Segregation Mutant Research Articles

Altered RNA processing and export lead to retention of mRNAs near transcription sites and nuclear pore complexes or within the nucleolus.

Many protein factors are required for mRNA biogenesis and nuclear export, which are central to the eukaryotic gene expression program. It is unclear, however, whether all factors have been identified. Here we report on a screen of >1000 essential gene mutants in Saccharomyces cerevisiae for defects in mRNA processing and export, identifying 26 mutants with defects in this process. Single-molecule FISH data showed that the majority of these mutants accumulated mRNA within specific regions of the nucleus, which included 1) mRNAs within the nucleolus when nucleocytoplasmic transport, rRNA biogenesis, or RNA processing and surveillance was disrupted, 2) the buildup of mRNAs near transcription sites in 3'-end processing and chromosome segregation mutants, and 3) transcripts being enriched near nuclear pore complexes when components of the mRNA export machinery were mutated. These data show that alterations to various nuclear processes lead to the retention of mRNAs at discrete locations within the nucleus.

Activity-dependent PI(3,5)P2 synthesis controls AMPA receptor trafficking during synaptic depression.

Dynamic regulation of phosphoinositide lipids (PIPs) is crucial for diverse cellular functions, and, in neurons, PIPs regulate membrane trafficking events that control synapse function. Neurons are particularly sensitive to the levels of the low abundant PIP, phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2], because mutations in PI(3,5)P2-related genes are implicated in multiple neurological disorders, including epilepsy, severe neuropathy, and neurodegeneration. Despite the importance of PI(3,5)P2 for neural function, surprisingly little is known about this signaling lipid in neurons, or any cell type. Notably, the mammalian homolog of yeast vacuole segregation mutant (Vac14), a scaffold for the PI(3,5)P2 synthesis complex, is concentrated at excitatory synapses, suggesting a potential role for PI(3,5)P2 in controlling synapse function and/or plasticity. PI(3,5)P2 is generated from phosphatidylinositol 3-phosphate (PI3P) by the lipid kinase PI3P 5-kinase (PIKfyve). Here, we present methods to measure and control PI(3,5)P2 synthesis in hippocampal neurons and show that changes in neural activity dynamically regulate the levels of multiple PIPs, with PI(3,5)P2 being among the most dynamic. The levels of PI(3,5)P2 in neurons increased during two distinct forms of synaptic depression, and inhibition of PIKfyve activity prevented or reversed induction of synaptic weakening. Moreover, altering neuronal PI(3,5)P2 levels was sufficient to regulate synaptic strength bidirectionally, with enhanced synaptic function accompanying loss of PI(3,5)P2 and reduced synaptic strength following increased PI(3,5)P2 levels. Finally, inhibiting PI(3,5)P2 synthesis alters endocytosis and recycling of AMPA-type glutamate receptors (AMPARs), implicating PI(3,5)P2 dynamics in AMPAR trafficking. Together, these data identify PI(3,5)P2-dependent signaling as a regulatory pathway that is critical for activity-dependent changes in synapse strength.

A dynamic meiotic SUN belt includes the zygotene-stage telomere bouquet and is disrupted in chromosome segregation mutants of maize (Zea mays L.).

The nuclear envelope (NE) plays an essential role in meiotic telomere behavior and links the cytoplasm and nucleoplasm during homologous chromosome pairing and recombination in many eukaryotic species. Resident NE proteins including SUN (Sad-1/UNC-84) and KASH (Klarsicht/ANC-1/Syne-homology) domain proteins are known to interact forming the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex that connects chromatin to the cytoskeleton. To investigate the possible cross-kingdom conservation of SUN protein functions in plant meiosis, we immunolocalized maize SUN2 using 3D microscopy of pollen mother cells from maize (Zea mays L.), a large-genome plant model with a canonical NE zygotene-stage telomere bouquet. We detected SUN2 at the nuclear periphery and found that it exhibited a distinct belt-like structure that transitioned to a half-belt during the zygotene stage and back to a full belt during and beyond the pachytene stage. The zygotene-stage half-belt SUN structure was shown by 3D immuno-FISH to include the NE-associated telomere cluster that defines the bouquet stage and coincides with homologous chromosome synapsis. Microtubule and filamentous actin staining patterns did not show any obvious belt or a retracted-like structure other than a general enrichment of tubulin staining distributed widely around the nucleus and throughout the cytoplasm. Genetic disruption of the meiotic SUN belt staining patterns with three different meiosis-specific mutants, desynaptic (dy1), asynaptic1 (as1), and divergent spindle1 (dv1) provides additional evidence for the role of the nuclear envelope in meiotic chromosome behavior. Taking into account all of the observations from this study, we propose that the maize SUN belt is directly or indirectly involved in meiotic telomere dynamics, chromosome synapsis, and possibly integration of signals and forces across the meiotic prophase nuclear envelope.

Meiotic chromosome segregation mutants identified by insertional mutagenesis of fission yeast Schizosaccharomyces pombe; tandem-repeat, single-site integrations

Identification of genes required for segregation of chromosomes in meiosis (scm) is difficult because in most organisms high-fidelity chromosome segregation is essential to produce viable meiotic products. The biology of fission yeast Schizosaccharomyces pombe facilitates identification of such genes. Insertional mutagenesis was achieved by electroporation of linear ura4+ DNA into cells harboring a ura4 deletion. Approximately 1000 stable transformants were screened individually for the production of elevated frequencies of aneuploid spore colonies. Twenty-two candidates were subjected to a secondary screen for cytological defects. Five mutants exhibited significant levels of aberrant meiotic chromosome segregation, but were proficient for mating and completion of meiosis. Each mutant's phenotype cosegregated with its respective ura4+ transgene. The mutations were recessive and defined five complementation groups, revealing five distinct genes (scm1, scm2, scm3, scm4 and scm5). Southern blotting revealed single-site integration in each transformant, indicating that insertional mutagenesis is useful for generating single-locus scm mutations linked to a selectable marker. The transgene insertion points were refractory to analysis by inverse-PCR. Molecular and real-time PCR analyses revealed the presence of multiple, truncated copies of ura4+ at each integration site. Thus, electroporation-mediated insertional mutagenesis in S.pombe is preceded by exonucleolytic processing and concatomerization of the transforming DNA.

Characterization of HCP-6, a C. elegans protein required to prevent chromosome twisting and merotelic attachment.

Previous studies of mitosis show that capture of single kinetochores by microtubules from both centrosomes (merotelic orientation) is a major cause of aneuploidy. We have characterized hcp-6, a temperature-sensitive chromosome segregation mutant in C. elegans that exhibits chromosomes attached to both poles via a single sister kinetochore. We demonstrate that the primary defect in this mutant is a failure to fully condense chromosomes during prophase. Although centromere formation and sister centromere resolution remain unaffected in hcp-6, the chromosomes lack the rigidity of wild-type chromosomes and twist around the long axis of the chromosome. As such, they are unable to establish a proper orientation at prometaphase, allowing individual kinetochores to be captured by microtubules from both poles. We therefore propose that chromosome rigidity plays an essential role in maintaining chromosome orientation to prevent merotelic capture.

Targeting and Processing of Nuclear-encoded Apicoplast Proteins in Plastid Segregation Mutants of Toxoplasma gondii

The apicoplast is a distinctive organelle associated with apicomplexan parasites, including Plasmodium sp. (which cause malaria) and Toxoplasma gondii (the causative agent of toxoplasmosis). This unusual structure (acquired by the engulfment of an ancestral alga and retention of the algal plastid) is essential for long-term parasite survival. Similar to other endosymbiotic organelles (mitochondria, chloroplasts), the apicoplast contains proteins that are encoded in the nucleus and post-translationally imported. Translocation across the four membranes surrounding the apicoplast is mediated by an N-terminal bipartite targeting sequence. Previous studies have described a recombinant "poison" that blocks plastid segregation during mitosis, producing parasites that lack an apicoplast and siblings containing a gigantic, nonsegregating plastid. To learn more about this remarkable phenomenon, we examined the localization and processing of the protein produced by this construct. Taking advantage of the ability to isolate apicoplast segregation mutants, we also demonstrated that processing of the transit peptide of nuclear-encoded apicoplast proteins requires plastid-associated activity.

Identification and characterization of a new prespore-specific regulatory gene, rsfA, of Bacillus subtilis.

Differential gene expression during Bacillus subtilis sporulation is controlled by sigma factors and other regulatory effectors. The first compartmentalized sigma factor, sigma(F), is active specifically in the prespore compartment. During our screening for new chromosome segregation mutants using a sigma(F)-dependent gpr-lacZ reporter as a probe, we identified a new gene (ywfN) required for maximal expression of the reporter and named it rsfA. The product of rsfA has features of gene regulatory proteins, and the protein colocalizes with DNA. The expression of rsfA is under the control of both sigma(F) and sigma(G). Null mutations in rsfA have different effects on the expression of sigma(F)-dependent genes, suggesting that the RsfA protein is a regulator of transcription that fine-tunes gene expression in the prespore.

Inactivation by gene disruption of 2‐cysteine‐peroxiredoxin in Synechocystis sp. PCC 6803 leads to increased stress sensitivity

2‐Cysteine‐peroxiredoxins (2‐CP) constitute a ubiquitous group of enzymes which reduce toxic alkyl hydroperoxides. In higher plants it was shown that the nuclear encoded 2‐CPs are posttranslationally imported into the chloroplasts, the site of most active oxidative metabolism in plants (Baier and Dietz 1997, Plant J. 12; 179‐190). The genome of the bluegreen alga Synechocystis (EMBL acc. # D64000) encodes a 2‐CP which shares 60% homology to higher plant 2‐CPs on the gene level and about 70% on the level of the mature protein. In order to elucidate the physiological significance of 2‐CPs for photosynthetic organisms, the 2‐CP gene was mutated in Synechocystis sp. PCC 6803 by insertion of a kanamycin gene cartridge. Following complete segregation mutant lines were analyzed for growth and photosynthetic parameters. The mutants revealed decreased growth rates as compared to the wild type. Growth inhibition was relieved after lowering the concentration of Fe or trace elements in the growth medium. Chlorophyll a fluorescence transients as induced by saturating light pulses were used as indicator for the state of photosynthesis. The effective quantum yield decreased at lower light intensities in the mutants as compared to the wild type Synechocystis. Simultaneously, electron transport rates saturated at lower light intensities in the mutants. These data provide the first evidence that 2‐CPs play a pivotal protective role in photosynthesis.

Vacuole segregation in the Saccharomyces cerevisiae vac2-1 mutant: structural and biochemical quantification of the segregation defect and formation of new vacuoles.

The conditional vacuolar segregation mutant vac2-1 [Shaw and Wickner (1991) EMBO J. 10, 1741-1748] shifted to non-permissive temperature (37 degrees C), forms large-budded cells without a vacuole in the bud, and daughter cells without an apparent vacuole. Some cells still contain normal segregation structures. Structural and biochemical quantification of the segregation defect showed that (i) about 10% of the full-grown buds did not contain a vacuole, (ii) about 15% of the small cells washed out of a population growing in an elutriation chamber at 37 degrees C, did not contain a visible vacuole, and (iii) 15% of the cells per generation lost carboxypeptidase Y activity after proteinase A depletion. Thus, 10-15% of the daughter cells did not inherit vacuolar structures or vacuolar proteolytic activity from the mother cell. To investigate the fate of vacuole-less daughters, these cells were isolated by optical trapping. The isolated cells formed colonies on agar plates that consisted of cells with normal vacuoles, both at 23 and 37 degrees C. Thus, the vacuole-less cells that failed to inherit proteolytic activities from the mother cell apparently give rise to progeny containing structurally normal vacuoles. Time-lapse experiments showed that vacuole-less daughter cells formed vacuolar vesicles that fused into a new vacuole within 30 min. Although new buds only emerged after a vacuole had formed in the mother cell, the temporary lack of a vacuole had little effect on growth rate. The results suggest that an alternative pathway for vacuole formation exists, and that yeast cells may require a vacuole of some minimal size to initiate a new round of budding.

Genetic and morphological characterization of an Escherichia coli chromosome segregation mutant.

The temperature-sensitive nucleoid segregation mutant of Escherichia coli, PAT32, formerly described as a parA mutant, has been shown to carry a mutation near 66 min on the genetic map. Fine mapping with phages from the collection of Kohara et al. is consistent with its being a parC allele. Observation by fluorescence microscopy revealed the formation, at a nonpermissive temperature, of filaments containing one or two large nucleoids and of normal-size anucleate cells. There was also a significant loss of viability.

Characterization of a cold-sensitive chromosome segregation mutant of Escherichia coli.

A cold-sensitive mutant defective in chromosome segregation, cs 2-68, has been isolated from E. coli after NTG mutagenesis. When an exponentially growing culture of cs2-68 at 37°C was shifted to 20°C, the number of viable cells increased 3.2~4.0-fold in about 6 hr and gradually decreased thereafter. During the incubation at 20°C, most of the cells formed filaments and lost colony forming ability at the same time. A big nuclear mass in the central region of the filamentous cell at 20°C was observed by Giemsa staining. Macromolecular syntheses of cs2-68 including DNA, RNA and protein proceeded at a normal rate at 20°C. The number of viable cells of cs2-68 increased exponentially at 30° and 37°C but not at 20° or 25°C. When the portion of the culture of stationary phase at 37°C was introduced into the fresh medium and incubated at 20°C, the increase of viable cells was only 1.7~1.9-fold. The mutation has been mapped between approximately 70 and 80 min on E. coli genetic map, a previously undescribed region i...

Genetic analysis of a cold-sensitive chromosome segregation mutant of Escherichia coli K12.

Genetic analysis of a cold-sensitive chromosome segregation mutant of Escherichia coli K12.