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.     

Effects of mitotic and tubulin mutations on microtubule architecture in actively growing protoplasts of Aspergillus nidulans

We used immunofluorescent microscopy to characterize microtubule (MT) architecture in wild-type and mutant protoplasts of Aspergillus nidulans at interphase and at mitosis. Because the visualization of MTs by immunofluorescence is technically difficult in intact hyphae of A. nidulans, we developed a method for removing the cell wall under conditions that do not perturb cell physiology, as evidenced by the fact that the resulting protoplasts undergo nuclear division at a normal rate and that cell cycle mutant phenotypes are expressed at restrictive temperature. Interphase cells exhibited an extensive network of cytoplasmic MTs. During mitosis the cytoplasmic MTs mostly disappeared and an intranuclear mitotic spindle appeared. We have previously shown that the benA 33 beta-tubulin mutation causes hyperstabilization of the mitotic spindle, and we have presented additional indirect evidence that suggested that the tubA1 and tubA4 alpha-tubulin mutations destabilize spindle MTs. In this paper, we show that the benA33 mutation increases the stability of cytoplasmic MTs as well as spindle MTs and that the tubA1 and tubA4 mutations destabilize both spindle and cytoplasmic MTs.     

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.     

Gamma-tubulin is a component of the spindle pole body that is essential for microtubule function in Aspergillus nidulans

We have recently discovered that the mipA gene of A. nidulans encodes gamma-tubulin, a new member of the tubulin superfamily. To determine the function of gamma-tubulin in vivo, we have created a mutation in the mipA gene by integrative transformation, maintained the mutation in a heterokaryon, and determined the phenotype of the mutation in spores produced by the heterokaryon. The mutation is lethal and recessive. It strongly inhibits nuclear division, less strongly inhibits nuclear migration, and, as judged by immunofluorescence microscopy, causes a reduction in the number and length of cytoplasmic microtubules and virtually a complete absence of mitotic apparatus. We conclude that gamma-tubulin is essential for microtubule function in general and nuclear division in particular. Immunofluorescence microscopy of wild-type hyphae with affinity-purified, gamma-tubulin-specific antibodies reveals that gamma-tubulin is a component of interphase and mitotic spindle pole bodies. We propose that gamma-tubulin attaches microtubules to the spindle pole body, nucleates microtubule assembly, and establishes microtubule polarity in vivo.     

The bimG gene of Aspergillus nidulans, required for completion of anaphase, encodes a homolog of mammalian phosphoprotein phosphatase 1

In Aspergillus nidulans, the temperature-sensitive, recessive cell cycle mutation bimG11 causes an elevated mitotic index at restrictive temperature and an inability to complete the anaphase separation of daughter nuclei. We have shown that this mutation has an abnormally high content of nuclear phosphoproteins and that the wild-type gene encodes a type 1 protein phosphatase. We conclude that dephosphorylation of a key protein(s) is required to complete mitosis.     

Identification of cut8+ and cek1+, a novel protein kinase gene, which complement a fission yeast mutation that blocks anaphase.

The fission yeast Schizosaccharomyces pombe [corrected] temperature sensitivity cut8-563 mutation causes chromosome overcondensation and short spindle formation in the absence of sister chromatid separation. The cut8-563 mutation allows cytokinesis before the completion of anaphase, thus producing cells with a cut phenotype. The cut8+ gene product may be required for normal progression of anaphase. Diploidization occurs at the restrictive temperature, and 60 to 70% of the cells surviving after two generations are diploid. These phenotypes are reminiscent of those of budding yeast (Saccharomyces cerevisiae) ctf13 and ctf14 (ndc10) mutations. The cut8+ gene, isolated by complementation of the mutant, predicts a 262-amino-acid protein; the amino and carboxy domains are hydrophilic, while the central domain contains several hydrophobic stretches. It has a weak overall similarity to the budding yeast DBF8 gene product. DBF8 is an essential gene whose mutations result in delay in mitotic progression and chromosome instability. Anti-cut8 antibodies detect a 33-kDa polypeptide. Two multicopy suppressor genes for cut8-563 are identified. They are the cut1+ gene essential for nuclear division, and a new gene (designated cek1+) which encodes a novel protein kinase. The cek1+ gene product is unusually large (1,309 amino acids) and has a 112-amino-acid additional sequence in the kinase domain. The cek1+ gene is not an essential gene. Protein phosphorylation by cek1 may facilitate the progression of anaphase through direct or indirect interaction with the cut8 protein.     

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.     

PDIP38 is a novel mitotic spindle-associated protein that affects spindle organization and chromosome segregation

In order to maintain genomic integrity during mitosis, cells assemble the mitotic spindle to separate sister chromosomes to the two daughter cells. A variety of motor- and non motor-proteins are involved in the organization and regulation of this complex apparatus. DNA polymerase δ-interacting protein 38 (PDIP38) is a highly conserved protein and has so far been shown to be a cytoplasmic and nuclear protein. Cell cycle dependent nuclear localization and the interaction with DNA polymerase δ and proliferating cell nuclear antigen (PCNA) indicate a role for PDIP38 in DNA modification and/or proliferation. Here, we show for the first time that PDIP38 localizes to the mitotic spindle throughout mitosis. Using anti-PDIP38 antibody injections and siRNA silencing, we demonstrate that PDIP38 loss-of-function causes problems with spindle organization, aberrant chromosome segregation, and multinucleated cells. Taken together, the data indicate different roles for PDIP38 in safeguarding a proper cell division at various stages of the cell cycle, including DNA synthesis and repair, organization of the mitotic spindle and chromosome segregation.     

A screen for genes involved in the anaphase proteolytic pathway identifies tsm1(+), a novel Schizosaccharomyces pombe gene important for microtubule integrity.

The growth of several mitotic mutants of Schizosaccharomyces pombe, including nuc2-663, is inhibited by the protease inhibitor N-Tosyl-L-Phenylalanine Chloromethyl Ketone (TPCK). Because nuc2(+) encodes a presumptive component of the Anaphase Promoting Complex, which is required for the ubiquitin-dependent proteolysis of certain proteins during exit from mitosis, we have used sensitivity to TPCK as a criterion by which to search for novel S. pombe mutants defective in the anaphase-promoting pathway. In a genetic screen for temperature-sensitive mitotic mutants that were also sensitive to TPCK at a permissive temperature, we isolated three tsm (TPCK-sensitive mitotic) strains. Two of these are alleles of cut1(+), but tsm1-512 maps to a novel genetic location. The tsm1-512 mutation leads to delayed nuclear division at restrictive temperatures, apparently as a result of an impaired ability to form a metaphase spindle. After shift of early G2 cells to 36 degrees, tsm1-512 arrests transiently in the second mitotic division and then exits mitosis, as judged by spindle elongation and septation. The chromosomes, however, often fail to segregate properly. Genetic interactions between tsm1-512 and components of the anaphase proteolytic pathway suggest a functional involvement of the Tsm1 protein in this pathway.     

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.     

A mutation in aspergillus nidulans that blocks the transition from interphase to prophase

In order to develop a method for obtaining mitotic synchrony in aspergillus nidulans, we have characterized previously isolated heat-sensitive nim mutations that block the nuclear division cycle in interphase at restrictive temperature. After 3.5 h at restrictive temperature the mitotic index of a strain carrying one of these mutations, nimA5, was 0, but when this strain was subsequently shifted from restrictive to permissive temperature the mitotic index increased rapidly, reaching a maximum of 78 percent after 7.5 min. When this strain was examined electron-microscopically, mitotic spindles were absent at restrictive temperature. From these data we conclude that at restrictive temperature nimA5 blocks the nuclear division cycle at a point immediately preceding the initiation of chromosomal condensation and mitotic microtubule assembly, and upon shifting to permissive control over the initiation of microtubule assembly and chromosomal condensation in vivo through a simple temperature shift and, consequently, nimA5 should be a powerful tool for studying these processes. Electron-microscopic examination of spindles of material synchronized in this manner reveals that spindle formation, although very rapid, is gradual in the sense that spindle microtubule numbers increase as spindle formation proceeds.     

Uncontrolled septation in a cell division cycle mutant of the fission yeast Schizosaccharomyces pombe.

A temperature-sensitive Schizosaccharomyces pombe mutant, cdc16-116, has been isolated which undergoes uncontrolled septation during its cell division cycle. The mutant accumulates two types of cells after 3 h of growth at the restrictive temperature: (i) type I cells (85% of the population), which complete nuclear division and then form up to five septa between the divided nuclei; and (ii) type II cells (15% of the population), which form an asymmetrically situated septum in the absence of any nuclear division. cdc16-116 is a monogenic recessive mutation unlinked to any previously known cdc gene of S. pombe. It is not affected in a previously reported control by which septation is dependent upon completion of nuclear division. We propose the cdc16-116 is unable to complete septum formation and proceed to cell separation and is also defective in a control which prevents the manufacture of more than one septum in each cell cycle.     

BIK1, a protein required for microtubule function during mating and mitosis in Saccharomyces cerevisiae, colocalizes with tubulin

BIK1 function is required for nuclear fusion, chromosome disjunction, and nuclear segregation during mitosis. The BIK1 protein colocalizes with tubulin to the spindle pole body and mitotic spindle. Synthetic lethality observed in double mutant strains containing a mutation in the BIK1 gene and in the gene for alpha- or beta-tubulin is consistent with a physical interaction between BIK1 and tubulin. Furthermore, over- or underexpression of BIK1 causes aberrant microtubule assembly and function, bik1 null mutants are viable but contain very short or undetectable cytoplasmic microtubules. Spindle formation often occurs strictly within the mother cell, probably accounting for the many multinucleate and anucleate bik1 cells. Elevated levels of chromosome loss in bik1 cells are indicative of defective spindle function. Nuclear fusion is blocked in bik1 x bik1 zygotes, which have truncated cytoplasmic microtubules. Cells overexpressing BIK1 initially have abnormally short or nonexistent spindle microtubules and long cytoplasmic microtubules. Subsequently, cells lose all microtubule structures, coincident with the arrest of division. Based on these results, we propose that BIK1 is required stoichiometrically for the formation or stabilization of microtubules during mitosis and for spindle pole body fusion during conjugation.     

The molecular cloning and identification of a gene product specifically required for nuclear movement in Aspergillus nidulans

A temperature-sensitive mutation in the nudC gene (nudC3) of Aspergillus nidulans specifically prevents the microtubule-based movement of nuclei in this organism at the restrictive temperature. The mutation does not affect short term growth, nuclear division, or the movement of other subcellular organelles. Immunofluorescence analysis of cells blocked at the restrictive temperature, using antitubulin antibodies, shows that the inability of nuclei to move under these conditions is not related to an inability of a particular class of microtubule to form. The inability to move nuclei in this mutant is also shown to be independent of both mitosis and the number of nuclei in the cell as a double mutant carrying both nudC3 and a cell cycle-specific mutation blocks with a single immotile nucleus at the restrictive temperature. The molecular cloning of the nudC gene and sequence analysis reveal that it encodes a previously unidentified protein of 22 kd. Affinity-purified antisera reactive to the nudC protein cross reacts to a single protein of 22 kD in Aspergillus protein extracts. This purified sera failed to reveal a subcellular location for the nudC protein at the level of indirect immunofluorescence. The data presented suggest that the 22-kD nudC gene product functions by interacting between microtubules and nuclei and/or is involved in the generation of force used to move nuclei during interphase.     

Fission yeast cells undergo nuclear division in the absence of spindle microtubules

Mitosis in eukaryotic cells employs spindle microtubules to drive accurate chromosome segregation at cell division. Cells lacking spindle microtubules arrest in mitosis due to a spindle checkpoint that delays mitotic progression until all chromosomes have achieved stable bipolar attachment to spindle microtubules. In fission yeast, mitosis occurs within an intact nuclear membrane with the mitotic spindle elongating between the spindle pole bodies. We show here that in fission yeast interference with mitotic spindle formation delays mitosis only briefly and cells proceed to an unusual nuclear division process we term nuclear fission, during which cells perform some chromosome segregation and efficiently enter S-phase of the next cell cycle. Nuclear fission is blocked if spindle pole body maturation or sister chromatid separation cannot take place or if actin polymerization is inhibited. We suggest that this process exhibits vestiges of a primitive nuclear division process independent of spindle microtubules, possibly reflecting an evolutionary intermediate state between bacterial and Archeal chromosome segregation where the nucleoid divides without a spindle and a microtubule spindle-based eukaryotic mitosis.     

Microtubule stability in budding yeast: characterization and dosage suppression of a benomyl-dependent tubulin mutant.

To better understand the dynamic regulation of microtubule structures in yeast, we studied a conditional-lethal beta-tubulin mutation tub2-150. This mutation is unique among the hundreds of tubulin mutations isolated in Saccharomyces cerevisiae in that it appears to cause an increase in the stability of microtubules. We report here that this allele is a mutation of threonine 238 to alanine, and that tub2-150 prevents the spindle from elongating during anaphase, suggesting a nuclear microtubule defect. To identify regulators of microtubule stability and/or anaphase, yeast genes were selected that, when overexpressed, could suppress the tub2-150 temperature-sensitive phenotype. One of these genes, JSN1, encodes a protein of 125 kDa that has limited similarity to a number of proteins of unknown function. Overexpression of the JSN1 gene in a TUB2 strain causes that strain to become more sensitive to benomyl, a microtubule-destabilizing drug. Of a representative group of microtubule mutants, only one other mutation, tub2-404, could be suppressed by JSN1 overexpression, showing that JSN1 is an allele-specific suppressor. As tub2-404 mutants are also defective for spindle elongation, this provides additional support for a role for JSN1 during anaphase.     

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.     

A Conditional Mutant Having Paralyzed Cilia and a Block in Cytokinesis Is Rescued by Cytoplasmic Exchange in Tetrahymena Thermophila

Nineteen mutants that are conditional for both the ability to regain motility following deciliation and the ability to grow were isolated. The mutations causing slow growth were placed into five complementation groups. None of the mutations appears to affect energy production as all mutants remained motile at the restrictive temperature. In three complementation groups protein synthesis and the levels of mRNA encoding alpha-tubulin or actin were largely unaffected at the restrictive temperature, consistent with the hypothesis that mutations in these three groups directly affect the assembly of functional cilia and growth. Complementation group 1 was chosen for further characterization. Both phenotypes were shown to be linked, suggesting they are caused by a single mutation. Group 1 mutants regenerated cilia at the restrictive temperature, but the cilia were nonmotile. This mutation also caused a block in cytokinesis at the restrictive temperature but did not affect nuclear divisions or DNA synthesis. The block in cell division was transiently rescued by wild-type cytoplasm exchanged when mutants were paired with wild-type cells during conjugation (round 1 of genomic exclusion). Thus, at least one mutation has been isolated that affects assembly of some microtubule-based structures in Tetrahymena (cilia during regeneration) but not others (nuclei divide at 38 degrees), and the product of this gene is likely to play a role in both ciliary function and in cytokinesis.     

daughterless-abo-like, a Drosophila maternal-effect mutation that exhibits abnormal centrosome separation during the late blastoderm divisions

daughterless-abo-like (dal) is a maternal-effect semilethal mutation in Drosophila. The nuclear divisions of embryos derived from homozygous dal females are normal through nuclear cycle 10. However, during nuclear cycles 11, 12 and 13, a total of about half of the nuclei in each embryo either fail to divide or fuse with a neighboring nucleus during telophase. These abnormal nuclei eventually sink into the interior of the embryo, leaving their centrosomes behind on the surface. The loss of about one-half of the peripheral nuclei into the interior of the embryo results in these embryos cellularizing during nuclear cycle 14 with about one-half the normal number of cells. Surprisingly, many of these embryos develop a nearly normal larval cuticle and 8% develop to adulthood. Observations of live embryos doubly injected with tubulin and histones that have been fluorescently labeled allows nuclear and centrosomal behavior to be directly followed as the embryo develops. We find that the abnormal nuclei arise from nuclei whose centrosomes have failed to separate normally in the previous interphase. These incompletely separated centrosomes can cause a non-functional spindle to form, leading to a nuclear division failure. Alternatively, they can form an abnormal spindle with a centrosome from a neighboring nucleus, causing two nuclei to share a common spindle pole. Such nuclei with a shared centrosome will undergo telophase fusions, unequal divisions, or division failures later in mitosis. These findings have helped us to understand the function of the centrosome in the Drosophila embryo.