Regulation of Dictyostelium morphogenesis by RapGAP3

Rap1 is a key regulator of cell adhesion and cell motility in Dictyostelium. Here, we identify a Rap1-specific GAP protein (RapGAP3) and provide evidence that Rap1 signaling regulates cell-cell adhesion and cell migration within the multicellular organism. RapGAP3 mediates the deactivation of Rap1 at the late mound stage of development and plays an important role in regulating cell sorting during apical tip formation, when the anterior-posterior axis of the organism is formed, by controlling cell-cell adhesion and cell migration. The loss of RapGAP3 results in a severely altered morphogenesis of the multicellular organism at the late mound stage. Direct measurement of cell motility within the mound shows that rapGAP3⁻ cells have a reduced speed of movement and, compared to wild-type cells, have a reduced motility towards the apex. rapGAP3⁻ cells exhibit some increased EDTA/EGTA sensitive cell-cell adhesion at the late mound stage. RapGAP3 transiently and rapidly translocates to the cell cortex in response to chemoattractant stimulation, which is dependent on F-actin polymerization. We suggest that the altered morphogenesis and the cell-sorting defect of rapGAP3⁻ cells may result in reduced directional movement of the mutant cells to the apex of the mound.     

Ca signaling regulates ecmB expression,cell differentiation and slug regeneration in Dictyostelium

Ca²⁺ regulates cell differentiation and morphogenesis in a diversity of organisms and dysregulation of Ca²⁺ signal transduction pathways leads to many cellular pathologies. In Dictyostelium Ca²⁺ induces ecmB expression and stalk cell differentiation in vitro. Here we have analyzed the pattern of ecmB expression in intact and bisected slugs and the effect of agents that affect Ca²⁺ levels or antagonize calmodulin (CaM) on this expression pattern. We have shown that Ca²⁺ and CaM regulate ecmB expression and pstAB/pstB cell differentiation in vivo. Agents that increase intracellular Ca²⁺ levels increased ecmB expression and/or pstAB and pstB cell differentiation, while agents that decrease intracellular Ca²⁺ or antagonize CaM decreased it. In isolated slug tips agents that affect Ca²⁺ levels and antagonize CaM had differential effect on ecmB expression and cell differentiation in the anterior versus posterior zones. Agents that increase intracellular Ca²⁺ levels increased the number of ecmB expressing cells in the anterior region of slugs, while agents that decrease intracellular Ca²⁺ levels or antagonize CaM activity increased the number of ecmB expressing cells in the posterior. We have also demonstrated that agents that affect Ca²⁺ levels or antagonize CaM affect cells motility and regeneration of shape in isolated slug tips and backs and regeneration of tips in isolated slug backs. To our knowledge, this is the first study detailing the pattern of ecmB expression in regenerating slugs as well as the role of Ca²⁺ and CaM in the regeneration process and ecmB expression.     

RapGAP9 regulation of the morphogenesis and development in Dictyostelium

Recent reports have demonstrated that the importance of Rap1-specific GTPase-activating proteins (GAPs) in the spatial and temporal regulation of Rap1 activity during cell migration and development in Dictyostelium. Here, we identified another putative Rap1 GAP-domain containing protein, showing high sequence homologies with those of human Rap1GAP and Dictyotelium RapGAP3, by bioinformatic search. Loss of RapGAP9 resulted in some defects in morphogenesis and development in Dicytostelium. rapGAP9 null cells were more flattened and spread, and highly multinucleated. Compared to wild-type cells, cells lacking RapGAP9 exhibited increased levels of F-actin and more filopodia. These results suggest that RapGAP9 is involved in the regulation of cytoskeleton reorganization and cytokinesis. rapGAP9 null cells showed a small increase of cell–substratum attachment and slightly lower moving speed and directionality compared to wild-type cells. In addition, the loss of RapGAP9 resulted in an altered morphology of fruiting body with a shorter length of stalk and spore. Identification and characterization of RapGAP9 in this study will provide further insights into the molecular mechanism by which Rap1 regulates cytoskeleton reorganization and morphogenesis in Dictyostelium.     

An EGF-like peptide sequence from Dictyostelium enhances cell motility and chemotaxis

Dictyostelium discoideum possesses more EGF-like (EGFL) domains than any other sequenced eukaryote. Here we show that a synthetic EGFL peptide (DdEGFL1) based upon an amino acid sequence from a cysteine-rich Dictyostelium protein, functions extracellularly to enhance random cell motility and cAMP-mediated chemotaxis in Dictyostelium by 625% and 85%, respectively, in strain NC4 and by 620% and 80% in strain AX3. Quinacrine inhibited peptide-enhanced random motility but not chemotaxis in strain AX3 providing evidence that PLA2 is the predominant regulator of this process. While LY294002 alone had no significant effect on either event, in combination with quinacrine it dramatically inhibited both processes suggesting that both PI3K and PLA2-mediated signaling are required for EGFL peptide-enhanced cell movement. DdEGFL1 also sustained the threonine phosphorylation of a 210kDa protein that is dephosphorylated during Dictyostelium starvation. Taken together, these results suggest an important role for certain EGFL peptides in Dictyostelium cell movement.     

The interplay between SUCLA2, SUCLG2, and mitochondrial DNA depletion

SUCLA2-related mitochondrial DNA (mtDNA) depletion syndrome is a result of mutations in the β subunit of the ADP-dependent isoform of the Krebs cycle succinyl-CoA synthase (SCS). The mechanism of tissue specificity and mtDNA depletion is elusive but complementation by the GDP-dependent isoform encoded by SUCLG2, and the association with mitochondrial nucleoside diphosphate kinase (NDPK), is a plausible link.We have investigated this relationship by studying SUCLA2 deficient fibroblasts derived from patients and detected normal mtDNA content and normal NDPK activity. However, knockdown of SUCLG2 by shRNA in both patient and control fibroblasts resulted in a significant decrease in mtDNA amount, decreased NDPK and cytochrome c oxidase activities, and a marked growth impairment. This suggests that, SUCLG2, to a higher degree than SUCLA2, is crucial for mtDNA maintenance and that mitochondrial NDPK is involved. Although results pertain to a cell culture system, the findings might explain the pathomechanism and tissue specificity in mtDNA depletion caused by defective SUCLA2.     

Role of Bmbuffy in hydroxycamptothecine-induced apoptosis in BmN-SWU1 cells of the silkworm, Bombyx mori

Bcl-2 family proteins have been reported previously to play important roles in the mitochondrial apoptotic pathway. Particularly, Bmbuffy has been identified as a key homologue of Bcl-2 in silkworm; however, its exact function is unknown. In this study, we investigated the role of Bmbuffy in hydroxycamptothecine (HCPT)-induced apoptosis of BmN-SWU1 cells. By conducting confocal microscopy studies, we found that Bmbuffy is located on the outer membrane of mitochondria and endoplasmic reticulum (ER). Furthermore, we discovered that the hydrophobic transmembrane domain at the COOH terminus is a putative anchor for the subcellular localization of Bmbuffy. Overexpression of Bmbuffy inhibited cytochrome c release, activation of caspase-3 and cell apoptosis, while RNAi-mediated silencing of Bmbuffy promoted apoptosis. In the absence of a hydrophobic membrane anchor, we revealed that Bmbuffy is unable to block apoptosis. These results indicate that Bmbuffy acts as an anti-apoptotic protein, located on the mitochondrial outer membrane and is involved in the mitochondrial apoptotic pathway. Moreover, in HCPT-induced apoptosis, we showed that the translocation of endogenous Bmp53 from the nucleus to the mitochondria is a slow and progressive process, followed by cytochrome c release. This suggests that mitochondrial Bmp53 accumulation may contribute to membrane permeability. The co-localization of Bmp53 and Bmbuffy suggests the interaction of the two proteins, which was further confirmed by Co-IP assay. In addition, overexpression of Bmp53 increased cytochrome c release and the cell apoptotic rate, whereas Bmbuffy overexpression blocked these. All the data suggest that Bmbuffy functions as an anti-apoptotic protein and interacts with Bmp53 in HCPT-induced apoptosis of silkworm cells.     

Genetic structure of Phlebotomus (Larroussius) ariasi populations, the vector of Leishmania infantum in the western Mediterranean: Epidemiological implications

In recent years there has been growing interest in analyzing the geographical variations between populations of different Phlebotomus spp. by comparing the sequences of various genes. However, little is known about the genetic structure of Phlebotomus ariasi. In this study, we were able to sequence a fragment of the mitochondrial Cyt b gene in 133 sandflies morphologically identified as P. ariasi and proceeding from a wide geographical range covering 35 locations in 11 different regions from five countries. The intra-specific diversity of P. ariasi is high, with 45 haplotypes differing from each other by one to 26 bases and they are distributed in two mitochondrial lineages, one limited geographically to Algeria and the other widely dispersed across Mediterranean countries. The Algerian lineage is characterized by having 13 fixed polymorphisms and is made up of one sole haplotype. The European/Moroccan P. ariasi lineage is characterized by being made up of a great diversity of haplotypes (44) which display some geographical structuring. This could be one of the multiple factors involved in the epidemiological heterogeneity of the foci of leishmaniasis. Phlebotomus chadlii is the sister group of European/Moroccan P. ariasi. The separation of the Algerian haplotype, H45, from the rest of the specimens, European/Moroccan P. ariasi and P. chadlii, is well supported by the bootstrap analysis.     

Targeted gene deletion in Aspergillus fumigatus using microbial machinery and a recyclable marker

The emerging invasive fungal pathogen Aspergillus fumigatus causes very serious infections among immunocompromised patient populations. While the genome of this pathogen has been sequenced, a major barrier to better understanding the complex biology of this eukaryotic organism is a lack of tools for efficient genetic manipulation. To improve upon this, we have generated a new gene deletion system for A. fumigatus using yeast recombinational cloning and Agrobacterium tumefaciens mediated transformation (ATMT) employing a recyclable marker system. This system reduced the time for generating a gene deletion strain in our hands by two-thirds (12 weeks to 3 weeks) using minimal human labor, and we demonstrate that it can be used to efficiently generate multiple gene deletions within a single strain.     

Phylogeny of Phaeomollisia piceae gen. sp. nov.: a dark, septate, conifer-needle endophyte and its relationships to Phialocephala and Acephala

Dark, septate endophytes (DSE) were isolated from roots and needles of dwarf Picea abies and from roots of Vaccinium spp. growing on a permafrost site in the Jura Mountains in Switzerland. Two of the isolates sporulated after incubation for more than one year at 4 °C. One of them was a hitherto undescribed helotialean ascomycete Phaeomollisia piceae gen. sp. nov., the other was a new species of Phialocephala, P. glacialis sp. nov. Both species are closely related to DSE of the Phialocephala fortinii s. lat.-Acephala applanata species complex (PAC) as revealed by phylogenetic analyses of the ITS and 18S rDNA regions. Morphologically dissimilar fungi, such as Vibrissea and Loramyces species, are phylogenetically also closely linked to the new species and the PAC. Cadophora lagerbergii and C. (Phialophora) botulispora are moved to Phialocephala because Phialocephala dimorphospora and P. repens are the closest relatives. Several Mollisia species were closely related to the new species and the PAC according to ITS sequence comparisons. One DSE from needles of Abies alba and one from shoots of Castanea sativa formed Cystodendron anamorphs in culture. Their identical 18S sequences and almost identical ITS sequences indicated Mollisia species as closest relatives, suggesting that Mollisia species are highly euryoecious.     

Diversity,host associations,and phylogeography of temperate aurofusarin-producing Hypomyces/Cladobotryum including causal agents of cobweb disease of cultivated mushrooms

Temperate species of Hypomyces and Cladobotryum that produce the red pigment aurofusarin are common on agaricoid and polyporoid basidiomata of species from five orders of Agaricomycetes. Several cause cobweb disease of commercially cultivated mushrooms resulting in serious losses. We sequenced rpb1, rpb2, tef1, and FG1093 regions in 90 wild strains and 30 strains from mushroom farms, isolated from Europe, North America, Africa, Asia, Australia, and New Zealand. Multigene analyses support the distinctness of five species but reveal Hypomyces rosellus to be paraphyletic, comprising several cryptic lineages. Hypomyces rosellus s. str. is characterised by wide dispersal and gene flow across Eurasia but does not occur in North America. Instead, the lineages from the West and the East Coast appear distinct, having given rise to species inhabiting the Southern Hemisphere. Our results reveal wide misuse of the name H. rosellus, especially for cobweb isolates. The majority of these belong to Hypomyces odoratus, including a weakly supported group of fungicide-resistant strains from Europe and North America sharing identical sequence data. New collections are presented for Cladobotryum rubrobrunnescens and Cladobotryum tenue as well as Cladobotryum multiseptatum and Hypomyces dactylarioides, all previously known only from their type material. The former species pair occurs in Europe and the latter in Australia and New Zealand. Separate lineages appear to be maintained by geographic isolation in North America and temperate Australasia but by host specialisation in the species occurring sympatrically in Europe and Asia. Both specialist and generalist host use strategies have evolved in the group. Although teleomorphs are known in most of the species and unnamed lineages, analyses of the five-gene regions suggest the prevalence of clonal reproduction in H. odoratus. This can be the reason for its success in mushroom farms, also facilitating the spread of fungicide resistance. While tef1 and rpb2 can be recommended for species delimitation, low variation, not exceeding 1 % in the whole ingroup, impeaches the use of ITS as a barcoding gene region in this group of fungi.     

Mycobiota associated with the ambrosia beetle Scolytodes unipunctatus (Coleoptera: Curculionidae, Scolytinae)

Ambrosia beetles (Coleoptera: Scolytinae) are associated with strictly entomochoric and mutualistic fungi. We studied the mycobiota associated with Scolytodes unipunctatus, ambrosia beetles that infest Cecropia trees in Central America. Isolates were characterized using morphology and rDNA sequences (ITS region, LSU, and SSU rDNA). Four species are described here: Raffaelea scolytodis sp. nov. (Ophiostomatales), Gondwanamyces scolytodis sp. nov., Custingophora cecropiae sp. nov., and Graphium sp. (Microascales). The genus Custingophora is emended to include Knoxdaviesia anamorphs of Gondwanamyces based on uniformity of DNA sequences and phenotype.     

Starch grain morphology of the seagrasses Halodule wrightii, Ruppia maritima, Syringodium filiforme, and Thalassia testudinum

Starch grains are a ubiquitous component of plants that have been used in tandem with phytoliths, pollen, and macrofossils to reconstruct past floral diversity. This tool has yet to be fully explored for aquatic plants, specifically seagrasses, which lack phytoliths and are rarely preserved as macrofossils or pollen. If starch grains in seagrasses are morphologically distinct, this method has the potential to improve seagrass identification in the fossil record in such cases where its starch is preserved (e.g. scratches and occlusal surfaces of tooth enamel from seagrass consumers). The goals of this study were twofold: (1) to determine if starch is present in seagrass material and (2) to assess how starch grain morphology differs between different seagrasses.This study focused on four abundant and ecologically distinct seagrasses from the Caribbean: Halodule wrightii, Ruppia maritima, Syringodium filiforme, and Thalassia testudinum. Starch grains were observed in all species except S. filiforme. Grains from H. wrightii are typically observed in side-on orientation, are sub-round to angular, and are fairly small (3-19 μm, end-on). Grains of R. maritima are small spherical grains (4-8 μm) that have a centric hilum and a straight extinction cross with a median angle between the arms of 90°. Grains from T. testudinum are large (9-31 μm, end-on), conical in side-on and round/sub-round in end-on orientation, have a slightly eccentric hilum with an obvious particle, and prominent lamellae.Visual assessment and comparative statistics demonstrate that the morphology of starch grains from T. testudinum, R. maritima, and H. wrightii are significantly different. With more extensive research, there is potential for the positive identification of starch grains from an unknown seagrass. The ability to identify seagrass from starch grains could facilitate the identification of seagrasses in the fossil record and supply information on seagrass evolution and distribution, climate effects on seagrass distribution, and the diets of seagrass consumers.     

Phylogenetic reassessment of Hyaloscyphaceae sensu lato (Helotiales, Leotiomycetes) based on multigene analyses

Hyaloscyphaceae is the largest family in Helotiales, Leotiomycetes. It is mainly characterized by minute apothecia with well-differentiated hairs, but its taxonomic delimitation and infrafamilial classification remain ambiguous. This study performed molecular phylogenetic analyses using multiple genes including the ITS-5.8S rDNA, the D1–D2 region of large subunit of rDNA, RNA polymerase II subunit 2, and the mitochondrial small subunit. The primary objective was to evaluate the phylogenetic utility of morphological characters traditionally used in the taxonomy of Hyaloscyphaceae through reassessment of the monophyly of this family and its genera. The phylogenetic analyses inferred Hyaloscyphaceae as being a heterogeneous assemblage of a diverse group of fungi and not supported as monophyletic. Among the three tribes of Hyaloscyphaceae only Lachneae formed a monophyletic lineage. The presence of hairs is rejected as a synapomorphy, since morphologically diversified hairs have originated independently during the evolution of Helotiales. The true- and false-subiculum in Arachnopezizeae are hypothesized to have evolved through different evolutionary processes; the true-subiculum is likely the product of a single evolutionary origin, while the false-subiculum is hypothesized to have originated multiple times. Since Hyaloscyphaceae sensu lato was not resolved as monophyletic, Hyaloscyphaceae sensu stricto is redefined and only applied to the genus Hyaloscypha.