A visual atlas of meiotic protein dynamics in living fission yeast.

Meiosis is a carefully choreographed dynamic process that re-purposes proteins from somatic/vegetative cell division, as well as meiosis-specific factors, to carry out the differentiation and recombination pathway common to sexually reproducing eukaryotes. Studies of individual proteins from a variety of different experimental protocols can make it difficult to compare details between them. Using a consistent protocol in otherwise wild-type fission yeast cells, this report provides an atlas of dynamic protein behaviour of representative proteins at different stages during normal zygotic meiosis in fission yeast. This establishes common landmarks to facilitate comparison of different proteins and shows that initiation of S phase likely occurs prior to nuclear fusion/karyogamy.

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy.

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Replication stress in early S phase generates apparent micronuclei and chromosome rearrangement in fission yeast.

DNA replication stress causes genome mutations, rearrangements, and chromosome missegregation, which are implicated in cancer. We analyze a fission yeast mutant that is unable to complete S phase due to a defective subunit of the MCM helicase. Despite underreplicated and damaged DNA, these cells evade the G2 damage checkpoint to form ultrafine bridges, fragmented centromeres, and uneven chromosome segregations that resembles micronuclei. These micronuclei retain DNA damage markers and frequently rejoin with the parent nucleus. Surviving cells show an increased rate of mutation and chromosome rearrangement. This first report of micronucleus-like segregation in a yeast replication mutant establishes underreplication as an important factor contributing to checkpoint escape, abnormal chromosome segregation, and chromosome instability.

Replication fork stability is essential for the maintenance of centromere integrity in the absence of heterochromatin.

The centromere of many eukaryotes contains highly repetitive sequences marked by methylation of histone H3K9 by Clr4(KMT1). This recruits multiple heterochromatin proteins, including Swi6 and Chp1, to form a rigid centromere and ensure accurate chromosome segregation. In the absence of heterochromatin, cells show an increased rate of recombination in the centromere, as well as chromosome loss. These defects are severely aggravated by loss of replication fork stability. Thus, heterochromatin proteins and replication fork protection mechanisms work in concert to prevent abnormal recombination, preserve centromere integrity, and ensure faithful chromosome segregation.

Fission yeast Hsk1 (Cdc7) kinase is required after replication initiation for induced mutagenesis and proper response to DNA alkylation damage.

Genome stability in fission yeast requires the conserved S-phase kinase Hsk1 (Cdc7) and its partner Dfp1 (Dbf4). In addition to their established function in the initiation of DNA replication, we show that these proteins are important in maintaining genome integrity later in S phase and G2. hsk1 cells suffer increased rates of mitotic recombination and require recombination proteins for survival. Both hsk1 and dfp1 mutants are acutely sensitive to alkylation damage yet defective in induced mutagenesis. Hsk1 and Dfp1 are associated with the chromatin even after S phase, and normal response to MMS damage correlates with the maintenance of intact Dfp1 on chromatin. A screen for MMS-sensitive mutants identified a novel truncation allele, rad35 (dfp1-(1-519)), as well as alleles of other damage-associated genes. Although Hsk1-Dfp1 functions with the Swi1-Swi3 fork protection complex, it also acts independently of the FPC to promote DNA repair. We conclude that Hsk1-Dfp1 kinase functions post-initiation to maintain replication fork stability, an activity potentially mediated by the C terminus of Dfp1.

Conditional switches for extracellular matrix patterning in Drosophila melanogaster.

An F(1) mutagenesis strategy was developed to identify conditional mutations affecting extracellular matrix (ECM) patterning. Tubulogenesis requires coordinated movement of epithelial cells and deposition of a multilayered ECM. In the Drosophila ovary, an epithelium of follicle cells creates the eggshells, including the paired tubular dorsal appendages (DAs) that act as breathing tubes for the embryo. A P-element mutagenesis strategy allowed for conditional overexpression of hundreds of genes in follicle cells. Conditional phenotypes were scored at the level of individual mutant (F(1)) female flies. ECM pattern regulators were readily identified including MAPK signaling gene ets domain lacking (fused DAs), Wnt pathway genes frizzled 3 and osa (long DAs), Hh pathway gene debra (branched DAs), and transcription factor genes sima/HIF-1alpha, ush, lilli, Tfb1, broad, and foxo. In moving cells the [Ca(2+)]/calcineurin pathway can regulate adhesion to ECM while adherens junctions link cells together. Accordingly, thin eggshell and DA phenotypes were identified for the calcineurin regulator calreticulin and the adherens junction component arc. Finally a tubulogenesis defect phenotype was identified for the gene pterodactyl, homologous to the mammalian serine/threonine receptor-associated protein (STRAP) that integrates the TGF-beta and PI3K/AKT signaling pathways. Because phenotypes can be scored in each mutant fly before and after gene induction, this F(1) conditional mutagenesis strategy should allow for increased scale in screens for mutations affecting repeated (reiterated) events in adult animals, including gametogenesis, movement, behavior, and learning.

The regulatory role of residues 226-232 in phosphoenolpyruvate carboxylase from maize.

The regulatory properties of maize phosphoenolpyruvate carboxylase were significantly altered by site-directed mutagenesis of residues 226 through 232. This conserved sequence element, RTDEIRR, is part of a surface loop at the dimer interface. Mutation of individual residues in this sequence caused various kinetic changes, including desensitization of the enzyme to key allosteric effectors or alteration of the K(0.5 PEP) for the substrate phosphoenolpyruvate. R231A, and especially R232Q, displayed decreased apparent affinity for the activator glucose-6-phosphate. Apparent affinity for the activator glycine was reduced in D228N and R232Q, while the maximum activation caused by glycine was greatly reduced in R226Q and E229A. R226Q and E229A also showed significantly lower sensitivity to the inhibitors malate and aspartate. E229A exhibited a low K(0.5 PEP), while the K(0.5 PEP )of R232Q was significantly higher than that of wild type. Thus these seven residues are critical determinants of the enzyme's kinetic responses to activators, inhibitors and substrate. The present results support an earlier suggestion that Arg 231 contributes to the binding site of the allosteric activator glucose-6-phosphate, and are consistent with other proposals that the substrate phosphoenolpyruvate allosterically activates the enzyme by binding at or near the glucose-6-phosphate site. The results also suggest that the glycine binding site may be contiguous with the glucose-6-phosphate binding site. Glu 229, which extends from this interface region through the interior of the protein and emerges near the aspartate binding site, may provide a physical link for propagating conformational changes between the allosteric activator and inhibitor binding regions.