Gamma-tubulin and the C-terminal motor domain kinesin-like protein, KLPA, function in the establishment of spindle bipolarity in Aspergillus nidulans

Previous research has found that a gamma-tubulin mutation in Schizosaccharomyces pombe is synthetically lethal with a deletion of the C-terminal motor domain kinesin-like protein gene pkl1, but the lethality of the double mutant prevents a phenotypic analysis of the synthetic interaction. We have investigated interactions between klpA1, a deletion of an Aspergillus nidulans homolog of pkl1, and mutations in the mipA, gamma-tubulin gene. We find that klpA1 dramatically increases the cold sensitivity and slightly reduces the growth rate at all temperatures, of three mipA alleles. In synchronized cells we find that klpA1 causes a substantial but transient inhibition of the establishment of spindle bipolarity. At a restrictive temperature, mipAD123 causes a slight, transient inhibition of spindle bipolarity and a more significant inhibition of anaphase A. In the mipAD123/klpA1 strain, formation of bipolar spindles is more strongly inhibited than in the klpA1 single mutant and many spindles apparently never become bipolar. These results indicate, surprisingly, that gamma-tubulin and the klpA kinesin have overlapping roles in the establishment of spindle bipolarity. We propose a model to account for these data.     

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.     

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.     

The kinesin-like protein KLP61F is essential for mitosis in Drosophila

We report here that disruption of a recently discovered kinesin-like protein in Drosophila melanogaster, KLP61F, results in a mitotic mutation lethal to the organism. We show that in the absence of KLP61F function, spindle poles fail to separate, resulting in the formation of monopolar mitotic spindles. The resulting phenotype of metaphase arrest with polyploid cells is reminiscent of that seen in the fungal bimC and cut7 mutations, where it has also been shown that spindle pole bodies are not segregated. KLP61F is specifically expressed in proliferating tissues during embryonic and larval development, consistent with a primary role in cell division. The structural and functional homology of the KLP61F, bimC, cut7, and Eg5 kinesin-like proteins demonstrates the existence of a conserved family of kinesin-like molecules important for spindle pole separation and mitotic spindle dynamics.     

Cloning and expression of a human kinesin heavy chain gene: interaction of the COOH-terminal domain with cytoplasmic microtubules in transfected CV-1 cells

To understand the interactions between the microtubule-based motor protein kinesin and intracellular components, we have expressed the kinesin heavy chain and its different domains in CV-1 monkey kidney epithelial cells and examined their distributions by immunofluorescence microscopy. For this study, we cloned and sequenced cDNAs encoding a kinesin heavy chain from a human placental library. The human kinesin heavy chain exhibits a high level of sequence identity to the previously cloned invertebrate kinesin heavy chains; homologies between the COOH-terminal domain of human and invertebrate kinesins and the nonmotor domain of the Aspergillus kinesin-like protein bimC were also found. The gene encoding the human kinesin heavy chain also contains a small upstream open reading frame in a G-C rich 5' untranslated region, features that are associated with translational regulation in certain mRNAs. After transient expression in CV-1 cells, the kinesin heavy chain showed both a diffuse distribution and a filamentous staining pattern that coaligned with microtubules but not vimentin intermediate filaments. Altering the number and distribution of microtubules with taxol or nocodazole produced corresponding changes in the localization of the expressed kinesin heavy chain. The expressed NH2-terminal motor and the COOH-terminal tail domains, but not the alpha-helical coiled coil rod domain, also colocalized with microtubules. The finding that both the kinesin motor and tail domains can interact with cytoplasmic microtubules raises the possibility that kinesin could crossbridge and induce sliding between microtubules under certain circumstances.     

Separate domains of KAR1 mediate distinct functions in mitosis and nuclear fusion

The yeast KAR1 gene is essential for mitotic growth and important for nuclear fusion. Mutations in KAR1 prevent duplication of the spindle pole body (SPB), and affect functions associated with both the nuclear and cytoplasmic microtubules. The localization of hybrid Kar1-lacZ proteins, described elsewhere (Vallen, E. A., T. Y. Scherson, T. Roberts, K. van Zee, and M. D. Rose. 1992. Cell. In press), suggest that the protein is associated with the SPB. In this paper, we report a deletion analysis demonstrating that the mitotic and karyogamy functions of KAR1 are separate and independent, residing in discrete functional domains. One region, here shown to be essential for mitosis, coincided with a part of the protein that is both necessary and sufficient to target Karl-lacZ hybrid proteins to the SPB (Vallen, E. A., T. Y. Scherson, T. Roberts, K. van Zee, and M. D. Rose. 1992. Cell. In press). Complementation testing demonstrated that deletions in this interval did not affect nuclear fusion. A second region, required only for karyogamy, was necessary for the localization of a Kar3-lacZ hybrid protein to the SPB. These data suggest a model for the roles of Kar1p and Kar3p, a kinesin-like protein, in nuclear fusion. Finally, a third region of KAR1 was found to be important for both mitosis and karyogamy. This domain included the hydrophobic carboxy terminus and is sufficient to target a lacZ-Kar1 hybrid protein to the nuclear envelope (Vallen E. A., T. Y. Scherson, T. Roberts, K. van Zee, and M. D. Rose. 1992. Cell. In press). Altogether, the essential mitotic regions of KAR1 comprised 20% of the coding sequence. We propose a model for Kar1p in which the protein is composed of several protein-binding domains tethered to the nuclear envelope via its hydrophobic tail.     

KAR3, a kinesin-related gene required for yeast nuclear fusion

The KAR3 gene is essential for yeast nuclear fusion during mating, and its expression is strongly induced by alpha factor. The predicted KAR3 protein sequence contains two globular domains separated by an alpha-helical coiled coil. The carboxy-terminal domain is homologous to the amino-terminal mechanochemical domain of Drosophila kinesin heavy chain. Mutation of the putative ATP binding site produces a dominant "poison" of nuclear fusion. The mutant protein shows enhanced microtubule association in vivo, as predicted for a kinesin-like protein in a state of rigor binding. Localization of hybrid proteins to cytoplasmic microtubules in shmoos indicates that the amino-terminal domain also contains determinants for microtubule association. Thus, KAR3 is a member of a larger family of kinesin-like proteins characterized by the presence of the mechanochemical domain tethered to different protein binding domains. The phenotypes of kar3 mutants suggest that the protein mediates microtubule sliding during nuclear fusion and possibly mitosis.     

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.     

Bundling of microtubules by motor and tail domains of a kinesin-like calmodulin-binding protein from Arabidopsis: regulation by Ca(2+)/Calmodulin

Kinesin-like calmodulin-binding protein (KCBP), a novel kinesin-like protein from plants, is unique among kinesins and kinesin-like proteins in having a calmodulin-binding domain adjacent to its motor domain. KCBP localizes to mitotic microtubule (MT) arrays including the preprophase band, the spindle apparatus, and the phragmoplast, suggesting a role for KCBP in establishing these MT arrays by bundling MTs. To determine if KCBP bundles MTs, we expressed C-terminal motor and N-terminal tail domains of KCBP, and used the purified proteins in MT bundling assays. The 1.5 C protein with the motor and calmodulin-binding domains induced MT bundling. The 1.5 C-induced bundles were dissociated in the presence of Ca(2+)/calmodulin. Similar results were obtained with a 1.4 C protein, which lacks much of the coiled-coil region present in 1.5 C protein and does not form dimers. The N-terminal tail of KCBP, which contains an ATP-independent MT binding site, is also capable of bundling MTs. These results, together with the KCBP localization data, suggest the involvement of KCBP in establishing mitotic MT arrays during different stages of cell division and that Ca(2+)/calmodulin regulates the formation of these MT arrays.     

A screen for dynein synthetic lethals in Aspergillus nidulans identifies spindle assembly checkpoint genes and other genes involved in mitosis.

Cytoplasmic dynein is a ubiquitously expressed microtubule motor involved in vesicle transport, mitosis, nuclear migration, and spindle orientation. In the filamentous fungus Aspergillus nidulans, inactivation of cytoplasmic dynein, although not lethal, severely impairs nuclear migration. The role of dynein in mitosis and vesicle transport in this organism is unclear. To investigate the complete range of dynein function in A. nidulans, we searched for synthetic lethal mutations that significantly reduced growth in the absence of dynein but had little effect on their own. We isolated 19 sld (synthetic lethality without dynein) mutations in nine different genes. Mutations in two genes exacerbate the nuclear migration defect seen in the absence of dynein. Mutations in six other genes, including sldA and sldB, show a strong synthetic lethal interaction with a mutation in the mitotic kinesin bimC and, thus, are likely to play a role in mitosis. Mutations in sldA and sldB also confer hypersensitivity to the microtubule-destabilizing drug benomyl. sldA and sldB were cloned by complementation of their mutant phenotypes using an A. nidulans autonomously replicating vector. Sequencing revealed homology to the spindle assembly checkpoint genes BUB1 and BUB3 from Saccharomyces cerevisiae. Genetic interaction between dynein and spindle assembly checkpoint genes, as well as other mitotic genes, indicates that A. nidulans dynein plays a role in mitosis. We suggest a model for dynein motor action in A. nidulans that can explain dynein involvement in both mitosis and nuclear distribution.     

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.     

Yeast Dam1p Is Required to Maintain Spindle Integrity during Mitosis and Interacts with the Mps1p Kinase

We have identified a mutant allele of the DAM1 gene in a screen for mutations that are lethal in combination with the mps1-1 mutation. MPS1 encodes an essential protein kinase that is required for duplication of the spindle pole body and for the spindle assembly checkpoint. Mutations in six different genes were found to be lethal in combination with mps1-1, of which only DAM1 was novel. The remaining genes encode a checkpoint protein, Bub1p, and four chaperone proteins, Sti1p, Hsc82p, Cdc37p, and Ydj1p. DAM1 is an essential gene that encodes a protein recently described as a member of a microtubule binding complex. We report here that cells harboring the dam1-1 mutation fail to maintain spindle integrity during anaphase at the restrictive temperature. Consistent with this phenotype, DAM1 displays genetic interactions with STU1, CIN8, and KAR3, genes encoding proteins involved in spindle function. We have observed that a Dam1p-Myc fusion protein expressed at endogenous levels and localized by immunofluorescence microscopy, appears to be evenly distributed along short mitotic spindles but is found at the spindle poles at later times in mitosis.     

Deletion of nudC, a nuclear migration gene of Aspergillus nidulans, causes morphological and cell wall abnormalities and is lethal.

Nuclear migration is required for normal development in both higher and lower eukaryotes. In fungi this process is mediated by cytoplasmic dynein. It is believed that this motor protein is anchored to the cell membrane and moves nuclei by capturing and pulling on spindle pole body microtubules. To date, four genes have been identified and shown to be required for this process in Aspergillus nidulans. The nudA and nudG genes, respectively, encode the heavy and light chains of cytoplasmic dynein, and the nudF and nudC gene products encode proteins of 49 and 22 kDa. The precise biochemical functions of the nudF and nudC genes have not yet been identified. In this report we further investigate NUDC protein function by deleting the nudC gene. Surprisingly, although deletion of nudA and nudF affect nuclear migration, deletion of nudC profoundly affected the morphology and composition of the cell wall. Spores of the strain deleted for nudC grew spherically and lysed. The thickness of the cell wall was increased in the deletion mutant and wall polymer composition was abnormal. This phenotype could be repressed by growth on osmotically buffered medium at low temperature. Similar, but less severe, effects were also noted in a strain depleted for NUDC by down-regulation. These results suggest a possible relationship between fungal cell wall biosynthesis and nuclear migration.     

A novel calcium/calmodulin-regulated kinesin-like protein is highly conserved between monocots and dicots

Recently, a novel kinesin-like protein (KCBP) that is regulated by Ca2+/calmodulin was isolated from dicot plants. A homolog of KCBP has not been reported in monocots. To determine if this motor protein is present in phylogenetically divergent flowering plants, Arabidopsis KCBP cDNA was used as a probe to screen a genomic library of maize, an evolutionarily divergent species. This screening resulted in isolation of a KCBP homolog. Comparison of the predicted amino acid sequence of the KCBP from maize (ZmKCBP), a monocot, with the previously reported KCBP sequences from dicot species showed that the amino acid sequence, domain organization, and gene structure are highly conserved between monocots and dicots. The C-terminal region of ZmKCBP, containing the motor domain and the calmodulin-binding domain, and the N-terminal tail, with a myosin tail homology region (MyTH4) and talin-like region, showed strong sequence similarity to the KCBP homolog from dicots. However, the coiled-coil region is less conserved between monocots and dicots. The ZmKCBP gene contained 22 exons and 21 introns. The location of 19 of the 21 introns of ZmKCBP is also conserved. The ZmKCBP protein is encoded by a single gene and expressed in all tissues. Affinity-purified antibody to the calmodulin-binding domain of Arabidopsis KCBP detected a protein in both the soluble and the microsomal fractions. The C-terminal region of ZmKCBP, containing the motor and calmodulin-binding domains, bound calmodulin in the presence of calcium and failed to bind in the presence of EGTA. The ZmKCBP, along with other KCBPs from dicots, was grouped into a distinct group in the C-terminal subfamily of kinesin-like proteins. These data suggest that the KCBP is ubiquitous and highly conserved in all flowering plants and the origin of KCBP predated the divergence of monocots and dicots.     

Ambient pH signal transduction in Aspergillus: completion of gene characterization

Completing the molecular analysis of the six pal genes of the ambient pH signal transduction pathway in Aspergillus nidulans , we report the characterization of palC and palH . The derived translation product of palH contains 760 amino acids with prediction of seven transmembrane domains in its N-terminal moiety. Remarkably, a palH frameshift mutant lacking just over half the PalH protein, including almost all of the long hydrophilic region C-terminal to the transmembrane domains, retains some PalH function. The palC -derived translation product contains 507 amino acids, and the null phenotype of a frameshift mutation indicates that at least one of the C-terminal 142 residues is essential for function. Uniquely among the A. nidulans pH-signalling pal genes, palC appears to have no Saccharomyces cerevisiae homologue, although it does have a Neurospora crassa expressed sequence tag homologue. In agreement with findings for the palA , palB and palI genes of this signalling pathway, levels of the palC and palH mRNAs do not appear to be pH regulated.     

Kinesin-related proteins required for assembly of the mitotic spindle

We identified two new Saccharomyces cerevisiae kinesin-related genes, KIP1 and KIP2, using polymerase chain reaction primers corresponding to highly conserved regions of the kinesin motor domain. Both KIP proteins are expressed in vivo, but deletion mutations conferred no phenotype. Moreover, kip1 kip2 double mutants and a triple mutant with kinesin-related kar3 had no synthetic phenotype. Using a genetic screen for mutations that make KIP1 essential, we identified another gene, KSL2, which proved to be another kinesin-related gene, CIN8. KIP1 and CIN8 are functionally redundant: double mutants arrested in mitosis whereas the single mutants did not. The microtubule organizing centers of arrested cells were duplicated but unseparated, indicating that KIP1 or CIN8 is required for mitotic spindle assembly. Consistent with this role, KIP1 protein was found to colocalize with the mitotic spindle.     

Saccharomyces cerevisiae kinesin- and dynein-related proteins required for anaphase chromosome segregation

The Saccharomyces cerevisiae kinesin-related gene products Cin8p and Kip1p function to assemble the bipolar mitotic spindle. The cytoplasmic dynein heavy chain homologue Dyn1p (also known as Dhc1p) participates in proper cellular positioning of the spindle. In this study, the roles of these motor proteins in anaphase chromosome segregation were examined. While no single motor was essential, loss of function of all three completely halted anaphase chromatin separation. As combined motor activity was diminished by mutation, both the velocity and extent of chromatin movement were reduced, suggesting a direct role for all three motors in generating a chromosome-separating force. Redundancy for function between different types of microtubule-based motor proteins was also indicated by the observation that cin8 dyn1 double- deletion mutants are inviable. Our findings indicate that the bulk of anaphase chromosome segregation in S. cerevisiae is accomplished by the combined actions of these three motors.     

Aspergillus nidulans DigA, a potential homolog of Saccharomyces cerevisiae Pep3 (Vps18), is required for nuclear migration, mitochondrial morphology and polarized growth

The fungal vacuole is an acidic organelle that is involved in a variety of physiological processes, such as protein turnover, ion and pH homeostasis and osmoregulation. The function of the vacuole largely depends on vesicle transport providing the organelle with enzymes and substrates. The process of vesicle transportation has been studied best in Saccharomyces cerevisiae, where several proteins that are crucial for intracellular vesicle sorting have been identified. One such protein is Pep3 (Vps18). In pep3 mutants vacuole function and vacuole morphology are affected. We cloned the gene for a potential homolog of Pep3 from the filamentous fungus Aspergillus nidulans. The gene, digA (for dichotomous growth), was identified in a screen for nuclear migration mutants. A. nidulans digA encodes a protein of 108.3 kDa, which includes a 122-amino acid clathrin repeat motif, two short coiled-coil regions, and a RING finger Zn-binding motif at the C-terminus. All three sequence motifs suggest interaction of DigA with other proteins. DigA is 25% identical to a homolog from Schizosaccharomyces pombe, 23% to a protein from human, 21% to the product of the Drosophila melanogaster gene deep orange (dor) and 18% to S. cerevisiae Pep3 (Vps18). We localized DigA as a HA-epitope-tagged protein in the cytoplasm of A. nidulans vegetative cells, by secondary immunofluorescence. A digA mutant displays a pleiotropic phenotype with clustered mitochondria, clustered nuclei and a defect in polarization of the actin cytoskeleton. These results suggest that vacuolar functions are required for organelle positioning and polarized growth.     

Cytoplasmic dynein's mitotic spindle pole localization requires a functional anaphase-promoting complex, gamma-tubulin, and NUDF/LIS1 in Aspergillus nidulans

In Aspergillus nidulans, cytoplasmic dynein and NUDF/LIS1 are found at the spindle poles during mitosis, but they seem to be targeted to this location via different mechanisms. The spindle pole localization of cytoplasmic dynein requires the function of the anaphase-promoting complex (APC), whereas that of NUDF does not. Moreover, although NUDF's localization to the spindle poles does not require a fully functional dynein motor, the function of NUDF is important for cytoplasmic dynein's targeting to the spindle poles. Interestingly, a gamma-tubulin mutation, mipAR63, nearly eliminates the localization of cytoplasmic dynein to the spindle poles, but it has no apparent effect on NUDF's spindle pole localization. Live cell analysis of the mipAR63 mutant revealed a defect in chromosome separation accompanied by unscheduled spindle elongation before the completion of anaphase A, suggesting that gamma-tubulin may recruit regulatory proteins to the spindle poles for mitotic progression. In A. nidulans, dynein is not apparently required for mitotic progression. In the presence of a low amount of benomyl, a microtubule-depolymerizing agent, however, a dynein mutant diploid strain exhibits a more pronounced chromosome loss phenotype than the control, indicating that cytoplasmic dynein plays a role in chromosome segregation.