A Novel Zn2-Cys6 Transcription Factor AtrR Plays a Key Role in an Azole Resistance Mechanism of Aspergillus fumigatus by Co-regulating cyp51A and cdr1B Expressions.

Successful treatment of aspergillosis caused by Aspergillus fumigatus is threatened by an increasing incidence of drug resistance. This situation is further complicated by the finding that strains resistant to azoles, the major antifungal drugs for aspergillosis, have been widely disseminated across the globe. To elucidate mechanisms underlying azole resistance, we identified a novel transcription factor that is required for normal azole resistance in Aspergillus fungi including A. fumigatus, Aspergillus oryzae, and Aspergillus nidulans. This fungal-specific Zn2-Cys6 type transcription factor AtrR was found to regulate expression of the genes related to ergosterol biosynthesis, including cyp51A that encodes a target protein of azoles. The atrR deletion mutant showed impaired growth under hypoxic conditions and attenuation of virulence in murine infection model for aspergillosis. These results were similar to the phenotypes for a mutant strain lacking SrbA that is also a direct regulator for the cyp51A gene. Notably, AtrR was responsible for the expression of cdr1B that encodes an ABC transporter related to azole resistance, whereas SrbA was not involved in the regulation. Chromatin immunoprecipitation assays indicated that AtrR directly bound both the cyp51A and cdr1B promoters. In the clinically isolated itraconazole resistant strain that harbors a mutant Cyp51A (G54E), deletion of the atrR gene resulted in a hypersensitivity to the azole drugs. Together, our results revealed that AtrR plays a pivotal role in a novel azole resistance mechanism by co-regulating the drug target (Cyp51A) and putative drug efflux pump (Cdr1B).

Putative orotate transporter of Cryptococcus neoformans, Oat1, is a member of the NCS1/PRT transporter super family and its loss causes attenuation of virulence.

It is well known that 5-fluoroorotic acid (5-FOA)-resistant mutants isolated from wild-type Cryptococcus neoformans are exclusively either ura3 or ura5 mutants. Unexpectedly, many of the 5-FOA-resistant mutants isolated in our selective regime were Ura+. We identified CNM00460 as the gene responsible for these mutations. Cnm00460 belongs to the nucleobase cation symporter 1/purine-related transporter (NCS1/PRT) super family of fungal transporters, representative members of which are uracil transporter, uridine transporter and allantoin transporter of Saccharomyces cerevisiae. Since the CNM00460 gene turned out to be involved in utilization of orotic acid, most probably as transporter, we designated this gene Orotic Acid Transporter 1 (OAT1). This is the first report of orotic acid transporter in this family. C. neoformans has four members of the NCS1/PRT family, including Cnm00460, Cnm02550, Cnj00690, and Cnn02280. Since the cnm02550∆ strain showed resistance to 5-fluorouridine, we concluded that CNM02550 encodes uridine permease and designated it URidine Permease 1 (URP1). We found that oat1 mutants were sensitive to 5-FOA in the medium containing proline as nitrogen source. A mutation in the GAT1 gene, a positive transcriptional regulator of genes under the control of nitrogen metabolite repression, in the genetic background of oat1 conferred the phenotype of weak resistance to 5-FOA even in the medium using proline as nitrogen source. Thus, we proposed the existence of another orotic acid utilization system (tentatively designated OAT2) whose expression is under the control of nitrogen metabolite repression at least in part. We found that the OAT1 gene is necessary for full pathogenic activity of C. neoformans var. neoformans.

Comparative transcriptome analysis revealing dormant conidia and germination associated genes in Aspergillus species: an essential role for AtfA in conidial dormancy.

BACKGROUND: Fungal conidia are usually dormant unless the extracellular conditions are right for germination. Despite the importance of dormancy, little is known about the molecular mechanism underlying entry to, maintenance of, and exit from dormancy. To gain comprehensive and inter-species insights, transcriptome analyses were conducted across Aspergillus fumigatus, Aspergillus niger, and Aspergillus oryzae. RESULTS: We found transcripts of 687, 694, and 812 genes were enriched in the resting conidia compared with hyphae in A. fumigatus, A. niger, and A. oryzae, respectively (conidia-associated genes). Similarly, transcripts of 766, 1,241, and 749 genes were increased in the 1 h-cultured conidia compared with the resting conidia (germination-associated genes). Among the three Aspergillus species, we identified orthologous 6,172 genes, 91 and 391 of which are common conidia- and germination-associated genes, respectively. A variety of stress-related genes, including the catalase genes, were found in the common conidia-associated gene set, and ribosome-related genes were significantly enriched among the germination-associated genes. Among the germination-associated genes, we found that calA-family genes encoding a thaumatin-like protein were extraordinary expressed in early germination stage in all Aspergillus species tested here. In A. fumigatus 63 % of the common conidia-associated genes were expressed in a bZIP-type transcriptional regulator AtfA-dependent manner, indicating that AtfA plays a pivotal role in the maintenance of resting conidial physiology. Unexpectedly, the precocious expression of the germination-associated calA and an abnormal metabolic activity were detected in the resting conidia of the atfA mutant, suggesting that AtfA was involved in the retention of conidial dormancy. CONCLUSIONS: A comparison among transcriptomes of hyphae, resting conidia, and 1 h-grown conidia in the three Aspergillus species revealed likely common factors involved in conidial dormancy. AtfA positively regulates conidial stress-related genes and negatively mediates the gene expressions related to germination, suggesting a major role for AtfA in Aspergillus conidial dormancy.

Dendritic cell-based immunization ameliorates pulmonary infection with highly virulent Cryptococcus gattii.

Cryptococcosis due to a highly virulent fungus, Cryptococcus gattii, emerged as an infectious disease on Vancouver Island in Canada and surrounding areas in 1999, causing deaths among immunocompetent individuals. Previous studies indicated that C. gattii strain R265 isolated from the Canadian outbreak had immune avoidance or immune suppression capabilities. However, protective immunity against C. gattii has not been identified. In this study, we used a gain-of-function approach to investigate the protective immunity against C. gattii infection using a dendritic cell (DC)-based vaccine. Bone marrow-derived dendritic cells (BMDCs) efficiently engulfed acapsular C. gattii (Δcap60 strain), which resulted in their expression of costimulatory molecules and inflammatory cytokines. This was not observed for BMDCs that were cultured with encapsulated strains. When Δcap60 strain-pulsed BMDCs were transferred to mice prior to intratracheal R265 infection, significant amelioration of pathology, fungal burden, and the survival rate resulted compared with those in controls. Multinucleated giant cells (MGCs) that engulfed fungal cells were significantly increased in the lungs of immunized mice. Interleukin 17A (IL-17A)-, gamma interferon (IFN-γ)-, and tumor necrosis factor alpha (TNF-α)-producing lymphocytes were significantly increased in the spleens and lungs of immunized mice. The protective effect of this DC vaccine was significantly reduced in IFN-γ knockout mice. These results demonstrated that an increase in cytokine-producing lymphocytes and the development of MGCs that engulfed fungal cells were associated with the protection against pulmonary infection with highly virulent C. gattii and suggested that IFN-γ may have been an important mediator for this vaccine-induced protection.

Effects of the F-actin inhibitor latrunculin A on the budding yeast Saccharomyces cerevisiae.

Our basic cell biology research was aimed at investigating the effect on eukaryotic cells of the sudden loss of the F-actin cytoskeleton. Cells treated with latrunculin A (LA) in yeast extract peptone dextrose (YEPD) medium were examined using phase-contrast and fluorescent microscopy, freeze-substitution, transmission and scanning electron microscopy, counted using a Bürker chamber and their absorbance measured. The cells responded to the presence of LA, an F-actin inhibitor, with the disappearance of actin patches, actin cables and actin rings. This resulted in the formation of larger spherical cells with irregular morphology in the cell walls and ultrastructural disorder of the cell organelles and secretory vesicles. Instead of buds, LA-inhibited cells formed only 'table-mountain-like' wide flattened swellings without apical growth with a thinner glucan cell-wall layer containing ß-1,3-glucan microfibrils. The LA-inhibited cells lysed. Actin cables and patches were required for bud formation and bud growth. In addition, actin patches were required for the formation of ß-1,3-glucan microfibrils in the bud cell wall. LA has fungistatic, fungicidal and fungilytic effects on the budding yeast Saccharomyces cerevisiae.

Positional cloning in Cryptococcus neoformans and its application for identification and cloning of the gene encoding methylenetetrahydrofolate reductase.

Cryptococcus neoformans, a basidiomycetous human pathogenic yeast, has been widely used in research fields in medical mycology as well as basic biology. Gene cloning or identification of the gene responsible for a mutation of interest is a key step for functional analysis of a particular gene. The availability therefore, of the multiple methods for cloning is desirable. In this study, we proposed a method for a mapping-based gene identification/cloning (positional cloning) method in C. neoformans. To this end, we constructed a series of tester strains, one of whose chromosomes was labeled with the URA5 gene. A heterozygous diploid constructed by crossing one of the tester strains to a mutant strain of interest loses a chromosome(s) spontaneously, which is the basis for assigning a recessive mutant gene to a particular chromosome in the mitotic mapping method. Once the gene of interest is mapped to one of the 14 chromosomes, classical genetic crosses can then be performed to determine its more precise location. The positional information thus obtained can then be used to significantly narrow down candidate genes by referring to the Cryptococcus genome database. Each candidate gene is then examined whether it would complement the mutation. We successfully applied this method to identify CNA07390 encoding methylenetetrahydrofolate reductase as the gene responsible for a methionine-requiring mutant in our mutant collection.

Identification of genes involved in the phosphate metabolism in Cryptococcus neoformans.

Cryptococcus neoformans is a pathogenic basidiomycetous yeast that can cause life-threatening meningoencephalitis in immuno-compromized patients. To propagate in the human body, this organism has to acquire phosphate that functions in cellular signaling pathways and is also an essential component of nucleic acids and phospholipids. Thus it is reasonable to assume that C. neoformans (Cn) possesses a phosphate regulatory system (PHO system) analogous to that of other fungi. By BLAST searches using the amino acid sequences of the components of the PHO system of Saccharomyces cerevisiae (Sc), we found potential counterparts to ScPHO genes in C. neoformans, namely, acid phosphatase (CnPHO2), the cyclin-dependent protein kinase (CDK) inhibitor (CnPHO81), Pho85-cyclin (CnPHO80), and CDK (CnPHO85). Disruption of each candidate gene, except CnPHO85, followed by phenotypic analysis, identified most of the basic components of the CnPHO system. We found that CnPHO85 was essential for the growth of C. neoformans, having regulatory function in the CnPHO system. Genetic screening and ChIP analysis, showed that CnPHO4 encodes a transcription factor that binds to the CnPHO genes in a Pi-dependent manner. By RNA-seq analysis of the wild-type and the regulatory mutants of the CnPHO system, we found C. neoformans genes whose expression is controlled by the regulators of the CnPHO system. Thus the CnPHO system shares many properties with the ScPHO system, but expression of those CnPHO genes that encode regulators is controlled by phosphate starvation, which is not the case in the ScPHO system (except ScPHO81). We also could identify some genes involved in the stress response of the pathogenic yeast, but CnPho4 appeared to be responsible only for phosphate starvation.

Genome sequence comparison of Aspergillus fumigatus strains isolated from patients with pulmonary aspergilloma and chronic necrotizing pulmonary aspergillosis.

Aspergillus fumigatus is the Aspergillus species most commonly associated with aspergillosis. Of the various presentations of aspergillosis, one of the most frequently observed in cases involving A. fumigatus pulmonary infections is aspergilloma (PA). In such infections one finds a fungus ball composed of fungal hyphae, inflammatory cells, fibrin, mucus, and tissue debris. Chronic necrotizing pulmonary aspergillosis (CNPA), also known as semi-invasive or invasive aspergillosis, is locally invasive and predominantly seen in patients with mild immunodeficiency or with a chronic lung disease. In the present study, with the aid of a next-generation sequencer, we conducted whole genome sequence (WGS) analyses of 17 strains isolated from patients in Japan with PA and CNPA. A total of 99,088 SNPs were identified by mapping the reads to A. fumigatus genome reference strain Af293, and according to genome-wide phylogenetic analysis, there were no correlations between the whole genome sequence typing results and pathologic conditions of patients. Here, we conducted the first multi-genome WGS study to focus on the A. fumigatus strains isolated from patients with PA and CNPA, and comprehensively characterized genetic variations of strains. WGS approach will help in better understanding of molecular mechanisms of aspergillosis cases caused by A. fumigatus.

Whole-genome comparison of Aspergillus fumigatus strains serially isolated from patients with aspergillosis.

The emergence of azole-resistant strains of Aspergillus fumigatus during treatment for aspergillosis occurs by a mutation selection process. Understanding how antifungal resistance mechanisms evolve in the host environment during infection is of great clinical importance and biological interest. Here, we used next-generation sequencing (NGS) to identify mutations that arose during infection by A. fumigatus strains sequentially isolated from two patients, one with invasive pulmonary aspergillosis (IPA) (five isolations) and the other with aspergilloma (three isolations). The serial isolates had identical microsatellite types, but their growth rates and conidia production levels were dissimilar. A whole-genome comparison showed that three of the five isolates from the IPA patient carried a mutation, while 22 mutations, including six nonsynonymous ones, were found among three isolates from the aspergilloma patient. One aspergilloma isolate carried the cyp51A mutation P216L, which is reported to confer azole resistance, and it displayed an MIC indicating resistance to itraconazole. This isolate harbored five other nonsynonymous mutations, some of which were found in the afyap1 and aldA genes. We further identified a large deletion in the aspergilloma isolate in a region containing 11 genes. This finding suggested the possibility that genomic deletions can occur during chronic infection with A. fumigatus. Overall, our results revealed dynamic alterations that occur in the A. fumigatus genome within its host during infection and treatment.

The role of AtfA and HOG MAPK pathway in stress tolerance in conidia of Aspergillus fumigatus.

Aspergillus fumigatus is a life-threatening pathogenic fungus, whose conidium is the infectious agent of aspergillosis. To better understand the mechanism underlying the long-term viability of conidia, we characterized a bZip transcription factor, AtfA, with special reference to stress-tolerance in conidia. The atfA deletion mutant conidia showed significant sensitivity to high temperature and oxidative stress. The trehalose content that accumulated in conidia was reduced in the mutant conidia. Transcriptome analysis revealed that AtfA regulated several stress-protection-related genes such as catA, dprA, scf1, and conJ at the conidiation stage. The upstream high-osmolarity glycerol pathway was also involved in conferring stress tolerance in conidia because ΔpbsB showed stress sensitivity and reduced trehalose in conidia. However, a mutant lacking the SakA mitogen-activated protein kinase (MAPK) produced normal conidia. We investigated another MAPK, MpkC, in relation with SakA, and the double deletion mutant, ΔsakA,mpkC, was defective in conidia stress tolerance. We concluded that MpkC is able to bypass SakA, and the two MAPKs redundantly regulate the conidia-related function of AtfA in A. fumigatus.

Functional characterization of PMT2, encoding a protein-O-mannosyltransferase, in the human pathogen Cryptococcus neoformans.

Diazobenzoic acid B (DBB), also known as diazonium blue B or fast blue B, can be used to distinguish basidiomycetous yeasts from ascomycetes. This chemical has long been used for the taxonomic study of yeast species at the phylum level, but the mechanism underlying the DBB staining remains unknown. To identify molecular targets of DBB staining, we isolated Agrobacterium tumefaciens-mediated insertional mutants of Cryptococcus neoformans, a basidiomycetous pathogenic yeast, which were negative to DBB staining. In one of these mutants, we found that the PMT2 gene, encoding a protein-O-mannosyltransferase, was interrupted by a T-DNA insertion. A complete gene knockout of the PMT2 gene revealed that the gene was responsible for DBB staining in C. neoformans, suggesting that one of the targets of Pmt2-mediated glycosylation is responsible for interacting with DBB. We also determined that Cryptococcus gattii, a close relative of C. neoformans, was not stained by DBB when the PMT2 gene was deleted. Our finding suggests that the protein-O-mannosylation by the PMT2 gene product is required for DBB staining in Cryptococcus species in general. We also showed that glycosylation in Cryptococcus by Pmt2 plays important roles in controlling cell size, resistance to high temperature and osmolarity, capsule formation, sexual reproduction, and virulence.

Heterologous expression of a gene of Magnaporthe oryzae chrysovirus 1 strain A disrupts growth of the human pathogenic fungus Cryptococcus neoformans.

Magnaporthe oryzae chrysovirus 1 strain A (MoCV1-A) is the causal agent of growth repression and attenuated virulence (hypovirulence) of the rice blast fungus, M. oryzae. We have previously reported that heterologous expression of MoCV1-A ORF4 in Saccharomyces cerevisiae results in growth defects, a large central vacuole and other cytological changes. In this study, the effects of open reading frame (ORF) 4 expression in Cryptococcus neoformans, a human pathogenic fungus responsible for severe opportunistic infection, were investigated. Cells expressing the ORF4 gene in C. neoformans showed remarkably enlarged vacuoles, nuclear diffusion and a reduced growth rate. In addition, expression of ORF4 apparently suppressed formation of the capsule that surrounds the entire cell wall, which is one of the most important components of expression of virulence. After 5-fluoroorotic acid treatment of ORF4-expressing cells to remove the plasmid carrying the ORF4 gene, the resultant plasmid-free cells recovered normal morphology and growth, indicating that heterologous expression of the MoCV1-A ORF4 gene induces negative effects in C. neoformans. These data suggest that the ORF4 product is a candidate for a pharmaceutical protein to control disease caused by C. neoformans.

The effects of the F-actin inhibitor latrunculin A on the pathogenic yeast Cryptococcus neoformans.

BACKGROUND: This basic research aimed to investigate the effects of the actin inhibitor latrunculin A (LA) on the human pathogen Cryptococcus neoformans, by freeze-substitution (FS) and electron microscopy (EM), to determine whether the actin cytoskeleton can become a new antifungal target for inhibition of cell division. METHODS: Cells treated with LA for 20 h in yeast-extract peptone dextrose medium were investigated by phase-contrast and fluorescent microscopy, FS and transmission EM, counted in a Bürker chamber and the absorbance was then measured. RESULTS: The disappearance of actin patches, actin cables and actin rings demonstrated the response of the cells of C. neoformans to the presence of the actin inhibitor LA. The removal of actin cables and patches arrested proliferation and led to the production of cells that had ultrastructural disorder, irregular morphology of the mitochondria and thick aberrant cell walls. Budding cells lysed in the buds and septa. CONCLUSION: LA exerts fungistatic, fungicidal and fungilytic effects on the human pathogenic yeast C. neoformans.

Towards understanding cell cycle control in Cryptococcus neoformans: structure-function relationship of G1 and G1/S cyclins homologue CnCln1.

We have previously reported that only a single Cdk1-related G1 and G1/S cyclin homologue was found in the genome sequence of the pathogenic basidiomycetous yeast Cryptococcus neoformans (C. neoformans) and designated it CnCln1. Surprisingly, CnCln1 was not only able to complement the function of the G1 cyclins of the ascomycetous budding yeast Saccharomyces cerevisiae (S. cerevisiae), such as ScCln3, but also the G1/S cyclins of S. cerevisiae, such as ScCln1 and ScCln2. In this study, we investigated how CnCln1 cooperates with the cyclin-dependent kinases of S. cerevisiae (ScCdk1) and substitutes the function of G1 and G1/S cyclins of S. cerevisia from a point of view of their structure-function relationship. Our in silico analysis demonstrated that the CnCln1/ScCdk1 complex was more stable than any of the yeast cyclin and ScCdk1complexes. Thus, these results are consistent with in vitro analysis that has revealed the flexible functional capacity of CnCln1 as a Cdk1-related G1 and G1/S cyclins of S. cerevisiae.

Scanning and negative-staining electron microscopy of protoplast regeneration of a wild-type and two chitin synthase mutants in the pathogenic yeast Candida glabrata.

Protoplast regeneration of a wild-type and two mutant strains of Candida glabrata defective in CHS3 homologues encoding class IV chitin synthase in Saccharomyces cerevisiae was examined by scanning and negative-staining electron microscopy. In the wild-type strain, small particles and short filaments appeared on the protoplast surface at 10 min, filamentous materials covered the entire surface of the protoplast at 1 h, granular materials started filling interspaces of filamentous materials at 2 h and regeneration was completed at 6 h. The filamentous materials consisted of microfibrils of various widths ranging from ≤5 to 40 nm, and composed of ß-glucan. Protoplasts of the two chitin synthase mutant strains of Δchs3A and Δchs3B completed regeneration essentially by the same process as wild-type strain, although it took more time. These results suggest that CHS3A and CHS3B genes may have important roles in cell wall formation during protoplast regeneration, but can be compensated by other cell wall enzymes.

Structome of Saccharomyces cerevisiae determined by freeze-substitution and serial ultrathin-sectioning electron microscopy.

The cell structure has been studied using light and electron microscopies for centuries, and it is assumed that the whole structure is clarified by now. Little quantitative and three-dimensional analysis of cell structure, however, has been undertaken. We have coined a new word, 'structome', by combining 'structure' and '-ome', and defined it as the 'quantitative and three-dimensional structural information of a whole cell at the electron microscopic level'. In the present study, we performed structome analysis of Saccharomyces cerevisiae, one of the most widely researched biological materials, by using freeze-substitution and serial ultrathin-sectioning electron microscopy. Our analysis revealed that there were one to three mitochondria, ~220 000 ribosomes in a cell, and 13-28 endoplasmic reticula/Golgi apparatus which do not form networks in the cytoplasm in the G1 phase. Nucleus occupied ~10.5% of the cell volume; cell wall occupied ~17%; vacuole occupied ~5.8%; cytoplasm occupied ~64%; and mitochondria occupied only ~1.7% in the G1 phase. Structome analysis of cells would form a base for the post-genome research.

The single Cdk1-G1 cyclin of Cryptococcus neoformans is not essential for cell cycle progression, but plays important roles in the proper commitment to DNA synthesis and bud emergence in this yeast.

The cell cycle pattern of the pathogenic basidiomycetous yeast Cryptococcus neoformans differs from that of the ascomycetous budding yeast Saccharomyces cerevisiae. To clarify the cell cycle control mechanisms at the molecular level, homologues of cell cycle control genes in C. neoformans were cloned and analyzed. Here, we report on the cloning and characterization of genes coding for CDK1 cyclin homologues, in particular, the C. neoformans G1 cyclin. We have identified three putative CDK1 cyclin homologues and two putative CDK5 (PHO85) cyclin homologues from the genome. Complementation tests in an S. cerevisiae G1 cyclin triple mutant confirmed that C. neoformans CLN1 is able to complement S. cerevisiae G1 cyclin deficiency, demonstrating that it is a G1 cyclin homologue. Interestingly, cells deleted of the single Cdk1-G1 cyclin were viable, demonstrating that this gene is not essential. However, it exhibited aberrant budding and cell division and a clear delay in the initiation of DNA synthesis as well as an extensive delay in budding. The fact that the mutant managed to traverse the G1 to M phase may be due to the activities of Pho85-related G1 cyclins. Also, that C. neoformans had only a single Cdk1-G1 cyclin highlighted the importance of keeping in order the commitment to the initiation of DNA synthesis first and then that of budding, as discussed.

Step-wise elimination of α-mitochondrial nucleoids and mitochondrial structure as a basis for the strict uniparental inheritance in Cryptococcus neoformans.

In most sexual eukaryotes, mitochondrial (mt) DNA is uniparentally inherited, although the detailed mechanisms underlying this phenomenon remain controversial. The most widely accepted explanations include the autophagic elimination of paternal mitochondria in the fertilized eggs and the active degradation of paternal mitochondrial DNA. To decode the precise program for the uniparental inheritance, we focused on Cryptococcus neoformans as a model system, in which mtDNA is inherited only from the a-parent, although gametes of a- and α-cells are of equal size and contribute equal amounts of mtDNA to the zygote. In this research, the process of preferential elimination of the mitochondria contributed by the α-parent (α-mitochondria) was studied by fluorescence microscopy and single cell analysis using optical tweezers, which revealed that α-mitochondria are preferentially reduced by the following three steps: (1) preferential reduction of α-mitochondrial (mt) nucleoids and α-mtDNA, (2) degradation of the α-mitochondrial structure and (3) proliferation of remaining mt nucleoids during the zygote development. Furthermore, AUTOPHAGY RELATED GENE (ATG) 8 and the gene encoding mitochondrial endonuclease G (NUC1) were disrupted, and the effects of their disruption on the uniparental inheritance were scrutinized. Disruption of ATG8 (ATG7) and NUC1 did not have severe effects on the uniparental inheritance, but microscopic examination revealed that α-mitochondria lacking mt nucleoids persisted in Δatg8 zygotes, indicating that autophagy is not critical for the uniparental inheritance per se but is responsible for the clearance of mitochondrial structures after the reduction of α-mt nucleoids.

The synaptic scaffolding protein Delphilin interacts with monocarboxylate transporter 2.

Delphilin, which interacts with a glutamate receptor (GluR) delta2-subunit, is a postsynaptic density scaffolding protein at the cerebellar parallel fiber-Purkinje cell synapses. Delphilin specifically interacts with the GluRdelta2 C-terminus via its postsynaptic density-95/discs-large/ZO-1 (PDZ) domain. As a number of PDZ-containing scaffolding proteins bind to several membrane proteins, we expected that Delphilin might also have other binding partners besides GluRdelta2. To search for the link between Delphilin and other binding proteins, we carried out screening among candidate membrane proteins localized in Purkinje cells by surface plasmon resonance analyses. As a result, we found that the C-terminus of the monocarboxylate transporter 2 binds specifically and significantly with Delphilin PDZ and there is a probable existence of GluRdelta2-Delphilin-monocarboxylate transporter 2 complex in synaptic membranes.

Cytological study of cell cycle of the pathogenic yeast Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic human pathogen belonging to basidiomycetous fungi and has unique properties in cell cycle progression. The purpose of this study was to measure the duration of the cell cycle in this yeast. Under standard liquid culture conditions (1% yeast extract, 1% polypeptone, and 1% glucose; 24 degrees C; and 150 rpm), the doubling time of exponentially growing C. neoformans was 132 +/- 16 min (mean +/- standard deviation), and the durations of the G1, S, G2, and M phases were about 71, 18, 25, and 18 min, respectively. DNA synthesis started before bud emergence, and finished by the time the size of the bud became 1/4 that of the mother cell. The doubling time of the daughter cells was about twice that of the mother cells. The spindle pole body was located on the outer nuclear envelope and showed a duplicated form from the G1 phase to the G2 phase. These data form a basis for further cell cycle study of C. neoformans.