Dictyostelium cell-fate bias is regulated antagonistically by the Retinoblastoma orthologue RblA and the cyclin-dependent kinase Cdk1

A Dictyostelium cell chooses its fate based on the time of its last division prior to the initiation of development. At the onset of development, freshly-divided cells tend to form the stalk of the fruiting body while late-G2 cells become the reproductive spores. How the phases of the cell cycle are linked to cell-type differentiation is unknown. To address this issue, we targeted for analysis two key cell-cycle regulators, Cdk1 and the Retinoblastoma orthologue RblA. Using RNA-blot analysis and reporter-gene studies, we showed that cdk1 is expressed shortly before mitosis then again during mid-development. We also generated a series of doxycycline-inducible cdk1 mutants. Cells expressing cdk1Y15F were blocked in mitosis and displayed defects in spindle assembly, suggesting that Cdk1 dephosphorylation on tyrosine15 is a pivotal step in the G2/M transition of Dictyostelium. Those expressing cdk1T14A were smaller than the control strain, implying that Cdk1T14 phosphorylation is one mechanism by which Dictyostelium coordinates cell growth with cell division. When given the choice between becoming spores or stalk cells, Cdk1T14A cells opted for the stalk fate. Importantly the developmental phenotype was rescued when cdk1T14A was expressed in RblA-deficient cells. By comparing the transcriptional profiles of wild-type and RblA-deficient cells, we found that RblA-repressed genes associated with cell proliferation. These genes were expressed in late G2 then again in mid-development. Collectively our findings support a model in which RblA and Cdk1 play opposing roles in the cell cycle. In this model RblA prevents cell-cycle progression by repressing mitosis and S-phase genes. Repression is relieved in late G2 and genes such as cdk1 are activated, thereby allowing cells to advance through the cell cycle. The antagonistic relationship between Retinoblastoma and Cdks is not new; it has long been established in other model systems. The novelty of our findings is that RblA and Cdk1 also influenced development. We found that cells with an active Cdk1 (or lacking a functional RblA) at the onset of development were ushered to the stalk pathway. This is important since it implies that these cell-cycle regulators are part of the intrinsic signalling pathway responsible for initial cell-type choice in Dictyostelium

mycoCLAP, the database for characterized lignocellulose-active proteins of fungal origin: resource and text mining curation support

Enzymes active on components of lignocellulosic biomass are used for industrial applications ranging from food processing to biofuels production. These include a diverse array of glycoside hydrolases, carbohydrate esterases, polysaccharide lyases and oxidoreductases. Fungi are prolific producers of these enzymes, spurring fungal genome sequencing efforts to identify and catalogue the genes that encode them. To facilitate the functional annotation of these genes, biochemical data on over 800 fungal lignocellulose-degrading enzymes have been collected from the literature and organized into the searchable database, mycoCLAP (http://mycoclap.fungalgenomics.ca). First implemented in 2011, and updated as described here, mycoCLAP is capable of ranking search results according to closest biochemically characterized homologues: this improves the quality of the annotation, and significantly decreases the time required to annotate novel sequences. The database is freely available to the scientific community, as are the open source applications based on natural language processing developed to support the manual curation of mycoCLAP. Database URL: http://mycoclap.fungalgenomics.ca

Investigation of inter- and intraspecies variation through genome sequencing of Aspergillus section Nigri

Aspergillus section Nigri comprises filamentous fungi relevant to biomedicine, bioenergy, health, and biotechnology. To learn more about what genetically sets these species apart, as well as about potential applications in biotechnology and biomedicine, we sequenced 23 genomes de novo, forming a full genome compendium for the section (26 species), as well as 6 Aspergillus niger isolates. This allowed us to quantify both inter-and intraspecies genomic variation. We further predicted 17,903 carbohydrateactive enzymes and 2,717 secondary metabolite gene clusters, which we condensed into 455 distinct families corresponding to compound classes, 49% of which are only found in single species. We performed metabolomics and genetic engineering to correlate genotypes to phenotypes, as demonstrated for the metabolite aurasperone, and by heterologous transfer of citrate production to Aspergillus nidulans. Experimental and computational analyses showed that both secondary metabolism and regulation are key factors that are significant in the delineation of Aspergillus species.Peer reviewe

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field

A Retinoblastoma Orthologue Is a Major Regulator of S-Phase, Mitotic, and Developmental Gene Expression in <em>Dictyostelium</em>

<div><h3>Background</h3><p>The retinoblastoma tumour suppressor, Rb, has two major functions. First, it represses genes whose products are required for S-phase entry and progression thus stabilizing cells in G1. Second, Rb interacts with factors that induce cell-cycle exit and terminal differentiation. <em>Dictyostelium</em> lacks a G1 phase in its cell cycle but it has a retinoblastoma orthologue, <em>rblA.</em></p> <h3>Methodology/Principal Findings</h3><p>Using microarray analysis and mRNA-Seq transcriptional profiling, we show that RblA strongly represses genes whose products are involved in S phase and mitosis. Both S-phase and mitotic genes are upregulated at a single point in late G2 and again in mid-development, near the time when cell cycling is reactivated. RblA also activates a set of genes unique to slime moulds that function in terminal differentiation.</p> <h3>Conclusions</h3><p>Like its mammalian counterpart <em>Dictyostelium</em>, RblA plays a dual role, regulating cell-cycle progression and transcriptional events leading to terminal differentiation. In the absence of a G1 phase, however, RblA functions in late G2 controlling the expression of both S-phase and mitotic genes.</p> </div

Major groups of RblA-repressed genes.

<p>These genes are at least twofold upregulated in the <i>rblA</i> disruptant. Details and links to the individual genes are in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039914#pone.0039914.s003" target="_blank">Table S2</a></b>. In the interest of clarity where the gene name is unclear or cumbersome the name of the corresponding protein is given.</p

Developmental regulation of cell-cycle genes.

<p>(A) Developmental regulation of the selected cell-cycle genes <i>cdk1</i>, <i>kif2</i>, <i>polA1</i>, <i>cycD</i>, and <i>rblA</i>. (B) Averaged transcriptional profiles during development of groups of cell-cycle genes including 5 general cell cycle controllers, 41 mitotic genes, 57 S-phase genes, and 22 DNA repair genes. These profiles were generated by reanalysing the raw data from Parikh <i>et al </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039914#pone.0039914-Parikh1" target="_blank">[8]</a>. The developmental time in hours (x-axis) and normalized mRNA levels (y-axis) are shown.</p

Cell-cycle regulation after cold synchronization.

<p>Cells were synchronized using cold arrest and released from the block at 0 h. RNA samples were collected at hourly intervals over a 12 hour period for mRNA sequencing. Cells were counted every 30 minutes (grey dashed line) and pulse-labelled every hour with bromodeoxyuridine to monitor passage through S-phase (grey line). Panel (A) shows the expression pattern of selected genes: <i>cdk1</i>, <i>kif2</i>, <i>polA</i>, and <i>rblA</i>. In panel (B), the expression profiles of genes which act at specific phases of the cell cycle are grouped. Genes with RblA-repression factors of 2 or greater whose products are known to play roles in the cell cycle, including 5 general cell cycle controllers, 41 M-phase genes, and 57 S-phase genes were averaged and graphed. Note that because of the incomplete synchronization, modulation of greater than threefold is not expected even if a gene is expressed exclusively at the G2/M transition. The time in hours (x-axis) and normalized regulation level (y-axis) are shown.</p

Developmental regulation of RblA-activated genes.

<p>Cells were developed for the amount of time indicated and analysed using mRNA-sequencing. The averaged-developmental profiles of 8 terminal-differentiation genes and 45 genes regulated during development by RblA, as well as the profiles for <i>rblA</i>, and <i>srfA</i>. Raw data from Parikh <i>et al</i> reanalysed <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039914#pone.0039914-Parikh1" target="_blank">[8]</a>. The developmental time in hours (x-axis) and normalized mRNA levels (y-axis) are shown.</p