The basidiomycete Coprinus cinereus has many advantages as a model organism for studying sexual development and meiosis, but it has been difficult to investigate using reverse-genetics methods, such as gene disruption by homologous recombination. Here, gene repression by dsRNA-mediated gene silencing was tried as an alternative method for reverse-genetics studies. It was shown that transformation of the LIM15/DMC1 dsRNA expression construct (LIM15dsRNA) resulted in genomic insertion of LIM15dsRNA and paucity of the LIM15/DMC1 transcript. First, LIM15dsRNA was transformed into the homothallic strain AmutBmut to generate a homozygote in which both nuclei had a copy of LIM15dsRNA. The LIM15/DMC1-repressed strain showed abnormal homologous chromosome synapsis during meiosis. Basidiospore production was reduced to 16 % by the induction of dsRNA. However, approximately 60 % of basidiospores were viable. Next, a heterozygote was generated in which one nucleus had a copy of LIM15dsRNA. The phenotype was similar to that of the homozygote. These results are not only the first demonstration of dsRNA-mediated gene silencing in a member of the homobasidiomycete fungi, to which 90 % of mushroom species belong, but also the first successful use of a reverse-genetics approach in C. cinereus research.

Lim15/Dmc1 is a meiosis specific RecA-like protein. Here we propose its participation in meiotic chromosome pairing-related events along with DNA topoisomerase II. Analysis of protein–protein interactions using in vitro binding assays provided evidence that Coprinus cinereus DNA topoisomerase II (CcTopII) specifically interacts with C.cinereus Lim15/Dmc1 (CcLim15). Co-immunoprecipitation experiments also indicated that the CcLim15 protein interacts with CcTopII in vivo. Furthermore, a significant proportion of CcLim15 and CcTopII could be shown to co-localize on chromosomes from the leptotene to the zygotene stage. Interestingly, CcLim15 can potently activate the relaxation/catenation activity of CcTopII in vitro, and CcTopII suppresses CcLim15-dependent strand transfer activity. On the other hand, while enhancement of CcLim15's DNA-dependent ATPase activity by CcTopII was found in vitro, the same enzyme activity of CcTopII was inhibited by adding CcLim15. The interaction of CcLim15 and CcTopII may facilitate pairing of homologous chromosomes.

Existing colorectal cancer biomarkers are insufficient for providing a quick and accurate diagnosis, which is critical for a good prognosis. More appropriate biomarkers are thus needed. To identify new colorectal cancer biomarker candidates, we conducted a comprehensive differential proteomic analysis of six cancer cell lines and a normal cell line, utilizing a fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS) approach. Two sets of intracellular biomarker candidates were identified: one for colorectal cancer, and the other for metastatic colorectal cancer. Our results suggest that cooperative expression of FABP5 and cyclophilin A might be linked to Her2 signaling. Upregulation of LDHB and downregulation of GAPDH suggest the existence of a specific nonglycolytic energy production pathway in metastatic colorectal cancer cells. Downregulation of 14-3-3ζ/δ, cystatin-B, Ran and thioredoxin could be a result of their secretion, which then stimulates metastasis via activity in the sera and ascitic fluids. We propose a possible flow scheme to describe the dynamics of protein expression in colorectal cancer cells leading to tumor progression and metastasis via cell proliferation, angiogenesis, disorganization of actin filaments and epithelial-mesenchymal transition. Our results suggest that colorectal tumor progression may be regulated by signaling mediated by Her2, hypoxia, and TGFβ.

Sumoylation is a post‐translational modification system that covalently attaches the small ubiquitin‐related modifier (SUMO) to target proteins. Ubc9 is required as the E2‐type enzyme for SUMO‐1 conjugation to targets. Here, we show that Ubc9 interacts with the meiosis‐specific RecA homolog, Lim15/Dmc1 in the basidiomycete Coprinus cinereus (CcLim15), and mediates sumoylation of CcLim15 during meiosis. In vitro protein–protein interaction assays revealed that CcUbc9 interacts with CcLim15 and binds to the C‐terminus (amino acids 105–347) of CcLim15, which includes the ATPase domain. Immunocytochemistry demonstrates that CcUbc9 and CcLim15 colocalize in the nuclei from the leptotene stage to the early pachytene stage during meiotic prophase I. Coimmunoprecipitation experiments indicate that CcUbc9 interacts with CcLim15 in vivo during meiotic prophase I. Furthermore, we show that CcLim15 is a target protein of sumoylation both in vivo and in vitro, and identify the C‐terminus (amino acids 105–347) of CcLim15 as the site of sumoylation in vitro. These results suggest that sumoylation is a candidate modulator of meiotic recombination via interaction between Ubc9 and Lim15/Dmc1.

Mer3 is an evolutionarily conserved DNA helicase that has crucial roles in meiotic recombination and crossover formation. We have identified the MER3 homolog in Coprinus cinereus (Ccmer3) and show that it is expressed in zygotene and pachytene meiocytes. Immunostaining analysis indicated that CcMer3 was localized on chromosomes at zygotene and pachytene and CcMer3 foci were more frequent on paired than unpaired chromosomes. We generated a C. cinereus mer3 mutant (#1) and found that it showed abnormal meiosis progression and underwent apoptosis after prophase I. Basidiospore production in #1 was reduced to 0.8% of the wild-type level; the spores showed slower germination at 25 degrees C but were similar to the wild type at 37 degrees C. Electron microscopic analysis of chromosome spreads revealed that axial elements were formed in the mutant but that synapsis was defective, resulting in a reduction in spore production. Our results demonstrate that CcMer3 is required for synaptonemal complex formation after axial elements align and is thus essential for homologous synapsis.

To understand accurate protein dynamics, a highly reproducible proteomics analytical method is required. The acquired thus knowledge will lead to the diagnosis, treatment and protection against diseases. This review deals with proteomics analysis from a view of sample pre-treatment, sensitivity and reproducibility of the methods, including two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), liquid chromatography-tandem mass spectrometry (LC-MS/MS). and fluorogenic derivatization (FD)-LC-MS/MS.

In this review, we describe the role of a small ubiquitin‐like protein modifier (SUMO)‐conjugating protein, Ubc9, in synaptonemal complex formation during meiosis in a basidiomycete, Coprinus cinereus. Because its meiotic cell cycle is long and naturally synchronous, it is suitable for molecular biological, biochemical and genetic studies of meiotic prophase events. In yeast two‐hybrid screening using the meiotic‐specific cDNA library of C. cinereus, we found that the meiotic RecA homolog CcLim15 interacted with CcUbc9, CcTopII and CcPCNA. Moreover, both TopII and PCNA homologs were known as Ubc9 interactors and the targets of sumoylation. Immunocytochemistry demonstrates that CcUbc9, CcTopII and CcPCNA localize with CcLim15 in meiotic nuclei during leptotene to zygotene when synaptonemal complex is formed and when homologous chromosomes pair. We discuss the relationships between Lim15/Dmc1 (CcLim15), TopII (CcTopII), PCNA (CcPCNA) and CcUbc9, and subsequently, the role of sumoylation in the stages. We speculate that CcLim15 and CcTopII work in cohesion between homologous chromatins initially and then, in the process of the zygotene events, CcUbc9 works with factors including CcLim15 and CcTopII as an inhibitor of ubiquitin‐mediated degradation and as a metabolic switch in the meiotic prophase cell cycle. After CcLim15–CcTopII dissociation, CcLim15 remains on the zygotene DNA and recruits CcUbc9, Rad54B, CcUbc9, Swi5‐Sfr1, CcUbc9 and then CcPCNA in rotation on the C‐terminus. Finally during zygotene, CcPCNA replaces CcLim15 on the DNA and the free‐CcLim15 is probably ubiquitinated and disappears. CcPCNA may recruit the polymerase. The idea that CcUbc9 intervenes in every step by protecting CcLim15 and by switching several factors at the C‐terminus of CcLim15 is likely. At the boundary of the zygotene and pachytene stages, CcPCNA would be sumoylated. CcUbc9 may also be involved with CcPCNA in the switch from the replicative polymerase being recruited at zygotene to the repair‐type DNA polymerases being recruited at pachytene.

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