase, Nek7, and its novel interacting protein RGS2, are involved in mitosis regulation and spindle formation. We found that RGS2 localizes to the mitotic spindle in a Nek7-dependent manner, and along with Nek7 contributes to spindle morphology and mitotic spindle pole integrity. RGS2-depletion leads to a mitotic-delay and severe defects in the chromosomes alignment and congression. Importantly, RGS2 or Nek7 depletion or even overexpression of wild-type or kinase-dead Nek7, reduced γ-tubulin from the mitotic spindle poles. In addition to causing a mitotic delay, RGS2 depletion induced mitotic spindle misorientation coinciding with astral MT-reduction. We propose that these phenotypes directly contribute to a failure in mitotic spindle alignment to the substratum. In conclusion, we suggest a molecular mechanism whereupon Nek7 and RGS2 may act cooperatively to ensure proper mitotic spindle organization.     

The Kip3-like kinesin KipB moves along microtubules and determines spindle position during synchronized mitoses in Aspergillus nidulans hyphae

Kinesins are motor proteins which are classified into 11 different families. We identified 11 kinesin-like proteins in the genome of the filamentous fungus Aspergillus nidulans. Relatedness analyses based on the motor domains grouped them into nine families. In this paper, we characterize KipB as a member of the Kip3 family of microtubule depolymerases. The closest homologues of KipB are Saccharomyces cerevisiae Kip3 and Schizosaccharomyces pombe Klp5 and Klp6, but sequence similarities outside the motor domain are very low. A disruption of kipB demonstrated that it is not essential for vegetative growth. kipB mutant strains were resistant to high concentrations of the microtubule-destabilizing drug benomyl, suggesting that KipB destabilizes microtubules. kipB mutations caused a failure of spindle positioning in the cell, a delay in mitotic progression, an increased number of bent mitotic spindles, and a decrease in the depolymerization of cytoplasmic microtubules during interphase and mitosis. Meiosis and ascospore formation were not affected. Disruption of the kipB gene was synthetically lethal in combination with the temperature-sensitive mitotic kinesin motor mutation bimC4, suggesting an important but redundant role of KipB in mitosis. KipB localized to cytoplasmic, astral, and mitotic microtubules in a discontinuous pattern, and spots of green fluorescent protein moved along microtubules toward the plus ends.