Isolation of Two Apsa Suppressor Strains in Aspergillus Nidulans

Aspergillus nidulans reproduces asexually with single nucleated conidia. In apsA (anucleate primary sterigmata) strains, nuclear positioning is affected and conidiation is greatly reduced. To get further insights into the cellular functions of apsA, aconidial apsA strains were mutagenized and conidiating suppressor strains were isolated. The suppressors fell into two complementation groups, samA and samB (suppressor of anucleate metulae). samA mapped on linkage group I close to pyrG. The mutant allele was dominant in diploids homozygous for apsA. Viability of conidia of samA suppressor strains (samA(-); apsA(-)) was reduced to 50% in comparison to wild-type conidia. Eighty percent of viable spores produced small size colonies that were temperature- and benomyl-sensitive. samB mapped to chromosome VIII and was recessive. Viability of conidia from samB suppressor strains (apsA(-); samB(-)) was also affected but no small size colonies were observed. Both suppressors produced partial defects in sexual reproduction and both suppressed an apsA deletion mutation. In wild-type background the mutant loci affected hyphal growth rate (samA) or changed the colony morphology (samB) and inhibited sexual spore formation (samA and samB). Only subtle effects on conidiation were found. We conclude that both suppressor genes bypass the apsA function and are involved in microtubule-dependent processes.     

Altering a gene involved in nuclear distribution increases the repeat-induced point mutation process in the fungus Podospora anserina

Repeat-induced point mutation (RIP) is a homology-dependent gene-silencing mechanism that introduces C:G-to-T:A transitions in duplicated DNA segments. Cis-duplicated sequences can also be affected by another mechanism called premeiotic recombination (PR). Both are active over the sexual cycle of some filamentous fungi, e.g., Neurospora crassa and Podospora anserina. During the sexual cycle, several developmental steps require precise nuclear movement and positioning, but connections between RIP, PR, and nuclear distributions have not yet been established. Previous work has led to the isolation of ami1, the P. anserina ortholog of the Aspergillus nidulans apsA gene, which is required for nuclear positioning. We show here that ami1 is involved in nuclear distribution during the sexual cycle and that alteration of ami1 delays the fruiting-body development. We also demonstrate that ami1 alteration affects loss of transgene functions during the sexual cycle. Genetically linked multiple copies of transgenes are affected by RIP and PR much more frequently in an ami1 mutant cross than in a wild-type cross. Our results suggest that the developmental slowdown of the ami1 mutant during the period of RIP and PR increases time exposure to the duplication detection system and thus increases the frequency of RIP and PR.     

A homologue of the yeast SHE4 gene is essential for the transition between the syncytial and cellular stages during sexual reproduction of the fungus Podospora anserina.

The Podospora anserina cro1 gene was identified as a gene required for sexual sporulation. Crosses homozygous for the cro1-1 mutation yield fruiting bodies which produce few asci due to the formation of giant plurinucleate cells instead of dikaryotic cells after fertilization. This defect does not impair karyogamy, but meioses of the resultant polyploid nuclei are most often abortive. Cytological studies suggest that the primary defect of the mutant is its inability to form septa between the daughter nuclei after each mitosis, a step specific for normal dikaryotic cell divisions. The cro1-1 mutant would thus be unable to leave the syncytial vegetative state while abiding by the meiotic programme. cro1-1 also shows defects in ascospore germination and growth rate. GFP-tagging of the CRO1 protein reveals that it is a cytosolic protein mainly expressed at the beginning of the dikaryotic stage and at the time of ascospore maturation. The CRO1 protein exhibits significant similarity to the SHE4 protein, which is required for asymmetric mating-type switching in budding yeast cells. Thus, a gene involved in asymmetric cell divisions in a unicellular organism plays a key role at the transition between the syncytial (vegetative) state and the cellular (sexual) state in a filamentous fungus.     

Increased nuclear traffic chaos in hyphae of Aspergillus nidulans: molecular characterization of apsB and in vivo observation of nuclear behaviour

Filamentous fungi are model microorganisms for studying nuclear migration in eukaryotic cells. Two genes, apsA and apsB (=anucleate primary sterigmata), were identified in Aspergillus nidulans that affect nuclear distribution in hyphae and specifically block conidiophore development at the metula stage when mutant. Here we describe the cloning, sequencing and molecular analysis of apsB. The gene encodes a 121 kDa coiled-coil, hydrophilic protein that was localized in the cytoplasm. No protein-protein interaction was detected between ApsB and ApsA, a membrane-associated, previously identified protein. An apsB null mutant was characterized by video epifluorescence microscopy using strains that express green fluorescent protein (GFP) in nuclei. With this novel approach, we have discovered a new mutant phenotype and have found that nuclei display an increased chaotic movement in older hyphal compartments that results in clustering and an uneven distribution of these organelles. These results suggest a regulatory role of ApsB in nuclear migration.     

Genetic interactions of smc, ftsK, and parB genes in Streptomyces coelicolor and their developmental genome segregation phenotypes

The mechanisms by which chromosomes condense and segregate during developmentally regulated cell division are of interest for Streptomyces coelicolor, a sporulating, filamentous bacterium with a large, linear genome. These processes coordinately occur as many septa synchronously form in syncytial aerial hyphae such that prespore compartments accurately receive chromosome copies. Our genetic approach analyzed mutants for ftsK, smc, and parB. DNA motor protein FtsK/SpoIIIE coordinates chromosome segregation with septum closure in rod-shaped bacteria. SMC (structural maintenance of chromosomes) participates in condensation and organization of the nucleoid. ParB/Spo0J partitions the origin of replication using a nucleoprotein complex, assembled at a centromere-like sequence. Consistent with previous work, we show that an ftsK-null mutant produces anucleate spores at the same frequency as the wild-type strain (0.8%). We report that the smc and ftsK deletion-insertion mutants (ftsK′ truncation allele) have developmental segregation defects (7% and 15% anucleate spores, respectively). By use of these latter mutants, viable double and triple mutants were isolated in all combinations with a previously described parB-null mutant (12% anucleate spores). parB and smc were in separate segregation pathways; the loss of both exacerbates the segregation defect (24% anucleate spores). For a triple mutant, deletion of the region encoding the FtsK motor domain and one transmembrane segment partially alleviates the segregation defect of the smc parB mutant (10% anucleate spores). Considerable redundancy must exist in this filamentous organism because segregation of some genomic material occurs 90% of the time during development in the absence of three functions with only a fourfold loss of spore viability. Furthermore, we report that scpA and scpAB mutants (encoding SMC-associated proteins) have spore nucleoid organization defects. Finally, FtsK-enhanced green fluorescent protein (EGFP) localized as bands or foci between incipient nucleoids, while SMC-EGFP foci were not uniformly positioned along aerial hyphae, nor were they associated with every condensing nucleoid.     

A role for the divergent actin gene, ACT2, in nuclear pore structure and function.

We have identified a temperature-sensitive allele of the yeast divergent actin gene ACT2, act2-1, which displays defects in nuclear pore complex (NPC) structure and nuclear import at the restrictive temperature. Although defective in nuclear import, act2-1 cells still selectively retain reporter proteins in the nucleus, and by indirect immunofluorescence the actin cytoskeleton appears normal. Previous studies in Acanthamoeba and Saccharomyces cerevisiae reported that the cellular location of Act2p partially overlaps that of conventional actin, indicating that it has a cytoskeletal function. In this study, both immunofluorescence localization and cellular fractionation of different epitope-tagged versions of Act2p also reveal an association with the nucleus, suggesting an independent nuclear function for Act2p. Analysis of act2-1 by electron microscopy, 30 min after a shift to the restrictive temperature (37 degrees C), reveals a striking aberration in NPC morphology; NPCs appear as abnormal densities on either side of, rather than spanning, the nuclear envelope. Immunoelectron microscopy confirms that these densities contain XFXFG nucleoporins. act2-1 is synthetically lethal in combination with a deletion in the XFXFG nucleoporin gene, NUP1, or a mutation in the nuclear localization sequence receptor gene, SRP1. Act2p and Srp1p co-immunoprecipitate, suggesting that the proteins exist in a complex. Together our data argue that Act2p plays an important role in NPC structure and function.     

The classical nuclear localization signal receptor, importin-alpha, is required for efficient transition through the G1/S stage of the cell cycle in Saccharomyces cerevisiae

There is significant evidence linking nucleocytoplasmic transport to cell cycle control. The budding yeast, Saccharomyces cerevisiae, serves as an ideal model system for studying transport events critical to cell cycle progression because the nuclear envelope remains intact throughout the cell cycle. Previous studies linked the classical nuclear localization signal (cNLS) receptor, importin-alpha/Srp1, to the G(2)/M transition of the cell cycle. Here, we utilize two engineered mutants of importin-alpha/Srp1 with specific molecular defects to explore how protein import affects cell cycle progression. One mutant, Srp1-E402Q, is defective in binding to cNLS cargoes that contain two clusters of basic residues termed a bipartite cNLS. The other mutant, Srp1-55, has defects in release of cNLS cargoes into the nucleus. Consistent with distinct in vivo functional consequences for each of the Srp1 mutants analyzed, we find that overexpression of different nuclear transport factors can suppress the temperature-sensitive growth defects of each mutant. Studies aimed at understanding how each of these mutants affects cell cycle progression reveal a profound defect at the G(1) to S phase transition in both srp1-E402Q and srp1-55 mutants as well as a modest G(1)/S defect in the temperature-sensitive srp1-31 mutant, which was previously implicated in G(2)/M. We take advantage of the characterized defects in the srp1-E402Q and srp1-55 mutants to predict candidate cargo proteins likely to be affected in these mutants and provide evidence that three of these cargoes, Cdc45, Yox1, and Mcm10, are not efficiently localized to the nucleus in importin-alpha mutants. These results reveal that the classical nuclear protein import pathway makes important contributions to the G(1)/S cell cycle transition.     

The Drosophila ATM homologue Mei-41 has an essential checkpoint function at the midblastula transition

BACKGROUND: Drosophila embryogenesis is initiated by 13 rapid syncytial mitotic divisions that do not require zygotic gene activity. This maternally directed cleavage phase of development terminates at the midblastula transition (MBT), at which point the cell cycle slows dramatically, membranes surround the cortical nuclei to form a cellular blastoderm, and zygotic gene expression is first required. RESULTS: We show that embryos lacking Mei-41, a Drosophila homologue of the ATM tumor suppressor, proceed through unusually short syncytial mitoses, fail to terminate syncytial division following mitosis 13, and degenerate without forming cells. A similar cleavage-stage arrest is produced by mutations in grapes, which encodes a homologue of the Checkpoint-1 kinase. We present biochemical, cytological and genetic data indicating that Mei-41 and Grapes are components of a conserved DNA-replication/damage checkpoint pathway that triggers inhibitory phosphorylation of the Cdc2 kinase and mediates resistance to replication inhibitors and DNA-damaging agents. This pathway is nonessential during postembryonic development, but it is required to terminate the cleavage stage at the MBT. Cyclins are required for Cdc2 kinase activity, and mutations in cyclin A and cyclin B bypass the requirement for mei-41 at the MBT. These mutations do not restore wild-type syncytial cell-cycle timing or the embryonic replication checkpoint, however, suggesting that Mei-41-mediated inhibition of Cdc2 has an additional essential function at the MBT. CONCLUSIONS: The Drosophila DNA-replication/damage checkpoint pathway can be activated by externally triggered DNA damage or replication defects throughout the life cycle, and under laboratory conditions this inducible function is nonessential. During early embryogenesis, however, this pathway is activated by developmental cues and is required for the transition from maternal to zygotic control of development at the MBT.     

Structural association of Bax with nuclear matrix and cytomatrix revealed by embedment-free immunogold electron microscopy

Bax is a cellular protein functioning as a promoter of apoptosis. It is ultrastructurally associated with mitochondrial membranes, where it participates in permeability transition pore formation. By employing embedment-free electron microscopy (EFEM), we present evidence that Bax is also associated with the nuclear matrix and cytomatrix of cultured human tumour cells (COLO 205, PA-1, U-373 MG). Extracted cellular scaffolds were probed with anti-Bax antibody using the immunogold electron microscopy technique. Bax immunoreactivity was found on 10-15 nm intermediate filaments of karyo- and cytoskeleton, stretched between the nucleus, nuclear lamina and cell periphery. Bax immunoreactivity was preferentially localized to certain areas of filaments (spot-like). The target molecules for Bax binding in the cellular matrix and their physiological significance remain to be established.     

Integrity of a Zn finger-like domain in SamB is crucial for morphogenesis in ascomycetous fungi.

Genetic features determine the site of polarized growth in filamentous fungi and lead to hyphal tip extension or subapical branching. We have isolated the samB gene (suppressor of anucleate metulae) of Aspergillus nidulans which encodes a 66 kDa protein carrying an atypical Cys4 and an additional Cys2/His/Cys Zn finger motif at the carboxy-terminus. Such novel Zn finger-like domains have recently been found in several other developmental regulators in organisms ranging from yeast to man. Deletion of this domain at the carboxy-terminus of SamB led to premature hyphal ramification, mislocalization of septa and suppression of the asporogenous phenotype of the developmental mutant aps (anucleate primary sterigmata). A DeltasamB deletion strain displayed an identical phenotype. A homologous gene in Saccharomyces cerevisiae was also characterized whose deletion resulted in a multi-budding phenotype; thus it was named MUB1. An underlying common mechanism for both genes in determination of the onset of polarized growth and its links to other cellular developmental processes is discussed.     

Cytological investigation of the mechanism of parthenogenesis in Drosophila mercatorum

Thelytokous parthenogenesis (female progeny only) in animals is believed to arise initially in unfertilized eggs produced by bisexual females via the fusion of two haploid nuclei following meiosis, to produce diploid female progeny. The transition from sexual to parthenogenetic mechanisms of reproduction requires that the egg replace the paternal contributions of a haploid genetic complement and the basal body, which is thought to be essential for centrosome formation. The transitional facultative parthenogenetic stage is usually associated with a high rate of failed or abortive development, but the molecular and mechanistic reasons for this failure remain unclear. We show that a facultative parthenogenetic strain of Drosophila mercatorum produces a high percentage of unfertilized eggs competent to restore diploidy and form centrosomes de novo following meiosis. The female meiotic products replicate and divide by an acentrosomal mechanism in most oocytes and cytoplasmic centrosomes form in 35% of the oocytes. However, after pronuclear replication the cytoplasmic centrosomes must "capture" two haploid nuclei in order to restore diploidy. In practice, this process frequently fails due to centrosome-mediated capture events of single or more than two haploid nuclei, as well as multiple nuclear capture events in a single embryo when excess free centrosomes are not inactivated following formation of the first zygotic nucleus. Additionally, as development proceeds, many of the centrosomes that initiate syncytial development do not remain functional, possibly due to centrosome maturation defects, and later stages of syncytial development fail. The combined effect of the high error rate associated with nuclear capture and the failure of centrosome maturation during later developmental prevents successful parthenogenesis in most of the eggs that initiate development. This shows that the high rate of failed development associated with the transition from sexual to parthenogenetic reproduction is limited by the low probability of the formation of a diploid zygotic nucleus with the correct complement of centrosomes in D. mercatorum.     

Drop out: a third chromosome maternal-effect locus required for formation of the Drosophila cellular blastoderm.

drop out (dop) is a recessive maternal-effect locus identified in a screen for female-sterile mutations in Drosophila polytene region 71C-F. Phenotypic analyses of the dop mutation indicate that the gene is required for proper formation of the cellular blastoderm. In embryos derived from either homozygous or hemizygous dop mothers, cytoplasmic clearing, nuclear migration and division, and pole cell formation appear normal. However, developmental defects are observed prior to and during cellularization of the blastoderm. At the beginning of nuclear cycle 14, the distinct separation of the internal yolk mass and the cortical cytoplasm breaks down. Subsequently, a population of somatic nuclei located at the periphery of the syncytial blastoderm becomes irregularly spaced and nonuniform in their distribution. Despite a somewhat regular formation of the cortical actin network, cellularization in mutant embryos is extremely variable. Such embryos fail to gastrulate normally and produce variable amounts of defective cuticle. Overall, our analyses suggest that the dop gene functions in maintaining the separation of yolk and cortical cytoplasm and in stabilizing the distribution of somatic nuclei in the Drosophila syncytial blastoderm.     

Isolation and characterization of a Drosophila gene essential for early embryonic development and formation of cortical cleavage furrows

We have isolated a new female sterile mutant from Drosophila melanogaster, which arrests the embryonic development during the transition from syncytial to cellular blastoderm. Cytological analysis of the mutant embryos indicates that pseudocleavage furrows in the syncytial blastoderm are abnormal but not completely disrupted. However, cleavage furrows during cellularization are totally disorganized, and no embryos can develop beyond this stage. Consistent with this observation, the expression of this gene peaks around the cellular blastoderm and not in any later developmental stages. Based on immunofluorescence experiments, the protein product of this gene is localized in both pseudocleavage furrows at the syncytial blastoderm and in the cleavage furrows during the cellularization stage. Sequence homology analysis demonstrates a modest, but statistically significant, similarity of this protein with the carboxyl-terminal domains of dystrophin and a family of proteins collectively known as apodystrophins. It is possible that this protein may play an essential role in organizing and maintaining a specialized cytoskeletal structure, a function also suggested for dystrophin and apodystrophins.