rough deal: a gene required for proper mitotic segregation in Drosophila

We describe a genetic locus rough deal (rod) in Drosophila melanogaster, identified by mutations that interfere with the faithful transmission of chromosomes to daughter cells during mitosis. Five mutant alleles were isolated, each associated with a similar set of mitotic abnormalities in the dividing neuroblasts of homozygous mutant larvae: high frequencies of aneuploid cells and abnormal anaphase figures, in which chromatids may lag, form bridges, or completely fail to separate. Surviving homozygous adults are sterile, and show cuticular defects associated with cell death, i.e., roughened eyes, sparse abdominal bristles, and notched wing margins. The morphological process of spermatogenesis is largely unaffected and motile sperm are produced, but meiocyte aneuploidy is common. The nature of the observed abnormalities in mitotic cells suggests that the reduced fidelity of chromosome transmission to the daughter cells is due to a failure in a mechanism involved in assuring the proper release of sister chromatids.     

MISP is a novel Plk1 substrate required for proper spindle orientation and mitotic progression

Precise positioning of the mitotic spindle determines the correct cell division axis and is crucial for organism development. Spindle positioning is mediated through a cortical machinery by capturing astral microtubules, thereby generating pushing/pulling forces at the cell cortex. However, the molecular link between these two structures remains elusive. Here we describe a previously uncharacterized protein, MISP (C19orf21), as a substrate of Plk1 that is required for correct mitotic spindle positioning. MISP is an actin-associated protein throughout the cell cycle. MISP depletion led to an impaired metaphase-to-anaphase transition, which depended on phosphorylation by Plk1. Loss of MISP induced mitotic defects including spindle misorientation accompanied by shortened astral microtubules. Furthermore, we find that MISP formed a complex with and regulated the cortical distribution of the +TIP binding protein p150^glued, a subunit of the dynein–dynactin complex. We propose that Plk1 phosphorylates MISP, thus stabilizing cortical and astral microtubule attachments required for proper mitotic spindle positioning.     

A new yeast gene,HTR1, required for growth at high temperature,is needed for recovery from mating pheromone-induced G1 arrest

A new temperature-sensitive mutant of Saccharomyces cerevisiae was isolated. Arrested cells grown at the nonpermissive temperature were of dumb-bell shape and contained large vacuoles. A DNA fragment was cloned based on its ability to complement this temperature sensitivity. The HTR1 gene encodes a putative protein of 93 kDa without significant homology to any known proteins. The gene was mapped between ade5 and lys5 on the left arm of chromosome VII. The phenotype of the gene disruptant appeared to be strain-specific; disruption of the gene in strain W303 caused the cells to become temperature sensitive. The arrested phenotype here was similar to that of the original is mutant and cells in G2/M phase predominated at high temperature. Another disruptant in a strain YPH background grew slowly at high temperature due to slow progression through G2/M phase, and morphologically abnormal (elongated) cells accumulated. A single-copy suppressor that alleviated the temperature-sensitive defects in both strains was identified as MCS1/SSD1. The wild-type strains W303 and YPH are known to carry defective MCS1/SSD1 alleles; hence HTR1 may function redundantly with MCS1/SSD1 to suppress the temperature-sensitive phenotypes. In addition, based on a halo bioassay, the disruptant strains appeared to be defective in recovery from, or adaptive response to G1 arrest mediated by mating pheromone, even at the permissive temperature. Thus the gene has at least two functions and is designated HTR1 (required for high temperature growth and recovery from G1 arrest induced by mating pheromone).     

Molecular structure of a yeast gene, PDI1, encoding protein disulfide isomerase that is essential for cell growth.

A genomic DNA clone for protein disulfide isomerase (PDI) of Saccharomyces cerevisiae was isolated by hybridization with synthesized oligonucleotide probes based on a partial amino acid sequence of yeast PDI. The introduction of a multiple copy plasmid carrying this fragment into yeast caused a tenfold increase in PDI specific activity and in the amount of PDI antigen in the extract. The gene on this fragment was named PDI1. The nucleotide sequence of the gene predicts a polypeptide of 522 amino acids with about 30% identity to mammalian PDIs. The predicted amino acid sequence contains an N-terminal signal peptide-like sequence, the C-terminal putative endoplasmic reticulum retention signal of yeast (HDEL), and two putative active site sequences of PDI (WCGHCK). The predicted polypeptide is acidic and contains five putative glycosylation sites, consistent with the molecular properties of the purified yeast PDI [T. Mizunaga et al. (1990) J. Biochem. 108, 846-851]. The PDI1 gene was mapped on chromosome III. A gene disruption experiment revealed that the PDI1 gene is essential for cell growth.     

Characterization of the END1 gene required for vacuole biogenesis and gluconeogenic growth of budding yeast.

The Saccharomyces cerevisiae END1 gene is required for formation or maintenance of the vacuole, for growth on non-fermentable carbon sources, for efficient mating and for growth at 37 degrees C. The END1 gene was cloned by complementation of the end1 mutation. Two end1 null mutants, constructed by disruption and deletion of the END1 gene, show features identical to the original end1 mutant. However, in this paper we correct a previous finding from our group that end1 is defective in internalization of the yeast pheromone alpha-factor. End1 mutants take up alpha-factor at the same rate as corresponding wild-type cells but the internalized pheromone is not degraded. Since whole cell respiration and respiratory control of end1 mitochondria are not impaired, it seems plausible that a defect in gluconeogenesis could partially account for the inability of end1 to grow on non-fermentable carbon sources. DNA sequence analysis of the END1 gene reveals a 3090-bp open reading frame capable of encoding a hydrophilic protein of 118 kd. The molecular mass of End1p was confirmed by immunoprecipitation. The predicted End1p sequence shows no significant similarity to other known protein sequences except for a short region of homology with the putative adenine nucleotide binding sites shared by a group of enzymes, notably ATPases.     

The yeast MYO1 gene encoding a myosin-like protein required for cell division.

A yeast gene MYO1 that contains regions of substantial sequence homology with the nematode muscle myosin gene (unc54) has been isolated and sequenced. Although the disruption of MYO1 is not lethal, it leads to aberrant nuclear migration and cytokinesis. The 200-kd myosin heavy chain-like protein, the product of MYO1, cross-reacts with anti-nematode myosin heavy chain IgG and is present in wild-type strains but not in strains carrying the disrupted gene. Instead, a truncated polypeptide with a molecular mass of 120 kd can be detected in some myo1 mutants.     

Targeting of RCC1 to chromosomes is required for proper mitotic spindle assembly in human cells

Ran GTPase is involved in several aspects of nuclear structure and function, including nucleocytoplasmic transport and nuclear envelope formation. Experiments using Xenopus egg extracts have shown that generation of Ran-GTP by the guanine nucleotide exchange factor RCC1 also plays roles in mitotic spindle assembly. Here, we have examined the localization and function of RCC1 in mitotic human cells. We show that RCC1, either the endogenous protein or that expressed as a fusion with green fluorescent protein (GFP), is localized predominantly to chromosomes in mitotic cells. This localization requires an N-terminal lysine-rich region that also contains a nuclear localization signal and is enhanced by interaction with Ran. Either mislocalization of GFP-RCC1 by removal of the N-terminal region or the expression of dominant Ran mutants that perturb the GTP/GDP cycle causes defects in mitotic spindle morphology, including misalignment of chromosomes and abnormal numbers of spindle poles. These results indicate that the generation of Ran-GTP in the vicinity of chromosomes by RCC1 is important for the fidelity of mitotic spindle assembly in human cells. Defects in this system may result in abnormal chromosome segregation and genomic instability, which are characteristic of many cancer cells.     

The dhp1(+) gene, encoding a putative nuclear 5'-->3' exoribonuclease, is required for proper chromosome segregation in fission yeast

The Schizosaccharomyces pombe dhp1⁺ gene is an ortholog of the Saccharomyces cerevisiae RAT1 gene, which encodes a nuclear 5′→3′ exoribonuclease, and is essential for cell viability. To clarify the cellular functions of the nuclear 5′→3′ exoribonuclease, we isolated and characterized a temperature-sensitive mutant of dhp1 (dhp1-1 mutant). The dhp1-1 mutant showed nuclear accumulation of poly(A)⁺ RNA at the restrictive temperature, as was already reported for the rat1 mutant. Interestingly, the dhp1-1 mutant exhibited aberrant chromosome segregation at the restrictive temperature. The dhp1-1 cells frequently contained condensed chromosomes, most of whose sister chromatids failed to separate during mitosis despite normal mitotic spindle elongation. Finally, chromosomes were displaced or unequally segregated. As similar mitotic defects were also observed in Dhp1p-depleted cells, we concluded that dhp1⁺ is required for proper chromosome segregation as well as for poly(A)⁺ RNA metabolism in fission yeast. Furthermore, we isolated a multicopy suppressor of the dhp1-1 mutant, referred to as din1⁺. We found that the gene product of dhp1-1 was unstable at high temperatures, but that reduced levels of Dhp1-1p could be suppressed by overexpressing Din1p at the restrictive temperature. Thus, Din1p may physically interact with Dhp1p and stabilize Dhp1p and/or restore its activity.     

MER1, a yeast gene required for chromosome pairing and genetic recombination, is induced in meiosis.

The yeast MER1 gene is required for the production of viable meiotic products and for meiotic recombination. Cytological analysis of chromosome spreads from a mer1 mutant indicates that the MER1 gene product is also required for normal chromosome pairing. mer1 strains make axial elements, precursors to the synaptonemal complex; however, the chromosomes in most nuclei do not become fully synapsed. The DNA sequence of the MER1 coding region was determined; the MER1 open reading frame encodes a 270-amino-acid protein with a molecular mass of 31.1 kilodaltons. The MER1 protein shows limited sequence similarity to calmodulin. Expression of the MER1 gene was examined by RNA blot hybridization analysis and through the construction and analysis of mer1::lacZ fusion genes. Expression of the MER1 gene is meiotically induced and required the IME1 gene product. Thus, expression of the MER1 gene early in meiosis is required for proper chromosome pairing and meiotic recombination.     

Fission yeast kinesin-8 Klp5 and Klp6 are interdependent for mitotic nuclear retention and required for proper microtubule dynamics

Fission yeast has two kinesin-8s, Klp5 and Klp6, which associate to form a heterocomplex. Here, we show that Klp5 and Klp6 are mutually dependent on each other for nuclear mitotic localization. During interphase, they are exported to the cytoplasm. In sharp contrast, during mitosis, Klp5 and Klp6 remain in the nucleus, which requires the existence of each counterpart. Canonical nuclear localization signal (NLS) is identified in the nonkinesin C-terminal regions. Intriguingly individual NLS mutants (NLSmut) exhibit loss-of-function phenotypes, suggesting that Klp5 and Klp6 enter the nucleus separately. Indeed, although neither Klp5-NLSmut nor Klp6-NLSmut enters the nucleus, wild-type Klp6 or Klp5, respectively, does so with different kinetics. In the absence of Klp5/6, microtubule catastrophe/rescue frequency and dynamicity are suppressed, whereas growth and shrinkage rates are least affected. Remarkably, chimera strains containing only the N-terminal Klp5 kinesin domains cannot disassemble interphase microtubules during mitosis, leading to the coexistence of cytoplasmic microtubules and nuclear spindles with massive chromosome missegregation. In this strain, a marked reduction of microtubule dynamism, even higher than in klp5/6 deletions, is evident. We propose that Klp5 and Klp6 play a vital role in promoting microtubule dynamics, which is essential for the spatiotemporal control of microtubule morphogenesis.     

Molecular characterization of VAC1, a gene required for vacuole inheritance and vacuole protein sorting.

Cell division requires an accurate partitioning of cytoplasmic organelles. The segregation of vacuoles in the budding yeast Saccharomyces cerevisaie occurs at a specific time in the cell cycle and is spatially targeted to the small bud. Several yeast vac mutants have been isolated which are defective in this process. We have now cloned the VAC1 gene, corresponding to the first of these mutants, vac1-1. This gene encodes a protein of 515 amino acids, without homolog in the current data bases. It contains neither long hydrophobic stretches nor a classical leader peptide. The most notable aspect of the sequence is the presence of three zinc fingers. Yeast in which the VAC1 gene has been entirely deleted are viable. However, they grow more slowly than wild-type cells and only form microcolonies when grown on glycerol at 37 degrees C. These yeast are defective in vacuole segregation at both the permissive and nonpermissive temperatures. The vac1 mutant was previously shown to mislocalize carboxypeptidase Y to the cell surface, suggesting that Vac1p is involved in more than one vesicular traffic pathway.     

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.