Journal: Nature Communications

Article Title: Filamin A organizes γ‑aminobutyric acid type B receptors at the plasma membrane

doi: 10.1038/s41467-022-35708-1

Figure Lengend Snippet: a Representative frame of a single-molecule TIRF image sequence of SNAP-GABA B1 labeled with SNAP-647 in CHO cells co-transfected with GABA B2 and DsRed-FLNA19-20 or DsRed-FLNA17-18 as negative control. Images are representative of at least three independent experiments. b Representative outcome of single-particle tracking from at least three independent experiments. Each detected particle is surrounded by a blue circle and particle trajectories are shown in magenta. c Diffusion coefficients of GABA B receptor particles in the four groups identified by the TAMSD analysis. Data are mean ± SEM from n = 16 and 15 cells (2809 and 2670 trajectories) for FLNA17-18 and FLNA19-20, respectively, examined over three independent experiments. d Single-molecule analysis of GABA B -FLNA interactions. CHO cells were co-transfected with SNAP-GABA B1 and CLIP-FLNA and labeled with SNAP-647 and CLIP-TMR, respectively. A representative example of a transient colocalization event between a GABA B and a FLNA molecule is shown. e – g Relative frequency ( e ), duration ( f ), and density of diffusivity states ( g ) of single-molecule colocalizations between FLNA and either GABA B1 or GABA B1 -IL1(mGluR2) under basal and stimulated conditions (GABA 100 µM; 5 min incubation). Data are mean ± SEM. n = 53 (GABA B1 basal), 20 (GABA B1 stimulated), 23 (GABA B1 -IL1(mGluR2) basal) and 12 (GABA B1 -IL1(mGluR2) stimulated) examined over three independent experiments. * p < 0.05, ** p < 0.01 by two-tailed Mann-Whitney U test. ns statistically not significant. Scale bars, 5 µm ( a ), 500 nm ( b , d ). Source data are provided as a Source Data file.

Article Snippet: The fluorescent benzylguanine derivative SNAP-Surface 549 (SNAP-549), SNAP-Surface 647 (SNAP-647), and the benzylcytosine derivative CLIP-Cell tetramethylrhodamine-Star (CLIP-TMR) were from New England Biolabs.

Techniques: Sequencing, Labeling, Transfection, Negative Control, Single-particle Tracking, Diffusion-based Assay, Incubation, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Rewarding capacity of optogenetically activating a giant GABAergic central-brain interneuron in larval Drosophila

doi: 10.1101/2022.12.19.521052

Figure Lengend Snippet: Confocal maximum projection images of stainings for mCD8::GFP, Syt1::SNAP, and TLN::CLIP , driven by the APL-specific intersectional driver APLi ( ; ) at the following developmental times: (A-A’’’) third-instar larva (L3); (B-C’’’) 6h after puparium formation (6h APF: calyx: B-B’’’; lobes: C-C’’’); (D-D’’’) 12h APF; (E-F’’’) adult (calyx: E-E’’’; lobes: F-F’’’). Brains were stained with a polyclonal chicken anti-GFP antibody to label the APL neuron (A-F). To label pre-synapses (A’-F’) and post- synapses (A’’-F’’), the pre-synaptic reporter synaptotagmin was fused to the chemical tag SNAPm (Syt1-SNAPm), and the post-synaptic reporter telencephalin was fused to CLIPm (TLN-CLIPm), respectively . Merged images are shown in (A’’’-F’’’). In the third-instar larva, pre-synaptic staining was largely restricted to the calyx (A’), whereas post- synaptic staining was distributed in both the calyx and the lobes (A’’). At 6h APF, both pre- and post-synaptic staining are similarly distributed to that in the larvae (B’-C’’’); notably, pre- synaptic structures seem to be more punctate (B’, C’), and fewer post-synaptic structures are detectable (B’’, C’’). As late as 12h APF, both pre- and post-synaptic structures are still detectable (D’, D’’); post-synaptic structures appear to be detached from the neurite (D’’, D’’’; yellow arrowhead). In adults, both pre- and post-synaptic markers are detectable in both the calyx and the lobes (E-F’’’). The data were acquired with a 40x oil objective; scale bars: 20 μm.

Article Snippet: The chemical substrates were SNAP- tag ligands (SNAP surface 549 - BG 549 [NEB, S9112S]) and CLIP-tag ligands (CLIP surface 647 - BC 647 [NEB, S9234S]) at final concentrations of 1 μM in 0.3% PBT.

Techniques: Staining

Journal: Function

Article Title: ER Redox Homeostasis Regulates Proinsulin Trafficking and Insulin Granule Formation in the Pancreatic Islet β-Cell

doi: 10.1093/function/zqac051

Figure Lengend Snippet: Delayed ER export of proinsulin is specific and independent of overt ER stress. (A) Islets isolated from 10–14 wks old C57BLKS/J db/+ vs. db/db ( n = 3) were examined for ultrastructure by electron microscopy and representative micrographs depicting the ER are shown. (B and E–G) INS1 832/3 cells were cultured for 72 h in control media supplemented with BSA, media containing oleate: palmitate (2:1 and 1 m m OP) or media containing oleate: palmitate (2:1 and 1 m m ) and elevated glucose (20 m m ; OPG) as indicated. (B) mRNA expression was examined by qRT-PCR ( n = 4–6). Cells were treated for 18 h with thapsigargin (50 n m ) as indicated. (C) INS1 832/3 cells treated with AdRIP-CgB-CLIP were analyzed by immunoblot with antibodies raised against CLIP or endogenous CgB as indicated. (D) INS1 832/3 cells co-expressing proCpepSNAP and CgB-CLIP were pulse-labeled with SNAP-505 (green) and CLIP-TMR (red) following a 2 h synthesis period, and chased for 2 h prior to fixation. Cells were immunostained for TGN38 (magenta), and counterstained with DAPI (blue). Representative images are shown (scale bar = 5 μm). (E–G) INS1 832/3 cells expressing proCpepSNAP and CgB-CLIP were pulse-labeled with SNAP-505 (green) and CLIP-TMR (red), chased for 15 min, fixed and immunostained for TGN38 (magenta), and counterstained with DAPI (blue). (E) Representative images are shown (scale bar = 5 μm). Mander’s correlation coefficient (MCC) was used to determine colocalization of labeled proCpepSNAP (SNAP) vs CgB-CLIP (CLIP) with TGN38 (F) or proCpepSNAP (SNAP) with CgB-CLIP (CLIP) as indicated (G) ( n = 3 independent experiments; 53–70 cells per condition). (B, F, and G) Data represent the mean ± SD * P < 0.05, ** P < 0.005, *** P < 0.0001 by two-way ANOVA with Sidak post-test analysis (B and F) or Student’s t- test (G).

Article Snippet: For studies utilizing CgB-CLIP, an identical labeling scheme was followed using CLIPcell block (10 µ m ; NEB) and CLIPcell-TMR (1 µ m ; NEB) where appropriate.

Techniques: Isolation, Electron Microscopy, Cell Culture, Expressing, Quantitative RT-PCR, Western Blot, Labeling

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0078745

Figure Lengend Snippet: (A) Chemically fixed cells expressing the respective self-labelling proteins targeted to the mitochondrial matrix (mtSNAP, mtCLIP, or mtHalo) were labelled. (B) Labelling of live yeast cells expressing the mitochondrial targeted self-labelling proteins using an electroporation protocol. (C) Live yeast cells expressing the indicated fusion proteins labelled by electroporation. Cells were labelled using commercially available TMR substrates. Yeast strains expressing Abp1-SNAP and Pil1-CLIP were created by epitope-tagging, while the other fusion constructs were plasmid encoded. Shown are maximum projections of confocal sections. Scale bar: 2 µm.

Article Snippet: For the evaluation of the staining procedure, the commercially available fluorescent ligands SNAP-Cell TMR-Star, CLIP-Cell TMR-Star (isomer mixtures; New England Biolabs, Ipswich, MA, USA) and HaloTag-TMR ligand (6′-carboxy-isomer; Promega, Madison, WI, USA), were used.

Techniques: Expressing, Electroporation, Construct, Plasmid Preparation

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0078745

Figure Lengend Snippet: (A) Living yeast cells expressing Pil1-CLIP at a near native level from the endogenous chromosomal locus were labelled by electroporation with Atto565-CLIP and imaged using confocal (left) and STED (right) microscopy. Inset: Intensity profile over the region marked with the arrow heads. (B) Dual colour labelling with the CLIP- and the Halo-tag. mtHalo was labelled with 6′-CR110-Halo and Pil1-CLIP was labelled with CLIP-Cell TMR-Star and imaged by epifluorescence microscopy. Scale bars: 2 µm.

Article Snippet: For the evaluation of the staining procedure, the commercially available fluorescent ligands SNAP-Cell TMR-Star, CLIP-Cell TMR-Star (isomer mixtures; New England Biolabs, Ipswich, MA, USA) and HaloTag-TMR ligand (6′-carboxy-isomer; Promega, Madison, WI, USA), were used.

Techniques: Expressing, Electroporation, Microscopy, Epifluorescence Microscopy

Journal: Genome Biology

Article Title: Insights into snoRNA biogenesis and processing from PAR-CLIP of snoRNA core proteins and small RNA sequencing

doi: 10.1186/gb-2013-14-5-r45

Figure Lengend Snippet: Summary of CLIPZ mapping statistics and annotation categories for PAR-CLIP samples.

Article Snippet: For one Fibrillarin targeted PAR-CLIP the immunoprecipitated complexes were treated with micrococcal nuclease (MNase, from New England Biolabs) for 5 min at 37°C [ ].

Techniques:

Journal: Genome Biology

Article Title: Insights into snoRNA biogenesis and processing from PAR-CLIP of snoRNA core proteins and small RNA sequencing

doi: 10.1186/gb-2013-14-5-r45

Figure Lengend Snippet: Annotation summary of the top 200 clusters inferred from PAR-CLIP experiments with snoRNA core proteins.

Article Snippet: For one Fibrillarin targeted PAR-CLIP the immunoprecipitated complexes were treated with micrococcal nuclease (MNase, from New England Biolabs) for 5 min at 37°C [ ].

Techniques:

Journal: Genome Biology

Article Title: Insights into snoRNA biogenesis and processing from PAR-CLIP of snoRNA core proteins and small RNA sequencing

doi: 10.1186/gb-2013-14-5-r45

Figure Lengend Snippet: Summary of PAR-CLIP data of snoRNP core proteins . (A) Profiles of sequencing reads obtained from PAR-CLIP experiments for selected snoRNAs. Black bars in the profiles indicate the number of T→C mutations observed in PAR-CLIP reads at a particular nucleotide. (B) Similarity of binding profiles of core proteins that associate with C/D box snoRNAs. (C) Comparison of protein binding profiles as inferred from RNase T1-treated and MNase-treated PAR-CLIP samples. (D, E) Preferential binding of Fibrillarin to box elements as inferred from PAR-CLIP samples prepared with T1 (D) and MNase ribonucleases (E). (F) Comparison of binding preferences at D'/D box elements and guide regions for snoRNAs with and without a known target. (G) Analysis of binding preferences of Dyskerin for H/ACA box snoRNA-specific elements. D, E, F and G show the cumulative distributions of CLIP read coverage z -scores for nucleotides located in various regions of the snoRNA relative to the overall coverage of the snoRNA. CLIP: cross-linking and immunoprecipitation; MNase: micrococcal nuclease; PAR-CLIP: photoactivatable-ribonucleoside-enhanced cross-linking and immunoprecipitation; snoRNA: small nucleolar RNA; snoRNP: small nucleolar ribonucleoprotein

Article Snippet: For one Fibrillarin targeted PAR-CLIP the immunoprecipitated complexes were treated with micrococcal nuclease (MNase, from New England Biolabs) for 5 min at 37°C [ ].

Techniques: Sequencing, Binding Assay, Protein Binding, Immunoprecipitation

Journal: Genome Biology

Article Title: Insights into snoRNA biogenesis and processing from PAR-CLIP of snoRNA core proteins and small RNA sequencing

doi: 10.1186/gb-2013-14-5-r45

Figure Lengend Snippet: Small RNA-seq and PAR-CLIP reads mapping to mini-snoRNAs . Mini-snoRNAs ZL77, ZL49, ZL103 and ZL63 are shown. Black bars in the panels corresponding to PAR-CLIP libraries indicate the number of T→C mutations observed at individual nucleotides. CLIP: cross-linking and immunoprecipitation; PAR-CLIP: photoactivatable-ribonucleoside-enhanced cross-linking and immunoprecipitation; snoRNA: small nucleolar RNA

Article Snippet: For one Fibrillarin targeted PAR-CLIP the immunoprecipitated complexes were treated with micrococcal nuclease (MNase, from New England Biolabs) for 5 min at 37°C [ ].

Techniques: RNA Sequencing Assay, Immunoprecipitation

Journal: bioRxiv

Article Title: Endosomal egress and intercellular transmission of hepatic ApoE-containing lipoproteins and its exploitation by the hepatitis C virus

doi: 10.1101/2022.12.08.519703

Figure Lengend Snippet: (A) Experimental approach. Schematic representations of SNAPf-tagged ApoE and the subgenomic replicon encoding CLIPf-tagged NS5A are shown on the top. Huh7-Lunet cells were lentivirally transduced with the ApoE expression vector and transfected with the subgenomic replicon RNA. ApoE and NS5A were detected by STED microscopy and CD63 by immunofluorescence confocal microscopy. (B) Colocalization of ApoE SNAPf and NS5A CLIPf . Huh7-Lunet/ApoE SNAPf cells were electroporated with subgenomic replicon RNA encoding NS5A CLIPf and after 48 h, cells were labeled with SNAP SiR647 and CLIP ATTO590 for 1 h, fixed, and subjected to confocal microscopy. Arrowheads: colocalized ApoE-NS5A signals. (C) Quantification of CD63-positive ApoE-NS5A double-positive foci. Cells from (B) harvested 72 h p.e were fixed, permeabilized, and incubated with anti-CD63 AF488 antibody. To determine the correlation between ApoE-NS5A double-positive foci and how many of them colocalized with CD63, 100 cells were analyzed. Each dot represents one cell and displays the number of ApoE-NS5A double-positive foci (x-axis) and the number of CD63-ApoE-NS5A triple-positive foci (y-axis). The R-squared value is given on the plot. (D) STED-resolved ApoE-NS5A double-positive structures colocalizing with the intraluminal vesicle marker CD63. Huh7-Lunet/ApoE SNAPf cells were electroporated with the subgenomic replicon RNA encoding NS5A CLIPf . After 48 h, cells were labeled with SNAP SiR647 and CLIP ATTO590 for 1 h, fixed, and incubated with anti-CD63 AF488 antibody. ApoE, NS5A, and CD63 fluorescent signals were sequentially imaged using confocal and STED microscopy, the latter to achieve a higher resolution of ApoE and NS5A signals that were deconvoluted using Huygens. Arrows: ∼100-200 nm-sized ApoE-NS5A-CD63 positive structures; star: ∼500 nm-sized ring-like NS5A positive structure.

Article Snippet: At 48 h post-electroporation, cells were sequentially incubated with CLIP ATTO590 (1:2500) and 5 µM SNAP SiR647 (NEB) in DMEMcplt for 1 h. Cells were washed intensively at least 3 times with DMEMcplt and cultured for 15 min.

Techniques: Transduction, Expressing, Plasmid Preparation, Transfection, Microscopy, Immunofluorescence, Confocal Microscopy, Labeling, Incubation, Marker

Journal: Cell reports

Article Title: Mechanisms of differential desensitization of metabotropic glutamate receptors

doi: 10.1016/j.celrep.2021.109050

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: SNAP-Surface Alexa Fluor 488 (BG-Alexa 488) , New England Biolabs , Cat#S9232S.

Techniques: Recombinant, Modification, Transfection, Software