Establishment of the monomeric yellow-green fluorescent protein mNeonGreen for life cell imaging in mycelial fungi

The engineered monomeric version of the lancelet Branchiostoma lanceolatum fluorescent protein, mNeonGreen (mNG), has several positive characteristics, such as a very bright fluorescence, high photostability and fast maturation. These features make it a good candidate for the utilization as fluorescent tool for cell biology and biochemical applications in filamentous fungi. We report the generation of plasmids for the expression of the heterologous mNG gene under the control of an inducible and a constitutive promoter in the filamentous ascomycete Sordaria macrospora and display a stable expression of mNG in the cytoplasm. To demonstrate its usefulness for labeling of organelles, the peroxisomal targeting sequence serine-lysine-leucine (SKL) was fused to mNG. Expression of this tagged version led to protein import of mNG into peroxisomes and their bright fluorescence in life cell imaging.

Palmitoylated mNeonGreen Protein as a Tool for Visualization and Uptake Studies of Extracellular Vesicles

Extracellular vesicles (EVs) are membranous nanoparticles released by cells as vital mediators of intercellular communication. As such, EVs have become an attractive target for pathogens and cancer cells, which can take control over their cargo composition, as well as their trafficking, shaping the pathogenesis. Despite almost four decades of research on EVs, the number of specific and efficient EV labeling methods is limited, and there is still no universal method for the visualization of their transport in living cells. Lipophilic dyes that non-specifically intercalate into the EVs membranes may diffuse to other membranes, leading to the misinterpretation of the results. Here, we propose a palmitoylated fluorescent mNeonGreen (palmNG) protein as an alternative to chemical dyes for EVs visualization. The Branchiostoma lanceolatum-derived mNeonGreen is a brighter, more stable, and less sensitive to laser-induced bleaching alternative to green fluorescent protein (GFP), which makes it a more potent tag in a variety of fluorescence-based techniques. A palmNG-expressing stable human melanoma cell line was generated using retrovirus gene transfer and cell sorting. This protein partially localizes to cellular membranes, and can be detected inside size-exclusion (SEC)-purified EVs. With the use of flow cytometry and fluorescent confocal microscopy, we performed qualitative and quantitative analyses of palmNG-EVs uptake in recipient human hepatoma cells, in comparison to PKH67-labeled vesicles. Our findings confirm that membrane-embedded mNeonGreen can be successfully applied as a tool in EVs transfer and uptake studies.

An updated staging system for cephalochordate development: one table suits them all

BackgroundThe chordates are divided into three subphyla: Vertebrata, Tunicata and Cephalochordata. Phylogenetically, the Cephalochordata, more commonly known as lancelets or amphioxus, constitute the sister group of Vertebrata plus Tunicata. Due to their phylogenetic position and their conserved morphology and genome architecture, lancelets are important models for understanding the evolutionary history of chordates. Lancelets are small, marine filter-feeders, and the few dozen species that have so far been described have been grouped into three genera: Branchiostoma, Epigonichthys and Asymmetron. Given their relevance for addressing questions about the evolutionary diversification of chordates, lancelets have been the subjects of study by generations of scientists, with the first descriptions of adult anatomy and developmental morphology dating back to the 19th century. Today, several different lancelet species are used as laboratory models, predominantly for developmental, molecular and genomic studies. It is thus very surprising that there is currently no universal staging system and no unambiguous nomenclature for developing lancelets.ResultsWe illustrated the development of the European amphioxus (Branchiostoma lanceolatum) using confocal microscopy and compiled a streamlined developmental staging system, from fertilization through larval life, with an unambiguous stage nomenclature. By tracing growth curves of the European amphioxus reared at different temperatures, we were able to show that our staging system permits the easy conversion of any developmental time into a defined stage name. Furthermore, comparisons of embryos and larvae from the European amphioxus (B. lanceolatum), the Florida amphioxus (B. floridae), the Chinese amphioxus (B. belcheri), the Japanese amphioxus (B. japonicum) and the Bahamas lancelet (Asymmetron lucayanum) demonstrated that our staging system can readily be applied to other lancelet species.ConclusionsHere, we propose an updated staging and nomenclature system for lancelets. Although the detailed staging description was carried out on developing B. lanceolatum, comparisons with other lancelet species strongly suggest that both staging and nomenclature are applicable to all extant lancelets. We thus believe that this description of embryonic and larval development can be of great use for the scientific community and hope that it will become the new standard for defining and naming developing lancelets.

New records of the lancelet Branchiostoma lanceolatum in Scottish waters

New records of the lancelet Branchiostoma lanceolatum from Scottish waters are presented. Most of the records originate from sublittoral monitoring around fish farms from Orkney, Shetland, the Western Isles, the Isles of Skye and Mull, but also from a distillery discharge in the Firth of Clyde and a plankton survey in the Sea of the Hebrides. Lancelets were recovered in sediment grab samples from 6 - 60 m depth. Some recent accounts of intertidal lancelets are also cited. The lancelets appear to prefer coarser sediments and in the fish farm surveys were found predominantly at reference sites, away from the immediate influence of farm deposition.

Morphological Stasis and Proteome Innovation in Cephalochordates

Lancelets, extant representatives of basal chordates, are prototypic examples of evolutionary stasis; they preserved a morphology and body-plan most similar to the fossil chordates from the early Cambrian. Such a low level of morphological evolution is in harmony with a low rate of amino acid substitution; cephalochordate proteins were shown to evolve slower than those of the slowest evolving vertebrate, the elephant shark. Surprisingly, a study comparing the predicted proteomes of Chinese amphioxus, Branchiostoma belcheri and the Florida amphioxus, Branchiostoma floridae has led to the conclusion that the rate of creation of novel domain combinations is orders of magnitude greater in lancelets than in any other Metazoa, a finding that contradicts the notion that high rates of protein innovation are usually associated with major evolutionary innovations. Our earlier studies on a representative sample of proteins have provided evidence suggesting that the differences in the domain architectures of predicted proteins of these two lancelet species reflect annotation errors, rather than true innovations. In the present work, we have extended these studies to include a larger sample of genes and two additional lancelet species, Asymmetron lucayanum and Branchiostoma lanceolatum. These analyses have confirmed that the domain architecture differences of orthologous proteins of the four lancelet species are because of errors of gene prediction, the error rate in the given species being inversely related to the quality of the transcriptome dataset that was used to aid gene prediction.

Functional lability of RNA-dependent RNA polymerases in animals

RNA interference (RNAi) requires RNA-dependent RNA polymerases (RdRPs) in many eukaryotes, and RNAi amplification constitutes the only known function for eukaryotic RdRPs. Yet in animals, classical model organisms can elicit RNAi without possessing RdRPs, and only nematode RNAi was shown to require RdRPs. Here we show that RdRP genes are much more common in animals than previously thought, even in insects, where they had been assumed not to exist. RdRP genes were present in the ancestors of numerous clades, and they were subsequently lost at a high frequency. In order to probe the function of RdRPs in a deuterostome (the cephalochordate Branchiostoma lanceolatum), we performed high-throughput analyses of small RNAs from various Branchiostoma developmental stages. Our results show that Branchiostoma RdRPs do not appear to participate in RNAi: we did not detect any candidate small RNA population exhibiting classical siRNA length or sequence features. Our results show that RdRPs have been independently lost in dozens of animal clades, and even in a clade where they have been conserved (cephalochordates) their function in RNAi amplification is not preserved. Such a dramatic functional variability reveals an unexpected plasticity in RNA silencing pathways.Author summaryRNA interference (RNAi) is a conserved gene regulation system in eukaryotes. In non-animal eukaryotes, it necessitates RNA-dependent RNA polymerases (”RdRPs”). Among animals, only nematodes appear to require RdRPs for RNAi. Yet additional animal clades have RdRPs and it is assumed that they participate in RNAi. Here, we find that RdRPs are much more common in animals than previously thought, but their genes were independently lost in many lineages. Focusing on a species with RdRP genes (a cephalochordate), we found that it does not use them for RNAi. While RNAi is the only known function for eukaryotic RdRPs, our results suggest additional roles. Eukaryotic RdRPs thus have a complex evolutionary history in animals, with frequent independent losses and apparent functional diversification.

Revisited Syllidae of the English Channel coarse sediment communities

Among the polychaetes, the Syllidae comprise numerous species whose study over many years has benefitted from valuable revisions and descriptions of new species in Europe. This abundant literature proves very useful for revisiting the taxonomy and distribution of the Syllidae in the English Channel (EC), mainly as regards existing studies on coarse sediment communities in the eastern part of the Channel. This habitat is one of the most widespread in the EC and is known as favourable for the small polychaete fauna including Syllidae. A 2-year survey (winter and summer sampling periods) covering 19 stations, associated with theBranchiostoma lanceolatumcoarse sand community offshore Dieppe-Le Tréport, led to the identification of 6537 individuals from 29 taxa including 27 species. Six fine sand stations were also sampled in which only 12 individuals were collected. Among these species, seven are new for the EC polychaete fauna and six others are observed for the first time in the eastern part of the EC. All the new species for the EC are warm temperate species previously only known south of the Bay of Biscay. The Syllidae list given in Dauvinet al. (2003) has been re-analysed and amended with our list and that of the Chausey Archipelago study (Olivieret al., 2012). To date, 91 Syllidae species have been recorded in the EC and are established mostly in coarse sediments.