Snap-, CLIP- and Halo-Tag Labelling of Budding Yeast Cells

Fluorescence microscopy of the localization and the spatial and temporal dynamics of specifically labelled proteins is an indispensable tool in cell biology. Besides fluorescent proteins as tags, tag-mediated labelling utilizing self-labelling proteins as the SNAP-, CLIP-, or the Halo-tag are widely used, flexible labelling systems relying on exogenously supplied fluorophores. Unfortunately, labelling of live budding yeast cells proved to be challenging with these approaches because of the limited accessibility of the cell interior to the dyes. In this study we developed a fast and reliable electroporation-based labelling protocol for living budding yeast cells expressing SNAP-, CLIP-, or Halo-tagged fusion proteins. For the Halo-tag, we demonstrate that it is crucial to use the 6′-carboxy isomers and not the 5′-carboxy isomers of important dyes to ensure cell viability. We report on a simple rule for the analysis of ¹H NMR spectra to discriminate between 6′- and 5′-carboxy isomers of fluorescein and rhodamine derivatives. We demonstrate the usability of the labelling protocol by imaging yeast cells with STED super-resolution microscopy and dual colour live cell microscopy. The large number of available fluorophores for these self-labelling proteins and the simplicity of the protocol described here expands the available toolbox for the model organism Saccharomyces cerevisiae.     

RNA at DNA Double-Strand Breaks: The Challenge of Dealing with DNA:RNA Hybrids

RNA polymerase II is recruited to DNA double-strand breaks (DSBs), transcribes the sequences that flank the break and produces a novel RNA type that has been termed damage-induced long non-coding RNA (dilncRNA). DilncRNAs can be processed into short, miRNA-like molecules or degraded by different ribonucleases. They can also form double-stranded RNAs or DNA:RNA hybrids. The DNA:RNA hybrids formed at DSBs contribute to the recruitment of repair factors during the early steps of homologous recombination (HR) and, in this way, contribute to the accuracy of the DNA repair. However, if not resolved, the DNA:RNA hybrids are highly mutagenic and prevent the recruitment of later HR factors. Here recent discoveries about the synthesis, processing, and degradation of dilncRNAs are revised. The focus is on RNA clearance, a necessary step for the successful repair of DSBs and the aim is to reconcile contradictory findings on the effects of dilncRNAs and DNA:RNA hybrids in HR.     

Oscillations in a white blood cell production model with multiple differentiation stages

Journal of Mathematical Biology - In this work we prove occurrence of a super-critical Hopf bifurcation in a model of white blood cell formation structured by three maturation stages. We provide an...     

Assessment of novel binocular colour, motion and contrast tests in glaucoma

The effects of glaucoma on binocular visual sensitivity for the detection of various stimulus attributes are investigated at the fovea and in four paracentral retinal regions. The study employed a number of visual stimuli designed to isolate the processing of various stimulus attributes. We measured absolute contrast detection thresholds and functional contrast sensitivity by using Landolt ring stimuli. This psychophysical Landolt C-based contrast test of detection and gap discrimination allowed us to test parafoveally at 6 ° from fixation and foveally by employing interleaved testing locations. First-order motion perception was examined by using moving stimuli embedded in static luminance contrast noise. Red/green (RG) and yellow/blue (YB) colour thresholds were measured with the Colour Assessment and Diagnosis (CAD) test, which utilises random dynamic luminance contrast noise (± 45 %) to ensure that only colour and not luminance signals are available for target detection. Subjects were normal controls (n?=?65) and glaucoma patients with binocular visual field defects (n?=?15) classified based on their Humphrey Field Analyzer mean deviation (MD) scores. The impairment of visual function varied depending on the stimulus attribute and location tested. Progression of loss was noted for all tests as the degree of glaucoma increased. For subjects with mild glaucoma (MD ?0.01 dB to ?6.00 dB) significantly more data points fell outside the normal age-representative range for RG colour thresholds than for any other visual test, followed by motion thresholds. This was particularly the case for the parafoveal data compared with the foveal data. Thus, a multifaceted measure of binocular visual performance, incorporating RG colour and motion test at multiple locations, might provide a better index for comparison with quality of life measures in glaucoma.     

The Collagen-binding Integrin α2β1 Is a Novel Interaction Partner of the Trimeresurus flavoviridis Venom Protein Flavocetin-A

Many snake venoms are known for their antithrombotic activity. They contain components that specifically target different platelet-activating receptors such as the collagen-binding integrin α2β1 and the von Willebrand factor receptor GPIb. In a search for an α2β1 integrin-blocking component from the venom of the habu snake (Trimeresurus flavoviridis), we employed two independent purification protocols. First, we used the integrin α2A domain, a major collagen-binding domain, as bait for affinity purification of an α2β1 integrin-binding toxin from the crude venom. Second, in parallel, we used classical protein separation protocols and tested for α2β1 integrin-inhibiting capabilities by ELISA. Using both approaches, we identified flavocetin-A as an inhibitor of α2β1 integrin. Hitherto, flavocetin-A has been reported as a GPIb inhibitor. However, flavocetin-A inhibited collagen-induced platelet aggregation even after GPIb was blocked with other inhibitors. Moreover, flavocetin-A antagonized α2β1 integrin-mediated adhesion and migration of HT1080 human fibrosarcoma cells, which lack any GPIb, on collagen. Protein chemical analyses proved that flavocetin-A binds to α2β1 integrin and its α2A domain with high affinity and in a cooperative manner, which most likely is due to its quaternary structure. Kinetic measurements confirmed the formation of a strong complex between integrin and flavocetin-A, which dissociates very slowly. This study proves that flavocetin-A, which has long been known as a GPIb inhibitor, efficiently targets α2β1 integrin and thus blocks collagen-induced platelet activation. Moreover, our findings suggest that the separation of GPIb- and α2β1 integrin-blocking members within the C-type lectin-related protein family is less strict than previously assumed.     

An ITS-based phylogenetic framework for the genus Vorticella: finding the molecular and morphological gaps in a taxonomically difficult group

Vorticella includes more than 100 currently recognized species and represents one of the most taxonomically challenging genera of ciliates. Molecular phylogenetic analysis of Vorticella has been performed so far with only sequences coding for small subunit ribosomal RNA (SSU rRNA); only a few of its species have been investigated using other genetic markers owing to a lack of similar sequences for comparison. Consequently, phylogenetic relationships within the genus remain unclear, and molecular discrimination between morphospecies is often difficult because most regions of the SSU rRNA gene are too highly conserved to be helpful. In this paper, we move molecular systematics for this group of ciliates to the infrageneric level by sequencing additional molecular markers—fast-evolving internal transcribed spacer (ITS) regions—in a broad sample of 66 individual samples of 28 morphospecies of Vorticella collected from Asia, North America and Europe. Our phylogenies all featured two strongly supported, highly divergent, paraphyletic clades (I, II) comprising the morphologically defined genus Vorticella. Three major lineages made up clade I, with a relatively well-resolved branching order in each one. The marked divergence of clade II from clade I confirms that the former should be recognized as a separate taxonomic unit as indicated by SSU rRNA phylogenies. We made the first attempt to elucidate relationships between species in clade II using both morphological and multi-gene approaches, and our data supported a close relationship between some morphospecies of Vorticella and Opisthonecta, indicating that relationships between species in the clade are far more complex than would be expected from their morphology. Different patterns of helix III of ITS2 secondary structure were clearly specific to clades and subclades of Vorticella and, therefore, may prove useful for resolving phylogenetic relationships in other groups of ciliates.     

Mechanistic insights into the superoxide–cytochrome c reaction by lysine surface scanning

This study summarizes results which have been obtained by a mutational study of human cytochrome c. The protein can be used as a recognition element in analytical assays and biosensors for superoxide radicals since ferricytochrome c reacts with superoxide to form ferrocytochrome c and oxygen. Here lysine mutagenesis of the distal surface (i.e., of exposed residues around the Met80 axial ligand) of human cytochrome c was pursued to evaluate the effect of the surface charges on the reaction rate with the superoxide anion radical and on the redox properties of the heme protein. The latter feature is particularly relevant when the protein is immobilized on a negatively charged self-assembled monolayer on an electrode to be used as a biosensor. The observed effects of the mutations are rationalized through structural investigations based on solution NMR spectroscopy and computational analysis of the surface electrostatics. The results suggest the presence of a specific path that guides superoxide toward an efficient reaction site. Localized positive charges at the rim of the entry channel are effective in increasing the reaction rate, whereas diffused positive charges or charges far from this area are not effective or are even detrimental, resulting in a misguided approach of the anion to the protein surface.     

Vegetation controls methane emissions in a coastal brackish fen

Climate change and associated sea level rise will likely affect coastal ecosystems and lead to more frequent inundations. Plants are an important control for methane (CH₄) emissions in peatlands because the metabolism of the living plant can either enhance or attenuate CH₄ emissions and plant litter supplies an easily available carbon source for methanogenesis. Here we compare the contribution of various dominant plant species to methane emissions in a degraded, rewetted coastal brackish fen at the southern Baltic Sea coast in Northeast Germany. We analyse one year of bi-weekly static closed chamber data gathered at measurement spots that were located in different mono-dominant vegetation stands (Bolboschoenus maritimus (L.) Palla, Schoenoplectus tabernaemontani (C.C.Gmel.) Palla, Carex acutiformis Ehrh.). Furthermore, data on water level, water temperature, conductivity (sulphate), and several peat characteristics were recorded. Generally, the annual methane emissions were low with an average across vegetation stands of 14 kg CH₄ ha^?1 a^?1, which we related to high decomposition of peat after drainage and to relatively low water levels in summer. Nevertheless, methane emissions varied between different vegetation types with significantly higher methane fluxes (31.8 ± 5.7 kg CH₄ ha^?1 a^?1) from Bolboschoenus maritimus stands compared to Carex acutiformis and Schoenoplectus tabernaemontani stands (4.3 ± 1.2 and 5.7 ± 2.4 kg CH₄ ha^?1 a^?1, respectively). None of the environmental variables that have been recorded can explain this difference. Thus, vegetation composition seems to be an important driver for methane emissions in coastal brackish fens and may therefore be crucial with regard to recreation measures.     

Hooked on fat: the role of lipid synthesis in cancer metabolism and tumour development

An increased rate of lipid synthesis in cancerous tissues has long been recognised as an important aspect of the rewired metabolism of transformed cells. However, the contribution of lipids to cellular transformation, tumour development and tumour progression, as well as their potential role in facilitating the spread of cancerous cells to secondary sites, are not yet fully understood. In this article, we review the recent findings that support the importance of lipid synthesis and metabolism in tumorigenesis. Specifically, we explore the role of aberrant lipid biosynthesis in cancer cell migration and invasion, and in the induction of tumour angiogenesis. These processes are crucial for the dissemination of tumour cells and formation of metastases, which constitute the main cause of cancer mortality.