Cell fusion, nuclear fusion, and zygote differentiation during sexual development of Dictyostelium discoideum.
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Cell Fusion↗
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Nuclear fusion. Evidence for nuclear reactions in imploding bubbles.
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Comment↗
Saccharomyces cerevisiae nuclear fusion requires prior activation by alpha factor.
We have developed a protocol for efficient fusion of spheroplasts of the same mating type. Nuclear fusion in this whole-cell system is also efficient and closely parallels nuclear fusion in heterosexual mating of intact cells. In the spheroplast fusion system, nuclear fusion is dependent on both the KAR1 gene and prior exposure to alpha factor. The major products of nuclear fusion in the spheroplast fusion assay were true diploids that were homozygous at the mating-type locus. An additional 10% of the products were cells of ploidy greater than diploid. The dependence of nuclear fusion on alpha factor treatment could not be replaced by synchronization in G1 by mutations in CDC28 and CDC35 or by prior arrest in stationary phase. These data suggest that nuclear fusion is not a constitutive function of the nucleus, but rather is specifically induced by mating hormone.
Cell Nucleus↗
KAR5 encodes a novel pheromone-inducible protein required for homotypic nuclear fusion.
KAR5 is required for membrane fusion during karyogamy, the process of nuclear fusion during yeast mating. To investigate the molecular mechanism of nuclear fusion, we cloned and characterized the KAR5 gene and its product. KAR5 is a nonessential gene, and deletion mutations produce a bilateral defect in the homotypic fusion of yeast nuclei. KAR5 encodes a novel protein that shares similarity with a protein in Schizosaccharomyces pombe that may play a similar role in nuclear fusion. Kar5p is induced as part of the pheromone response pathway, suggesting that this protein uniquely plays a specific role during mating in nuclear membrane fusion. Kar5p is a membrane protein with its soluble domain entirely contained within the lumen of the endoplasmic reticulum. In pheromone-treated cells, Kar5p was localized to the vicinity of the spindle pole body, the initial site of fusion between haploid nuclei during karyogamy. We propose that Kar5p is required for the completion of nuclear membrane fusion and may play a role in the organization of the membrane fusion complex.
Amino Acid Sequence↗
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.
Adenosine Triphosphatases↗
ER membrane protein complex required for nuclear fusion.
Diploid cells of the yeast Saccharomyces cerevisiae form after the mating of two haploid cells of the opposite mating type. After fusion of the two plasma membranes of the mating cells, a dinucleated cell forms initially in which the two haploid nuclei then rapidly fuse to form a single diploid nucleus. This latter event, called karyogamy, can be divided into two distinct steps: the microtubule-based movement that causes the two nuclei to become closely juxtaposed and the fusion of the nuclear membranes. For the membrane fusion step, one required component, the ER luminal protein Kar2p (BiP), has been identified. For topological reasons, however, it has been unclear how Kar2p could function in this role. Kar2p is localized to the luminal (i.e., noncytoplasmic) face of the ER membrane, yet nuclear fusion must initiate from the cytosolic side of the outer nuclear membrane or the ER membrane with which it is contiguous. There is both genetic and biochemical evidence that Kar2p interacts with Sec63p, an ER membrane protein containing both luminal and cytosolic domains that is involved in protein translocation across the membrane. We have isolated novel sec63 mutant alleles that display severe karyogamy defects. Disruption of the genes encoding other Sec63p-associated proteins (Sec71p and Sec72p) also results in karyogamy defects. A suppressor mutant (sos1-1) partially corrects the translocation defect but does not alleviate the karyogamy defect. sec61 and sec62 mutant alleles that cause similar or more severe protein translocation defects show no karyogamy defects. Taken together, these results suggest a direct role for Sec63p, Sec71p, and Sec72p in nuclear membrane fusion and argue against the alternative interpretation that the karyogamy defects result as an indirect consequence of the impaired membrane translocation of another component(s) required for the process. We propose that an ER/nuclear membrane protein complex composed of Sec63p, Sec71p, and Sec72p plays a central role in mediating nuclear membrane fusion and requires ER luminally associated Kar2p for its function.
Endoplasmic Reticulum↗
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.
Alleles↗
The role of S. cerevisiae cell division cycle genes in nuclear fusion.
Forty temperature-sensitive cell division cycle (cdc) mutants of Saccharomyces cerevisiae were examined for their ability to complete nuclear fusion during conjugation in crosses to a CDC parent strain at the restrictive temperature. Most of the cdc mutant alleles behaved as the CDC parent strain from which they were derived, in that zygotes produced predominantly diploid progeny with only a small fraction of zygotes giving rise to haploid progeny (cytoductants) that signalled a failure in nuclear fusion. However, cdc4 mutants exhibited a strong nuclear fusion (karyogamy) defect in crosses to a CDC parent and cdc28, cdc34 and cdc37 mutants exhibited a weak karyogamy defect. For all four mutants, the karyogamy defect and the cell cycle defect cosegregated, suggesting that both defects resulted from a single lesion for each of these cdc mutants. Therefore, the cdc 4, 28, 34 and 37 gene products are required in both cell division and karyogamy.
Cell Cycle↗
Nuclear fusion in the yeast Saccharomyces cerevisiae.
Karyogamy, or nuclear fusion, is the process during mating by which two haploid yeast nuclei fuse to produce a single diploid nucleus. Karyogamy occurs in two major steps: microtubule-dependent nuclear congression followed by fusion of the nuclear envelope membranes. Many of the proteins required for karyogamy have been discovered to act in related processes during mitotic growth. Accordingly, yeast karyogamy has become an important model system to investigate critical functions of the cytoplasmic microtubules and the microtubule organizing center, the nuclear envelope, and the endoplasmic reticulum.
Cell Fusion↗
kem mutations affect nuclear fusion in Saccharomyces cerevisiae.
We have identified mutations in three genes of Saccharomyces cerevisiae, KEM1, KEM2 and KEM3, that enhance the nuclear fusion defect of kar1-1 yeast during conjugation. The KEM1 and KEM3 genes are located on the left arm of chromosome VII. Kem mutations reduce nuclear fusion whether the kem and the kar1-1 mutations are in the same or in different parents (i.e., in both kem kar1-1 X wild-type and kem X kar 1-1 crosses). kem 1 X kem 1 crosses show a defect in nuclear fusion, but kem 1 X wild-type crosses do not. Mutant kem 1 strains are hypersensitive to benomyl, lose chromosomes at a rate 10-20-fold higher than KEM+ strains, and lose viability upon nitrogen starvation. In addition, kem 1/kem 1 diploids are unable to sporulate. Cells containing a kem 1 null allele grow very poorly, have an elongated rod-shape and are defective in spindle pole body duplication and/or separation. The KEM 1 gene, which is expressed as a 5.5-kb mRNA transcript, contains a 4.6-kb open reading frame encoding a 175-kD protein.
Amino Acid Sequence↗
Benomyl prevents nuclear fusion in Saccharomyces cerevisiae.
Benomyl prevents nuclear fusion in mating mixtures of Saccharomyces cerevisiae. Cytoductants, heterokaryons and diploids may be recovered from these mixtures.
Benomyl↗
Genetic interactions between KAR7/SEC71, KAR8/JEM1, KAR5, and KAR2 during nuclear fusion in Saccharomyces cerevisiae.
During mating of Saccharomyces cerevisiae, two nuclei fuse to produce a single diploid nucleus. Two genes, KAR7 and KAR8, were previously identified by mutations that cause defects in nuclear membrane fusion. KAR7 is allelic to SEC71, a gene involved in protein translocation into the endoplasmic reticulum. Two other translocation mutants, sec63-1 and sec72Delta, also exhibited moderate karyogamy defects. Membranes from kar7/sec71Delta and sec72Delta, but not sec63-1, exhibited reduced membrane fusion in vitro, but only at elevated temperatures. Genetic interactions between kar7 and kar5 mutations were suggestive of protein-protein interactions. Moreover, in sec71 mutants, Kar5p was absent from the SPB and was not detected by Western blot or immunoprecipitation of pulse-labeled protein. KAR8 is allelic to JEMI, encoding an endoplasmic reticulum resident DnaJ protein required for nuclear fusion. Overexpression of KAR8/JEM1 (but not SEC63) strongly suppressed the mating defect of kar2-1, suggesting that Kar2p interacts with Kar8/Jem1p for nuclear fusion. Electron microscopy analysis of kar8 mutant zygotes revealed a nuclear fusion defect different from kar2, kar5, and kar7/sec71 mutants. Analysis of double mutants suggested that Kar5p acts before Kar8/Jem1p. We propose the existence of a nuclear envelope fusion chaperone complex in which Kar2p, Kar5p, and Kar8/Jem1p are key components and Sec71p and Sec72p play auxiliary roles.
Alleles↗
Genetic screens and selections for cell and nuclear fusion mutants.
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Cell Cycle↗
Genes involved in the control of nuclear fusion during the sexual cycle of Saccharomyces cerevisiae.
Mutants of Saccharomyces cerevisiae defective for nuclear fusion have been isolated. Their mutations have been characterized by meiotic analysis, dominance-recessivity and complementation. Twelve of the mutations are allelic to the previously described kar 1-1; five affect a second gene designated KAR 2 and three affect a third gene designated KAR 3. There is evidence suggesting that other two mutants are affected in a gene different from the three mentioned. Mutations in KAR 1 and KAR 2 genes are recessive and do not cause obvious effects other than the failure of the karyogamy. Mutations in KAR 3 are semidominant and do cause pleiotropic effects affecting both a mitosis and meiosis.
Cell Fusion↗
Nuclear fusion induced by Coulomb explosion of heteronuclear clusters.
We propose a new mechanism for the production of high-energy ( E>3 keV) deuterons, suitable to induce dd nuclear fusion, based on multielectron ionization and Coulomb explosion of heteronuclear deuterium containing molecular clusters, e.g., (D2O)n, in intense ( 10(16)-2x10(18) W/cm2) laser fields. Cluster size equations for E, in conjunction with molecular dynamics simulations, reveal important advantages of Coulomb explosion of (D2O)n heteronuclear clusters, as compared with (D)n clusters. These involve the considerably increased D+ kinetic energy and a narrow, high-energy distribution of deuterons.
Journal Article↗
Trypanosome hybrids generated in tsetse flies by nuclear fusion.
Genetic exchange may occur between two particular Trypanosoma brucei clones simultaneously transmitted by the same tsetse fly. We report here that this exchange takes place in the fly, through nuclear fusion. The resulting hybrids appear to be sub-tetraploid, some particular DNA sequences from one of the parental stocks being lost before enough cloned hybrid trypanosomes could be harvested for DNA analysis. A further reduction of the DNA content of these hybrids occurs gradually upon growth and yields near diploid value in a major part of the population. This mode of hybrid generation is different from the fusion of haploid gametes, which is thought to occur normally upon inoculation of metacyclic trypanosomes in their mammalian host. In this respect, the sub-tetraploid hybrids appear to undergo meiosis in the fly, generating sub-diploid metacyclic forms, then fusion in the mammalian blood.
Animals↗
A mutant of Saccharomyces cerevisiae defective for nuclear fusion.
A mutant unable to fuse nuclei during mating has been isolated from standard wild-type Saccharomyces cerevisiae. Tetrad analysis of the mutation responsible for this defect (kar1-1) shows that it segregates as a single Mendelian factor. The defect kn kaf1-1 appears to be nuclear limited. Cytological and genetic evidence shows that in this mutant the events associated with zygote formation are normal until the point of nuclear fusion. The consequence of this defect is the formation of a multinucleate zygote which in subsequent divisions can segregate heterokaryons and haploid heterplasmons.
Cell Fusion↗
Nuclear fusion driven by coulomb explosions of large deuterium clusters
Recent experiments on the interaction of intense, ultrafast laser pulses with large van der Waals bonded clusters have shown that these clusters can explode with substantial kinetic energy. By driving explosions in deuterium clusters with a 35 fs laser pulse, we have accelerated ions to sufficient kinetic energy to produce DD nuclear fusion. By diagnosing the fusion yield through measurements of 2.45 MeV fusion neutrons, we have found that the fusion yield from these exploding clusters varies strongly with the cluster size, consistent with acceleration of deuterons via Coulomb explosion forces.
Journal Article↗