Perception of riverscapes: sensual experience and knowledge

International audienc

Perception of riverscapes: sensual experience and knowledge

International audienc

Difficulties in Generating Specific Antibodies for Immunohistochemical Detection of Nitrosylated Tubulins

<div><p>Protein S-nitrosylation, the covalent attachment of a nitroso moiety to thiol groups of specific cysteine residues, is one of the major pathways of nitric oxide signaling. Hundreds of proteins are subject to this transient post-translational modification and for some the functional consequences have been identified. Biochemical assays for the analysis of protein S-nitrosylation have been established and can be used to study if and under what conditions a given protein is S-nitrosylated. In contrast, the equally desirable subcellular localization of specific S-nitrosylated protein isoforms has not been achieved to date. In the current study we attempted to specifically localize S-nitrosylated α- and β-tubulin isoforms in primary neurons after fixation. The approach was based on <i>in situ</i> replacement of the labile cysteine nitroso modification with a stable tag and the subsequent use of antibodies which recognize the tag in the context of the tubulin polypeptide sequence flanking the cysteine residue of interest. We established a procedure for tagging S-nitrosylated proteins in cultured primary neurons and obtained polyclonal anti-tag antibodies capable of specifically detecting tagged proteins on immunoblots and in fixed cells. However, the antibodies were not specific for tubulin isoforms. We suggest that different tagging strategies or alternative methods such as fluorescence resonance energy transfer techniques might be more successful.</p></div

Characterization of antisera raised against tagged tubulin peptides.

<p>(A) Schematic representation of immunogens and inhibitor-T. Antibodies directed against an epitope contained within the area shaded in green would detect either just the tag or tagged cysteines in a context-unspecific manner. The reactivity of these antibodies can be blocked by inhibitor-T, an unrelated peptide carrying the biotin tag on its internal cysteine. (B) Lysates of differentiated N2a cells were treated with 100 µM GSNO for 20 min and then subjected to the biotin switch protocol. 17.4 µg of biotinylated lysate was loaded in each lane, fractionated by non-reducing SDS-PAGE and transferred to a nitrocellulose membrane. Following blocking the membrane was cut into stripes and each stripe was then probed with the indicated antiserum diluted at 1∶500 pre-incubated in the presence (<i>+</i>) or absence (−) of inhibitor-T. Successful application of the biotin switch protocol was confirmed with streptavidin-HRP (<i>streptavidin</i>) and the positions of tubulin bands were located with both anti-α- (<i>α-tubulin)</i> and anti-β-tubulin (<i>β-tubulin</i>) antibodies as indicated. Loss in staining after pre-incubation with inhibitor-T of serum #2, #3 and #4 shows that these antisera contain mostly context-unspecific antibodies directed against the tag or tagged cysteine. However, antisera #1 and #4 also bind to a 60-kDa band, the detection of which is insensitive to pre-incubation of the antisera with the non-specific inhibitor-T. This band co-migrated with the upper band detected with pan anti-β-tubulin antibodies in lysates that were subjected to S-nitrosylation and the biotin switch protocol.</p

Immunological detection of protein S-nitrosylation in fixed cells.

<p>Differentiated N2a cells were treated for 20 min with 500 µM GSNO or left untreated as indicated. Following treatment, cells were fixed with cold methanol, subjected to the <i>in situ</i> biotin switch protocol and stained either with antiserum #4 (<i>AS #4</i>) or streptavidin-rhodamine red (<i>streptavidin</i>) as indicated. As a negative control, GSNO treated cells were subjected to the <i>in situ</i> biotin switch protocol without the biotin-HPDP tag (<i>GSNO w/o tag</i>). (A) Optical slices of stained cells. To visualize both high and low intensity areas, staining is shown using an orange-hot LUT. For each detection type (either streptavidin-rhodamine red or antiserum #4) the corresponding high-low intensity color legend is shown. (B) Median intensity per cell normalized to average intensity of the untreated group (error bars 95% of confidence interval). Cells treated with GSNO show a significant elevation of the signal when stained with antiserum #4, indicating the ability of the serum to detect labeled proteins. However, there is high background staining with antiserum #4 in negative control cells that were not labeled with tag. Scale bars, 50 µm.</p

Detection of the 60-kDa band is insensitive to blocking of the antisera with specific inhibitor-TP.

<p>(A) Schematic representation of immunogen-α and the corresponding inhibitor-TPα. Pink shading outlines the epitope of interest. (B) Lysates of differentiated N2a cells were treated with 100 µM GSNO and then subjected to the biotin switch protocol. The resulting biotinylated lysates were fractionated by non-reducing SDS-PAGE and transferred to a nitrocellulose membrane. Following blocking the membrane was cut into stripes and each stripe was then probed with the indicated antiserum (<i>AS</i>) diluted at 1∶500 pre-incubated in the presence (<i>+ inh-TP</i>) or absence of inhibitor-TP. Successful application of the biotin switch protocol was confirmed with anti-biotin antibodies (<i>anti-biotin</i>) or streptavidin-HRP (<i>streptavidin</i>) and the position of tubulin bands was located with anti-β-tubulin antibodies (<i>β-tubulin</i>) as indicated.</p

The <i>in situ</i> biotin switch assay reveals rapid increase in intracellular protein S-nitrosylation after treatment with NO donors.

<p>(A, B) <i>In situ</i> biotin switch assay on fixed cells of established cell lines. Differentiated N2a cells and COS7 cells were treated for 20 min with NO donor (SNAP or GSNO at the indicated µM concentrations), treated with solvent (control for SNAP treatment) or were left untreated (control for GSNO treatment). Following treatment, cells were fixed with cold methanol and subjected to the <i>in situ</i> biotin switch protocol. The resulting biotin-HPDP-tagged proteins were detected with streptavidin-rhodamine red. (A) Optical slices of stained cells. To visualize both high and low intensity areas, streptavidin-rhodamine red is shown using an orange-hot lookup table (LUT). For each cell type the corresponding high-low intensity color legend is shown. (B) Average intensity per cell normalized to average intensity of the control group (error bars 95% of confidence interval). Both cell types show a significant increase in the amount of S-nitrosylated proteins after treatment with NO donors (up to 6-fold). p values are indicated where the effect was significant. (C, D) <i>In situ</i> biotin switch assay in primary neurons. Primary cultures of adult DRG neurons (<i>DRG</i>), P0 cortical neurons (<i>Cortical</i>) or P0 hippocampal neurons (<i>Hippocampal</i>) were treated for 20 min with 500 µM GSNO or with 500 µM GSH as a control. Following treatment, cells were fixed with cold methanol, subjected to the <i>in situ</i> biotin switch protocol and stained with streptavidin-rhodamine red. (C) Optical slices of stained cells. To visualize both high and low intensity areas, streptavidin-rhodamine red is shown using an orange-hot LUT. For each cell type the corresponding high-low intensity color legend is shown. (D) Average intensity per cell normalized to average intensity of the control group (error bars 95% of confidence interval). All cell types show a significant increase in the amount of S-nitrosylated proteins after treatment with NO donor (up to 5-fold). p values are indicated.</p

Concept for specific detection of S-nitrosylated tubulin by a combination of the biotin switch protocol and immunodetection.

<p>(A) 3D structure of the α/β-tubulin heterodimer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068168#pone.0068168-Nogales1" target="_blank">[20]</a>. Positions of cysteine residues found to be S-nitrosylated in murine brain are marked. Cysteine residues are localized on the surface of the dimer (filled circles) as well as inside the polypeptides (open circles). Cys 295 and Cys 303 of α- and β-tubulin, respectively, were chosen for further analysis. (B) Tagging of S-nitrosylated tubulin. Endogenously or exogenously S-nitrosylated cysteine residues (shown here for Cys 295 of α-tubulin) are stably tagged with biotin-HPDP using the biotin switch protocol. (C) Structure of immunogen-α. Pink shading outlines the epitope of interest.</p

BMC Bioinformatics / Quantification of experimentally induced nucleotide conversions in high-throughput sequencing datasets

Background Methods to read out naturally occurring or experimentally introduced nucleic acid modifications are emerging as powerful tools to study dynamic cellular processes. The recovery, quantification and interpretation of such events in high-throughput sequencing datasets demands specialized bioinformatics approaches. Results Here, we present Digital Unmasking of Nucleotide conversions in K-mers (DUNK), a data analysis pipeline enabling the quantification of nucleotide conversions in high-throughput sequencing datasets. We demonstrate using experimentally generated and simulated datasets that DUNK allows constant mapping rates irrespective of nucleotide-conversion rates, promotes the recovery of multimapping reads and employs Single Nucleotide Polymorphism (SNP) masking to uncouple true SNPs from nucleotide conversions to facilitate a robust and sensitive quantification of nucleotide-conversions. As a first application, we implement this strategy as SLAM-DUNK for the analysis of SLAMseq profiles, in which 4-thiouridine-labeled transcripts are detected based on T>C conversions. SLAM-DUNK provides both raw counts of nucleotide-conversion containing reads as well as a base-content and read coverage normalized approach for estimating the fractions of labeled transcripts as readout. Conclusion Beyond providing a readily accessible tool for analyzing SLAMseq and related time-resolved RNA sequencing methods (TimeLapse-seq, TUC-seq), DUNK establishes a broadly applicable strategy for quantifying nucleotide conversions.(VLID)489159

Semisynthetic LC3 Probes for Autophagy Pathways Reveal a Noncanonical LC3 Interacting Region Motif Crucial for the Enzymatic Activity of Human ATG3

Macroautophagy is one of two major degradation systems in eukaryotic cells. Regulation and control of autophagy are often achieved through the presence of short peptide sequences called LC3 interacting regions (LIR) in autophagy-involved proteins. Using a combination of new protein-derived activity-based probes prepared from recombinant LC3 proteins, along with protein modeling and X-ray crystallography of the ATG3-LIR peptide complex, we identified a noncanonical LIR motif in the human E2 enzyme responsible for LC3 lipidation, ATG3. The LIR motif is present in the flexible region of ATG3 and adopts an uncommon beta-sheet structure binding to the backside of LC3. We show that the beta-sheet conformation is crucial for its interaction with LC3 and used this insight to design synthetic macrocyclic peptide-binders to ATG3. CRISPR-enabled in cellulo studies provide evidence that LIRATG3 is required for LC3 lipidation and ATG3 similar to LC3 thioester formation. Removal of LIRATG3 negatively impacts the rate of thioester transfer from ATG7 to ATG3.ISSN:2374-795