tmr star  (New England Biolabs)


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    Name:
    CLIP Cell TMR Star
    Description:
    CLIP Cell TMR Star 30 nmol
    Catalog Number:
    s9219s
    Price:
    344
    Size:
    30 nmol
    Category:
    Fluorochromes
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    Structured Review

    New England Biolabs tmr star
    CLIP Cell TMR Star
    CLIP Cell TMR Star 30 nmol
    https://www.bioz.com/result/tmr star/product/New England Biolabs
    Average 95 stars, based on 42 article reviews
    Price from $9.99 to $1999.99
    tmr star - by Bioz Stars, 2020-09
    95/100 stars

    Images

    1) Product Images from "Single molecule super-resolution imaging of proteins in living Salmonella enterica using self-labelling enzymes"

    Article Title: Single molecule super-resolution imaging of proteins in living Salmonella enterica using self-labelling enzymes

    Journal: Scientific Reports

    doi: 10.1038/srep31601

    Labelling specificity probed by single molecule localization. ( A ) MvP818 [p3725], the invC deletion strain expressing invC ::HaloTag, was treated with 20 nM HTL-TMR or 150 mM HTL-Atto655. MvP818 [p3723], the invC deletion strain expressing invC ::SNAP-tag, was treated with 30 nM TMR-Star. Labeling reactions were performed for 45 min at 37 °C. For the shape of the bacteria, the maximum intensity projection with bilinear interpolation of all 500 acquired frames is shown (left panel). After localization distinct patches of InvC are visible for each condition. The white dots in the localized images show the localized molecules of all 500 acquired frames (right panel). Clusters are indicated with red arrows. ( B ) Salmonella WT cells were treated with 20 nM HTL-TMR, 150 nM HTL-Atto655 or 30 nM TMR-Star for 45 min at 37 °C. 500 frames were acquired as described before with 5 mW laser power at the focal plane. The autofluorescence of bacteria upon excitation with a 488 nm laser is shown (left panel). SRM images rendered after localization of individual emitters (right panel) confirmed labelling specificity in all cases. Scale bars, 1 μm.
    Figure Legend Snippet: Labelling specificity probed by single molecule localization. ( A ) MvP818 [p3725], the invC deletion strain expressing invC ::HaloTag, was treated with 20 nM HTL-TMR or 150 mM HTL-Atto655. MvP818 [p3723], the invC deletion strain expressing invC ::SNAP-tag, was treated with 30 nM TMR-Star. Labeling reactions were performed for 45 min at 37 °C. For the shape of the bacteria, the maximum intensity projection with bilinear interpolation of all 500 acquired frames is shown (left panel). After localization distinct patches of InvC are visible for each condition. The white dots in the localized images show the localized molecules of all 500 acquired frames (right panel). Clusters are indicated with red arrows. ( B ) Salmonella WT cells were treated with 20 nM HTL-TMR, 150 nM HTL-Atto655 or 30 nM TMR-Star for 45 min at 37 °C. 500 frames were acquired as described before with 5 mW laser power at the focal plane. The autofluorescence of bacteria upon excitation with a 488 nm laser is shown (left panel). SRM images rendered after localization of individual emitters (right panel) confirmed labelling specificity in all cases. Scale bars, 1 μm.

    Techniques Used: Expressing, Labeling

    Effect of enzyme tags and ligands on functionality of protein secretion systems in Salmonella . Invasion of polarized epithelial cell line MDCK by various Salmonella strains was quantified. Salmonella WT, mutant strains, and strains expressing HaloTag or HaloTag and SNAP-tag fusions as indicated were used to infect MDCK cells at an multiplicity of infection (MOI) of 5. Non-internalized bacteria were removed by washing and remaining bacteria killed by addition of gentamicin for 1 h. Subsequently, the cells were lysed and serial dilutions were plated onto agar plates for colony-forming units (CFU) determination. Invasion is expressed as percentage of the inoculum that was internalized by host ells. After adding the inoculum, infection was synchronized by centrifugation ( A ), or no centrifugation was performed ( B,C ). Mutant strains siiF, spaS and fliM served as SPI4-T1SS-defective, SPI1-T3SS-defective and non-motile control strains, respectively ( C ). ( D,E ) Prior to invasion, the cells were mock-treated (−) or 20 nM HTL-TMR ( D , +, hatched bars) or 30 nM TMR-Star was added ( E , +, hatched bars). Cells were infected without centrifugation. Statistical significance as determined by Student’s t test is indicated as ns, not significant; * P
    Figure Legend Snippet: Effect of enzyme tags and ligands on functionality of protein secretion systems in Salmonella . Invasion of polarized epithelial cell line MDCK by various Salmonella strains was quantified. Salmonella WT, mutant strains, and strains expressing HaloTag or HaloTag and SNAP-tag fusions as indicated were used to infect MDCK cells at an multiplicity of infection (MOI) of 5. Non-internalized bacteria were removed by washing and remaining bacteria killed by addition of gentamicin for 1 h. Subsequently, the cells were lysed and serial dilutions were plated onto agar plates for colony-forming units (CFU) determination. Invasion is expressed as percentage of the inoculum that was internalized by host ells. After adding the inoculum, infection was synchronized by centrifugation ( A ), or no centrifugation was performed ( B,C ). Mutant strains siiF, spaS and fliM served as SPI4-T1SS-defective, SPI1-T3SS-defective and non-motile control strains, respectively ( C ). ( D,E ) Prior to invasion, the cells were mock-treated (−) or 20 nM HTL-TMR ( D , +, hatched bars) or 30 nM TMR-Star was added ( E , +, hatched bars). Cells were infected without centrifugation. Statistical significance as determined by Student’s t test is indicated as ns, not significant; * P

    Techniques Used: Mutagenesis, Expressing, Infection, Centrifugation

    SMT of TMR-labelled SiiF, SpaS, FliN and HilA. ( A ) SiiF-HaloTag, HilA-HaloTag, SpaS-HaloTag, or FliN-HaloTag were labelled with TMR-Star and imaged as described for Fig. 4 . Selected frames from series of 500 frames are shown (frame rate: 15 frames per second) and frame numbers are indicated. Inserts in frame 2 show localized images. Each trajectory has a different colour. Using pooled trajectories for proteins in at least 20 bacteria recorded under the same conditions, the diffusion coefficient D was calculated using the Jaqaman algorithm 5 20 61 . ( B ) Values for the diffusion coefficients. Scale bars, 0.5 μm (SiiF-HaloTag), 1 μm (SpaS-HaloTag, FliN-HaloTag, HilA-HaloTag). The sequences of 500 frames are shown in Movie S1 for (SiiF-HaloTag), Movie S2 (SpaS-HaloTag), Movie S3 (FliN-HaloTag), and Movie S4 (HilA-HaloTag).
    Figure Legend Snippet: SMT of TMR-labelled SiiF, SpaS, FliN and HilA. ( A ) SiiF-HaloTag, HilA-HaloTag, SpaS-HaloTag, or FliN-HaloTag were labelled with TMR-Star and imaged as described for Fig. 4 . Selected frames from series of 500 frames are shown (frame rate: 15 frames per second) and frame numbers are indicated. Inserts in frame 2 show localized images. Each trajectory has a different colour. Using pooled trajectories for proteins in at least 20 bacteria recorded under the same conditions, the diffusion coefficient D was calculated using the Jaqaman algorithm 5 20 61 . ( B ) Values for the diffusion coefficients. Scale bars, 0.5 μm (SiiF-HaloTag), 1 μm (SpaS-HaloTag, FliN-HaloTag, HilA-HaloTag). The sequences of 500 frames are shown in Movie S1 for (SiiF-HaloTag), Movie S2 (SpaS-HaloTag), Movie S3 (FliN-HaloTag), and Movie S4 (HilA-HaloTag).

    Techniques Used: Diffusion-based Assay

    Dual colour dSTORM of Salmonella using fusion proteins to HaloTag and SNAP-tag. Flagellar motor subunit FliN and SPI1-T3SS subunit SpaS were visualized within one cell. ( A ) FliN-SNAP-tag and SpaS-HaloTag were stained with 30 nM TMR-Star (green) and 150 nM HTL-Atto655 (red), respectively. ( B ) SpaS-SNAP-tag and FliN-HaloTag were stained with 30 nM TMR-Star (green) and 150 nM HTL-Atto655 (red), respectively. After staining, cells were washed and fixed with 3% PFA and immobilized on glass slides. For dSTORM imaging, cells were incubated in a buffer containing 100 mM β-Mercaptoethylamine, 4.5 mg × ml −1 D-Glucose, 40 μg × ml −1 Catalase and 0.5 mg × ml −1 Glucose-Oxidase and maximum laser power was used for excitation. SR images were rendered from single emitter localizations obtained within 500 frames. SR images of the TMR channel (i), of the Atto655 channel (ii) and merged images of both channels (iii) are shown. For the shape of the bacteria, the maximum intensity projection with bilinear interpolation of all 500 acquired frames is shown in the lower corner in (iii). Scale bar, 0.5 μm.
    Figure Legend Snippet: Dual colour dSTORM of Salmonella using fusion proteins to HaloTag and SNAP-tag. Flagellar motor subunit FliN and SPI1-T3SS subunit SpaS were visualized within one cell. ( A ) FliN-SNAP-tag and SpaS-HaloTag were stained with 30 nM TMR-Star (green) and 150 nM HTL-Atto655 (red), respectively. ( B ) SpaS-SNAP-tag and FliN-HaloTag were stained with 30 nM TMR-Star (green) and 150 nM HTL-Atto655 (red), respectively. After staining, cells were washed and fixed with 3% PFA and immobilized on glass slides. For dSTORM imaging, cells were incubated in a buffer containing 100 mM β-Mercaptoethylamine, 4.5 mg × ml −1 D-Glucose, 40 μg × ml −1 Catalase and 0.5 mg × ml −1 Glucose-Oxidase and maximum laser power was used for excitation. SR images were rendered from single emitter localizations obtained within 500 frames. SR images of the TMR channel (i), of the Atto655 channel (ii) and merged images of both channels (iii) are shown. For the shape of the bacteria, the maximum intensity projection with bilinear interpolation of all 500 acquired frames is shown in the lower corner in (iii). Scale bar, 0.5 μm.

    Techniques Used: Staining, Imaging, Incubation

    Labelling of distinct subunits of the SPI4-T1SS. Salmonella expressing siiF ::HaloTag and siiC ::SNAP-tag was subcultured for 2 h 45 min in LB and incubation was continued for 45 min at 37 °C cells with labelling by 30 nM TMR-Star (red), and 20 nM HTL-SiR (green). Subsequently, cells were applied to agarose pads and 750 images were recorded with excitation with 561 nm and 642 nm lasers at 15% power at the focal plane. After applying threshold settings as described in Fig. S7 , localization using modulated MTT was performed. DIC images were recorded in order to outline the bacterial cell body of three representative cells. Cluster of individual SiiF and SiiC subunits, as well as complexes containing both subunits are observed. Of the events analysed, 21.4% and 42.8% represented cluster of only SiiF or only SiiC, respectively, and co-cluster of SiiC and SiiF were observed for 35.8% of the events. Scale bar, 1 μm.
    Figure Legend Snippet: Labelling of distinct subunits of the SPI4-T1SS. Salmonella expressing siiF ::HaloTag and siiC ::SNAP-tag was subcultured for 2 h 45 min in LB and incubation was continued for 45 min at 37 °C cells with labelling by 30 nM TMR-Star (red), and 20 nM HTL-SiR (green). Subsequently, cells were applied to agarose pads and 750 images were recorded with excitation with 561 nm and 642 nm lasers at 15% power at the focal plane. After applying threshold settings as described in Fig. S7 , localization using modulated MTT was performed. DIC images were recorded in order to outline the bacterial cell body of three representative cells. Cluster of individual SiiF and SiiC subunits, as well as complexes containing both subunits are observed. Of the events analysed, 21.4% and 42.8% represented cluster of only SiiF or only SiiC, respectively, and co-cluster of SiiC and SiiF were observed for 35.8% of the events. Scale bar, 1 μm.

    Techniques Used: Expressing, Incubation, MTT Assay

    2) Product Images from "CAS-LiveFISH: Simple and versatile imaging of genomic loci in live mammalian cells and early pre-implantation embryos"

    Article Title: CAS-LiveFISH: Simple and versatile imaging of genomic loci in live mammalian cells and early pre-implantation embryos

    Journal: bioRxiv

    doi: 10.1101/2020.08.25.265306

    Pools of pre-assembled RNPs enable visualizing the MYC locus in live HeLa cells. ( A ) Organization of the extended MYC locus though proximity ligation (ChIA-PET) and ChIP-Seq assays. HeLa Pol II ChIA-PET data (GSM832461) ( Li et al., 2012 ),as well as ChIP-Seq data for p300 (GSM935500), H3K27ac (GSM733684) and Pol2-Ser2Ph (GSM935383), are visualized using the WashU Epigenome Browser ( Zhou et al., 2011 ). ( B ) Visualization of the HPV integration site using Alexa 647-labeled dCas9 RNPs, assembled with a pool of 36 distinct gRNAs. The right cell contains doublets of closely-spaced puncta, indicative of replicated loci in S/G2. ( C ) Distribution of number of discernible Alexa 647-HPV spots per cell. ( D ) Two-color imaging of HPV integration site (Alexa 647) and MYC (TMR) using co-delivery of preassembled RNPs in live HeLa cells. ( E ) 2D scatter plot of relative positions and statistics (histogram and box plot) of 2D distances between Alexa 647-HPV and TMR-MYC in live HeLa cells. 2D distance is 177±155 nm (mean±S.D.; n =472 measurements from > 30 loci).
    Figure Legend Snippet: Pools of pre-assembled RNPs enable visualizing the MYC locus in live HeLa cells. ( A ) Organization of the extended MYC locus though proximity ligation (ChIA-PET) and ChIP-Seq assays. HeLa Pol II ChIA-PET data (GSM832461) ( Li et al., 2012 ),as well as ChIP-Seq data for p300 (GSM935500), H3K27ac (GSM733684) and Pol2-Ser2Ph (GSM935383), are visualized using the WashU Epigenome Browser ( Zhou et al., 2011 ). ( B ) Visualization of the HPV integration site using Alexa 647-labeled dCas9 RNPs, assembled with a pool of 36 distinct gRNAs. The right cell contains doublets of closely-spaced puncta, indicative of replicated loci in S/G2. ( C ) Distribution of number of discernible Alexa 647-HPV spots per cell. ( D ) Two-color imaging of HPV integration site (Alexa 647) and MYC (TMR) using co-delivery of preassembled RNPs in live HeLa cells. ( E ) 2D scatter plot of relative positions and statistics (histogram and box plot) of 2D distances between Alexa 647-HPV and TMR-MYC in live HeLa cells. 2D distance is 177±155 nm (mean±S.D.; n =472 measurements from > 30 loci).

    Techniques Used: Ligation, ChIA Pet Assay, Chromatin Immunoprecipitation, Labeling, Imaging

    Imaging genomic loci in live cells with CAS-LiveFISH. ( A ) Schematic of cell delivery of in vitro transcribed gRNAs. ( B, C ) Visualization of telomeres in U-2 OS cells stably expressing dCas9-EGFP, by electroporation ( B ) or microinjection ( C ) of an in vitro transcribed gRNA (sgTelomere). A non-targeting control (sgGal4) shows no discernible nuclear puncta ( B ), demonstrating locus-specific tagging. In ( B ), cells are imaged 20hrs after electroporation. Co-injection of a BFP-LifeAct-expressing plasmid ( B ) or Alexa 647-Benzylguanine ( C ) results in simultaneous visualization of actin filaments and RNA Polymerase II (SNAP-Pol II), respectively. ( D ) Schematic of recombinant fluorescent dCas9:gRNA RNP assembly and cell delivery. ( E, F, G ) Visualization of genomic loci in U-2 OS ( E ) and HeLa ( F, G ) cells using pre-assembled dCas9-gRNA RNPs. ( F ) Co-delivery of TMR-RNPs targeting telomeres and Alexa 647-RNPs targeting a-satellite sequences shows distinct, non-overlapping nuclear puncta. ( G ) Co-injection of telomere-targeting Atto 488- or TMR-RNPs and non-targeting control Alexa 647-RNPs (sgGal4) shows no discernible nuclear puncta in the Alexa 647 channel, demonstrating locus-specific tagging and minimal cross-talk between colors.
    Figure Legend Snippet: Imaging genomic loci in live cells with CAS-LiveFISH. ( A ) Schematic of cell delivery of in vitro transcribed gRNAs. ( B, C ) Visualization of telomeres in U-2 OS cells stably expressing dCas9-EGFP, by electroporation ( B ) or microinjection ( C ) of an in vitro transcribed gRNA (sgTelomere). A non-targeting control (sgGal4) shows no discernible nuclear puncta ( B ), demonstrating locus-specific tagging. In ( B ), cells are imaged 20hrs after electroporation. Co-injection of a BFP-LifeAct-expressing plasmid ( B ) or Alexa 647-Benzylguanine ( C ) results in simultaneous visualization of actin filaments and RNA Polymerase II (SNAP-Pol II), respectively. ( D ) Schematic of recombinant fluorescent dCas9:gRNA RNP assembly and cell delivery. ( E, F, G ) Visualization of genomic loci in U-2 OS ( E ) and HeLa ( F, G ) cells using pre-assembled dCas9-gRNA RNPs. ( F ) Co-delivery of TMR-RNPs targeting telomeres and Alexa 647-RNPs targeting a-satellite sequences shows distinct, non-overlapping nuclear puncta. ( G ) Co-injection of telomere-targeting Atto 488- or TMR-RNPs and non-targeting control Alexa 647-RNPs (sgGal4) shows no discernible nuclear puncta in the Alexa 647 channel, demonstrating locus-specific tagging and minimal cross-talk between colors.

    Techniques Used: Imaging, In Vitro, Stable Transfection, Expressing, Electroporation, Injection, Plasmid Preparation, Recombinant

    3) Product Images from "Optimization of fluorophores for chemical tagging and immunohistochemistry of Drosophila neurons"

    Article Title: Optimization of fluorophores for chemical tagging and immunohistochemistry of Drosophila neurons

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0200759

    Detail comparison of SS02565 neuronal membrane labeling. All samples show the same region of projections crossing from the left optic lobe to the central brain. Only the neuronal membrane channel is shown, and is labeled via antibodies in (A-B) and CLIP-tag in (C-D). (A) SS02565 was crossed to w;; 5XUAS-IVS-myr :: smFLAG in VK00005 , pJFRC51-3XUAS-IVS-Syt :: smHA in su(Hw)attP1 and brains were labeled with pure IHC, including rat anti-FLAG and ATTO 647N goat anti-rat antibodies over a period of 13 days. (B) SS02565 was crossed to w; brp-SNAP; 5XUAS-IVS-myr :: smFLAG in VK00005 , pJFRC51-3XUAS-IVS-Syt :: smHA in su(Hw)attP1 and brains were labeled with hybrid IHC, including rat anti-FLAG and ATTO 647N goat anti-rat antibodies over a period of 6 days. (C) SS02565 was crossed to w; brp-SNAP; UAS-myr :: 4xCLIPf in VK00005 , UAS-Syt :: Halo7 in VK0027 and brains were labeled for 15 minutes with pure chemical tags, including TMR CLIP-tag ligand. (D) SS02565 was crossed to w; brp-SNAP; UAS-myr :: 4xCLIPf in VK00005 , UAS-Syt :: Halo7 in VK0027 and brains were labeled for 15 minutes with pure chemical tags, including JF 549 CLIP-tag ligand.
    Figure Legend Snippet: Detail comparison of SS02565 neuronal membrane labeling. All samples show the same region of projections crossing from the left optic lobe to the central brain. Only the neuronal membrane channel is shown, and is labeled via antibodies in (A-B) and CLIP-tag in (C-D). (A) SS02565 was crossed to w;; 5XUAS-IVS-myr :: smFLAG in VK00005 , pJFRC51-3XUAS-IVS-Syt :: smHA in su(Hw)attP1 and brains were labeled with pure IHC, including rat anti-FLAG and ATTO 647N goat anti-rat antibodies over a period of 13 days. (B) SS02565 was crossed to w; brp-SNAP; 5XUAS-IVS-myr :: smFLAG in VK00005 , pJFRC51-3XUAS-IVS-Syt :: smHA in su(Hw)attP1 and brains were labeled with hybrid IHC, including rat anti-FLAG and ATTO 647N goat anti-rat antibodies over a period of 6 days. (C) SS02565 was crossed to w; brp-SNAP; UAS-myr :: 4xCLIPf in VK00005 , UAS-Syt :: Halo7 in VK0027 and brains were labeled for 15 minutes with pure chemical tags, including TMR CLIP-tag ligand. (D) SS02565 was crossed to w; brp-SNAP; UAS-myr :: 4xCLIPf in VK00005 , UAS-Syt :: Halo7 in VK0027 and brains were labeled for 15 minutes with pure chemical tags, including JF 549 CLIP-tag ligand.

    Techniques Used: Labeling, Cross-linking Immunoprecipitation, Immunohistochemistry

    Comparison of Polarity IHC and chemical tag labeling methods. All samples show the Drosophila left optic lobe imaged at 63X. Each image is independently scaled for optimal intensity. (A). Polarity pure IHC : Split GAL4 SS02565 was crossed to w;; 5XUAS-IVS-myr :: smFLAG in VK00005 , pJFRC51-3XUAS-IVS-Syt :: smHA in su(Hw)attP1 and was labeled over a period of 13 days with nc82 mouse anti-Brp/Cy2 anti-mouse, rabbit anti-HA/Cy3 anti-rabbit, and rat anti-FLAG/Cy5 anti-rat. (B). Polarity hybrid IHC : SS02565 was crossed to w; brp-SNAP; 5XUAS-IVS-myr :: smFLAG in VK00005 , pJFRC51-3XUAS-IVS-Syt :: smHA in su(Hw)attP1 and labeled with Cy2 SNAP-tag ligand for 15 minutes, followed by rabbit anti-HA/Cy3 anti-rabbit, and rat anti-FLAG/Cy5 anti-rat over 6 days. Arrowheads indicate bleed-through of Cy5 into Cy2 channel. (C). Polarity pure chemical tag : SS02565 was crossed to w; brp-SNAP; UAS-myr :: 4xCLIPf in VK00005 , UAS-Syt :: Halo7 in VK0027 and labeled for 15 minutes with Cy2 SNAP-tag ligand, TMR CLIP-tag ligand, and ATTO 647N HaloTag ligand. (D). Polarity ATTO 647N pure IHC : As in (A) but with ATTO 647N instead of Cy5. (E). Polarity ATTO 647N hybrid IHC : As in (B) but with ATTO 647N instead of Cy5.
    Figure Legend Snippet: Comparison of Polarity IHC and chemical tag labeling methods. All samples show the Drosophila left optic lobe imaged at 63X. Each image is independently scaled for optimal intensity. (A). Polarity pure IHC : Split GAL4 SS02565 was crossed to w;; 5XUAS-IVS-myr :: smFLAG in VK00005 , pJFRC51-3XUAS-IVS-Syt :: smHA in su(Hw)attP1 and was labeled over a period of 13 days with nc82 mouse anti-Brp/Cy2 anti-mouse, rabbit anti-HA/Cy3 anti-rabbit, and rat anti-FLAG/Cy5 anti-rat. (B). Polarity hybrid IHC : SS02565 was crossed to w; brp-SNAP; 5XUAS-IVS-myr :: smFLAG in VK00005 , pJFRC51-3XUAS-IVS-Syt :: smHA in su(Hw)attP1 and labeled with Cy2 SNAP-tag ligand for 15 minutes, followed by rabbit anti-HA/Cy3 anti-rabbit, and rat anti-FLAG/Cy5 anti-rat over 6 days. Arrowheads indicate bleed-through of Cy5 into Cy2 channel. (C). Polarity pure chemical tag : SS02565 was crossed to w; brp-SNAP; UAS-myr :: 4xCLIPf in VK00005 , UAS-Syt :: Halo7 in VK0027 and labeled for 15 minutes with Cy2 SNAP-tag ligand, TMR CLIP-tag ligand, and ATTO 647N HaloTag ligand. (D). Polarity ATTO 647N pure IHC : As in (A) but with ATTO 647N instead of Cy5. (E). Polarity ATTO 647N hybrid IHC : As in (B) but with ATTO 647N instead of Cy5.

    Techniques Used: Immunohistochemistry, Labeling, Cross-linking Immunoprecipitation

    4) Product Images from "Ran-GTP is non-essential to activate NuMA for spindle pole focusing, but dynamically polarizes HURP to control mitotic spindle length"

    Article Title: Ran-GTP is non-essential to activate NuMA for spindle pole focusing, but dynamically polarizes HURP to control mitotic spindle length

    Journal: bioRxiv

    doi: 10.1101/473538

    HURP dynamically accumulates on metaphase k-fibers in an importin-β-dependent manner. (A) Live fluorescent images of HURP-SNAP visualized with TMR-star (magenta) and SiR-tubulin (TUB) in control (top) and importin-β-depleted cells (bottom). Fluorescent signals were bleached in the indicated box region at t = 0, and the fluorescence recoveries were monitored for 120 sec. (B) A graph showing fluorescence recovery after photobleaching. An average of 7 samples was plotted. Bars indicate SDs. (C) Schematic diagram of the metaphase degradation assay. Following release from RO-3336-mediated G2 arrest, proTAME and Apcin were added to arrest cells in metaphase. Auxin (IAA) was added (indicated by the red line) to induce RCC1 degradation during metaphase. (D) Live fluorescent images of SiR-DNA, importin-β-mAC, HURP-mCh, and SiR-700-tubulin (TUB). IAA was added at t = 0. Arrows indicate a cell showing a reduction of importin-β signal during metaphase. (E) Enlarged images from (D) showing a re-localization of HURP-mCh from k-fibers (t = 0) to the spindle (t =90). Scale bars = 10 μm.
    Figure Legend Snippet: HURP dynamically accumulates on metaphase k-fibers in an importin-β-dependent manner. (A) Live fluorescent images of HURP-SNAP visualized with TMR-star (magenta) and SiR-tubulin (TUB) in control (top) and importin-β-depleted cells (bottom). Fluorescent signals were bleached in the indicated box region at t = 0, and the fluorescence recoveries were monitored for 120 sec. (B) A graph showing fluorescence recovery after photobleaching. An average of 7 samples was plotted. Bars indicate SDs. (C) Schematic diagram of the metaphase degradation assay. Following release from RO-3336-mediated G2 arrest, proTAME and Apcin were added to arrest cells in metaphase. Auxin (IAA) was added (indicated by the red line) to induce RCC1 degradation during metaphase. (D) Live fluorescent images of SiR-DNA, importin-β-mAC, HURP-mCh, and SiR-700-tubulin (TUB). IAA was added at t = 0. Arrows indicate a cell showing a reduction of importin-β signal during metaphase. (E) Enlarged images from (D) showing a re-localization of HURP-mCh from k-fibers (t = 0) to the spindle (t =90). Scale bars = 10 μm.

    Techniques Used: Fluorescence, Degradation Assay

    5) Product Images from "Membrane proteins follow multiple pathways to the basolateral cell surface in polarized epithelial cells"

    Article Title: Membrane proteins follow multiple pathways to the basolateral cell surface in polarized epithelial cells

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200901021

    Newly synthesized Na pump trafficking imaged via the SNAP tag. (A) Live SNAP cells were preincubated with nonfluorescent BG (BG-Block) for 30 min, fixed, stained with TMR-STAR (red), and processed for immunofluorescence with the indicated antibodies (green). (B and C) SNAP cells pretreated with BG-Block were washed and allowed to synthesize new protein for the indicated times at 37°C before fixation and labeling as in A. Arrows highlight examples of colocalization with the Golgi marker GM130 or with the lateral membrane stained with anti-HA. TMR, tetramethylrhodamine. Bars, 10 µm.
    Figure Legend Snippet: Newly synthesized Na pump trafficking imaged via the SNAP tag. (A) Live SNAP cells were preincubated with nonfluorescent BG (BG-Block) for 30 min, fixed, stained with TMR-STAR (red), and processed for immunofluorescence with the indicated antibodies (green). (B and C) SNAP cells pretreated with BG-Block were washed and allowed to synthesize new protein for the indicated times at 37°C before fixation and labeling as in A. Arrows highlight examples of colocalization with the Golgi marker GM130 or with the lateral membrane stained with anti-HA. TMR, tetramethylrhodamine. Bars, 10 µm.

    Techniques Used: Synthesized, Blocking Assay, Staining, Immunofluorescence, Labeling, Marker

    Na pump and VSV-G exit the TGN in separate PGTIs en route to the lateral membrane. SNAP cells were transfected with a plasmid encoding Ts 045 VSV-G–YFP and incubated for 24 h at 40°C to accumulate newly synthesized VSV-G in the ER. Next, samples were BG blocked at 40°C, washed, and incubated for a short 10-min recovery period to permit synthesis of new SNAP-tagged Na pump protein. (A and B) Samples were subjected to 19°C Golgi block and fixed immediately (19°C; A) or warmed to 31°C for 15 min (B). Samples were labeled with TMR-STAR (red) and imaged for YFP (green). The boxed areas are shown at higher magnification in the panels below. The green arrow highlights a PGTI containing only VSV-G, whereas the red arrow highlights a carrier containing only the Na pump. Bars, 5 µm.
    Figure Legend Snippet: Na pump and VSV-G exit the TGN in separate PGTIs en route to the lateral membrane. SNAP cells were transfected with a plasmid encoding Ts 045 VSV-G–YFP and incubated for 24 h at 40°C to accumulate newly synthesized VSV-G in the ER. Next, samples were BG blocked at 40°C, washed, and incubated for a short 10-min recovery period to permit synthesis of new SNAP-tagged Na pump protein. (A and B) Samples were subjected to 19°C Golgi block and fixed immediately (19°C; A) or warmed to 31°C for 15 min (B). Samples were labeled with TMR-STAR (red) and imaged for YFP (green). The boxed areas are shown at higher magnification in the panels below. The green arrow highlights a PGTI containing only VSV-G, whereas the red arrow highlights a carrier containing only the Na pump. Bars, 5 µm.

    Techniques Used: Transfection, Plasmid Preparation, Incubation, Synthesized, Blocking Assay, Labeling

    The Na,K-ATPase does not pass through REs en route to the lateral membrane. (A) Cells were infected with adenovirus expressing the human Tfn-R, after which they were BG blocked, and newly synthesized Na pump was accumulated in the TGN (red) for 2 h at 19°C. Alexa Fluor 488–Tfn was added to the medium during the Golgi block to label REs (green). Golgi-blocked cells were fixed (19°C) or warmed for the indicated period and stained with anti-HA (blue). (B) Cells were infected to express the human Tfn-R and VSV-G–YFP Ts 045 (green) and incubated overnight at 40°C. Cells were BG blocked at 40°C and incubated for 40 min at 40°C with Tfn-HRP along with 1 µM TMR-STAR to live label newly synthesized pump (red). Next, cells were washed and incubated for a further 20 min at 40°C to remove excess TMR-STAR and to allow for the accumulation of Tfn-HRP into REs. Tfn-HRP–loaded REs were ablated on ice by exposing cells to DAB and H 2 O 2 for 1 h, whereas control cells were treated with DAB alone. After the ablation reaction, cells were fixed or incubated in media plus CHX for 1.5 h at 31°C to allow trafficking of the proteins to the PM. (C) The percentage of protein that reached the lateral membrane 1.5 h after mock or ablation treatment was quantified using the enhanced colocalization tool (LSM Image Examiner software; Carl Zeiss, Inc.). 30 VSV-G–infected cells were analyzed, and data are represented as mean ± SD. (D) Samples were treated as in B and processed for immunofluorescence with a cocktail of Golgi antibodies (GM130, Vti1a, and GS15) or antibody against the Tfn-R shown in blue. The bottom panel depicts an individual confocal slice highlighting the colocalization of VSV-G with Tfn-R. TMR, tetramethylrhodamine. Bars, 5 µm.
    Figure Legend Snippet: The Na,K-ATPase does not pass through REs en route to the lateral membrane. (A) Cells were infected with adenovirus expressing the human Tfn-R, after which they were BG blocked, and newly synthesized Na pump was accumulated in the TGN (red) for 2 h at 19°C. Alexa Fluor 488–Tfn was added to the medium during the Golgi block to label REs (green). Golgi-blocked cells were fixed (19°C) or warmed for the indicated period and stained with anti-HA (blue). (B) Cells were infected to express the human Tfn-R and VSV-G–YFP Ts 045 (green) and incubated overnight at 40°C. Cells were BG blocked at 40°C and incubated for 40 min at 40°C with Tfn-HRP along with 1 µM TMR-STAR to live label newly synthesized pump (red). Next, cells were washed and incubated for a further 20 min at 40°C to remove excess TMR-STAR and to allow for the accumulation of Tfn-HRP into REs. Tfn-HRP–loaded REs were ablated on ice by exposing cells to DAB and H 2 O 2 for 1 h, whereas control cells were treated with DAB alone. After the ablation reaction, cells were fixed or incubated in media plus CHX for 1.5 h at 31°C to allow trafficking of the proteins to the PM. (C) The percentage of protein that reached the lateral membrane 1.5 h after mock or ablation treatment was quantified using the enhanced colocalization tool (LSM Image Examiner software; Carl Zeiss, Inc.). 30 VSV-G–infected cells were analyzed, and data are represented as mean ± SD. (D) Samples were treated as in B and processed for immunofluorescence with a cocktail of Golgi antibodies (GM130, Vti1a, and GS15) or antibody against the Tfn-R shown in blue. The bottom panel depicts an individual confocal slice highlighting the colocalization of VSV-G with Tfn-R. TMR, tetramethylrhodamine. Bars, 5 µm.

    Techniques Used: Infection, Expressing, Synthesized, Blocking Assay, Staining, Incubation, Software, Immunofluorescence

    TGN-accumulated Na pump is rapidly trafficked to the lateral membrane. (A) SNAP cells were subjected to BG-Block, incubated at 37°C for 30 min to begin synthesis of new Na pump, and placed at 19°C for 2 h to accumulate newly synthesized protein in the TGN. TMR-STAR is depicted in red, and Golgi markers are shown in green. (B) SNAP cells were treated as in A and either fixed immediately (19°C) or warmed to 31°C for the indicated times. Samples were labeled with TMR-STAR (red) and with anti-HA (blue). Bars, 5 µm.
    Figure Legend Snippet: TGN-accumulated Na pump is rapidly trafficked to the lateral membrane. (A) SNAP cells were subjected to BG-Block, incubated at 37°C for 30 min to begin synthesis of new Na pump, and placed at 19°C for 2 h to accumulate newly synthesized protein in the TGN. TMR-STAR is depicted in red, and Golgi markers are shown in green. (B) SNAP cells were treated as in A and either fixed immediately (19°C) or warmed to 31°C for the indicated times. Samples were labeled with TMR-STAR (red) and with anti-HA (blue). Bars, 5 µm.

    Techniques Used: Blocking Assay, Incubation, Synthesized, Labeling

    TGN-accumulated Na pump is trafficked directly to and randomly throughout the lateral membrane. SNAP cells were BG blocked, and newly synthesized Na,K-ATPase was accumulated in the TGN for 2 h at 19°C. After the Golgi block, samples were fixed immediately (19°C) or warmed to 31°C for the indicated times. (A) Samples labeled for TMR-STAR (red) and the apical membrane marker gp135 (blue). (B) Samples labeled with TMR-STAR (red), the junctional marker ZO-1 (blue), and for lateral membrane via anti-HA (green). Arrows denote areas of relatively high intensity TMR-STAR labeling along the lateral membrane. Bars, 5 µm.
    Figure Legend Snippet: TGN-accumulated Na pump is trafficked directly to and randomly throughout the lateral membrane. SNAP cells were BG blocked, and newly synthesized Na,K-ATPase was accumulated in the TGN for 2 h at 19°C. After the Golgi block, samples were fixed immediately (19°C) or warmed to 31°C for the indicated times. (A) Samples labeled for TMR-STAR (red) and the apical membrane marker gp135 (blue). (B) Samples labeled with TMR-STAR (red), the junctional marker ZO-1 (blue), and for lateral membrane via anti-HA (green). Arrows denote areas of relatively high intensity TMR-STAR labeling along the lateral membrane. Bars, 5 µm.

    Techniques Used: Synthesized, Blocking Assay, Labeling, Marker

    Detection of Na pump localization via the SNAP tag. (A) Diagram of the SNAP tag labeling reaction for the N-terminally tagged Na,K-ATPase α subunit. TMR, tetramethylrhodamine. (B) Stably transfected MDCK cells expressing both SNAP–Na,K-ATPase α subunit and Na,K-ATPase β subunit (SNAP cells) were fixed and stained with TMR-STAR (red) and processed for immunofluorescence with anti-HA or gp58 (blue). Bars, 10 µm.
    Figure Legend Snippet: Detection of Na pump localization via the SNAP tag. (A) Diagram of the SNAP tag labeling reaction for the N-terminally tagged Na,K-ATPase α subunit. TMR, tetramethylrhodamine. (B) Stably transfected MDCK cells expressing both SNAP–Na,K-ATPase α subunit and Na,K-ATPase β subunit (SNAP cells) were fixed and stained with TMR-STAR (red) and processed for immunofluorescence with anti-HA or gp58 (blue). Bars, 10 µm.

    Techniques Used: Labeling, Stable Transfection, Transfection, Expressing, Staining, Immunofluorescence

    Na pump trafficking is not regulated by the same small GTPases as AP-1B–dependent cargo and does not require E-cadherin. (A) SNAP cells were grown on clear polyester filters for 4 d to form a fully polarized monolayer and microinjected with plasmids expressing human LDLR and one of the following: GFP, GFP–Rab8 Q67L, GFP–Rab10 Q68L, GFP–Rab11 Q70L, or GFP–CDC42 T17N. Cells were then allowed to recover for 45 min before BG-Block addition. After BG blocking, cells were washed and allowed to synthesize new SNAP-tagged Na pump for 3 h, followed by incubation in the presence of CHX for 45 min. Samples were labeled with TMR-STAR (red) after being processed for surface immunofluorescence for LDLR (blue). (B). SNAP cells were transfected with shRNA expression plasmids directed against E-cadherin, Cadherin-6, or luciferase (Luc) as a negative control and cultured to allow monolayer polarization as previously described ( Capaldo and Macara, 2007 ). 72 h after transfection, samples were stained with antibodies directed against E-cadherin (green) and ZO-1 (blue) and were labeled with TMR-STAR. (C) Cells were transfected and grown as in B and then subjected to the Golgi block protocol described in Fig. 3 . Images depict samples fixed immediately after Golgi block or after incubation for 20 min at 31°C to allow Na pump exit from the TGN. Newly synthesized Na,K-ATPase traffics directly to the basolateral surface in cadherin-depleted cells. Arrows highlight lateral membrane delivery for each sample. Bars, 5 µm.
    Figure Legend Snippet: Na pump trafficking is not regulated by the same small GTPases as AP-1B–dependent cargo and does not require E-cadherin. (A) SNAP cells were grown on clear polyester filters for 4 d to form a fully polarized monolayer and microinjected with plasmids expressing human LDLR and one of the following: GFP, GFP–Rab8 Q67L, GFP–Rab10 Q68L, GFP–Rab11 Q70L, or GFP–CDC42 T17N. Cells were then allowed to recover for 45 min before BG-Block addition. After BG blocking, cells were washed and allowed to synthesize new SNAP-tagged Na pump for 3 h, followed by incubation in the presence of CHX for 45 min. Samples were labeled with TMR-STAR (red) after being processed for surface immunofluorescence for LDLR (blue). (B). SNAP cells were transfected with shRNA expression plasmids directed against E-cadherin, Cadherin-6, or luciferase (Luc) as a negative control and cultured to allow monolayer polarization as previously described ( Capaldo and Macara, 2007 ). 72 h after transfection, samples were stained with antibodies directed against E-cadherin (green) and ZO-1 (blue) and were labeled with TMR-STAR. (C) Cells were transfected and grown as in B and then subjected to the Golgi block protocol described in Fig. 3 . Images depict samples fixed immediately after Golgi block or after incubation for 20 min at 31°C to allow Na pump exit from the TGN. Newly synthesized Na,K-ATPase traffics directly to the basolateral surface in cadherin-depleted cells. Arrows highlight lateral membrane delivery for each sample. Bars, 5 µm.

    Techniques Used: Expressing, Blocking Assay, Incubation, Labeling, Immunofluorescence, Transfection, shRNA, Luciferase, Negative Control, Cell Culture, Staining, Synthesized

    6) Product Images from "Spt6 is a maintenance factor for centromeric CENP-A"

    Article Title: Spt6 is a maintenance factor for centromeric CENP-A

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16695-7

    dCENP-A abundance at the centromere is affected by phosphorylation. a Stably transfected cells with SNAP-tagged wildtype (WT) or S77D mutant dCENP-A were visualized by staining with TMR Star. Boxes indicate the 2.5 times enlarged inset. b Quantification of centromeric signal intensities of wildtype ( n = 196) and S77A CENP-A ( n = 182 centromeres). Representative images ( a ) and quantification ( b ) of one out of N = 3 independent experiments are shown. c SNAP-tagged wildtype and S77A-mutant dCENP-A staining by TMR Star. d Quantification of centromeric signal intensities of wildtype ( n = 110) and S77D CENP-A ( n = 112). Representative images ( c ) and quantification ( d ) of one out of N = 5 independent experiments. Values are normalized relative to the wildtype mean (set to 100%). Data are represented as scatter plots with mean and 95% CI. Statistical significance: quadruple dots P
    Figure Legend Snippet: dCENP-A abundance at the centromere is affected by phosphorylation. a Stably transfected cells with SNAP-tagged wildtype (WT) or S77D mutant dCENP-A were visualized by staining with TMR Star. Boxes indicate the 2.5 times enlarged inset. b Quantification of centromeric signal intensities of wildtype ( n = 196) and S77A CENP-A ( n = 182 centromeres). Representative images ( a ) and quantification ( b ) of one out of N = 3 independent experiments are shown. c SNAP-tagged wildtype and S77A-mutant dCENP-A staining by TMR Star. d Quantification of centromeric signal intensities of wildtype ( n = 110) and S77D CENP-A ( n = 112). Representative images ( c ) and quantification ( d ) of one out of N = 5 independent experiments. Values are normalized relative to the wildtype mean (set to 100%). Data are represented as scatter plots with mean and 95% CI. Statistical significance: quadruple dots P

    Techniques Used: Stable Transfection, Transfection, Mutagenesis, Staining

    Depletion of human SPT6 leads to the loss of CENP-A maintenance. a Experimental setup. HeLa cells expressing SNAP-tagged CENP-A were treated with TMR-star to detect previously incorporated CENP-A and siRNA-treated to deplete proteins indicated in ( b , c ). Cells were then synchronized in S phase by a thymidine block and released. Cells were allowed transit through G1 phase and were collected at the next G1/S boundary by re-addition of thymine. b Cells were treated with indicated siRNAs for 48 h and extracts were processed for immunoblotting and probed with indicated antibodies. CC CENP-C, M Marker. N = 3 independent experiments. c Representative images of siRNA-treated cells, 48 h after TMR pulse labeling and mRNA depletion. Cells were counter stained with DAPI and anti-CENP-B antibodies to label DNA and centromeres, respectively. Scale bar represents 10 μm. d Quantification of experiments shown in ( a , c ). Mean and ± SEM of N = 3 independent experiments is shown normalized to median control siRNA (ctrl). Images are quantified with CrAQ2. n Crtl = 3989, n CC = 4097, n Spt6_1 = 3635, n Spt6_1 = 3559 centromeres. Statistical significance: triple dots P CC = 0.0001, single dots P 1 = 0,0374, single dots P 2 = 0.019. (One-way ANOVA, Dunnett’s multiple comparisons test). Source data are provided as a Source Data file.
    Figure Legend Snippet: Depletion of human SPT6 leads to the loss of CENP-A maintenance. a Experimental setup. HeLa cells expressing SNAP-tagged CENP-A were treated with TMR-star to detect previously incorporated CENP-A and siRNA-treated to deplete proteins indicated in ( b , c ). Cells were then synchronized in S phase by a thymidine block and released. Cells were allowed transit through G1 phase and were collected at the next G1/S boundary by re-addition of thymine. b Cells were treated with indicated siRNAs for 48 h and extracts were processed for immunoblotting and probed with indicated antibodies. CC CENP-C, M Marker. N = 3 independent experiments. c Representative images of siRNA-treated cells, 48 h after TMR pulse labeling and mRNA depletion. Cells were counter stained with DAPI and anti-CENP-B antibodies to label DNA and centromeres, respectively. Scale bar represents 10 μm. d Quantification of experiments shown in ( a , c ). Mean and ± SEM of N = 3 independent experiments is shown normalized to median control siRNA (ctrl). Images are quantified with CrAQ2. n Crtl = 3989, n CC = 4097, n Spt6_1 = 3635, n Spt6_1 = 3559 centromeres. Statistical significance: triple dots P CC = 0.0001, single dots P 1 = 0,0374, single dots P 2 = 0.019. (One-way ANOVA, Dunnett’s multiple comparisons test). Source data are provided as a Source Data file.

    Techniques Used: Expressing, Blocking Assay, Marker, Labeling, Staining

    7) Product Images from "Spt6 is a maintenance factor for centromeric CENP-A"

    Article Title: Spt6 is a maintenance factor for centromeric CENP-A

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16695-7

    dCENP-A abundance at the centromere is affected by phosphorylation. a Stably transfected cells with SNAP-tagged wildtype (WT) or S77D mutant dCENP-A were visualized by staining with TMR Star. Boxes indicate the 2.5 times enlarged inset. b Quantification of centromeric signal intensities of wildtype ( n = 196) and S77A CENP-A ( n = 182 centromeres). Representative images ( a ) and quantification ( b ) of one out of N = 3 independent experiments are shown. c SNAP-tagged wildtype and S77A-mutant dCENP-A staining by TMR Star. d Quantification of centromeric signal intensities of wildtype ( n = 110) and S77D CENP-A ( n = 112). Representative images ( c ) and quantification ( d ) of one out of N = 5 independent experiments. Values are normalized relative to the wildtype mean (set to 100%). Data are represented as scatter plots with mean and 95% CI. Statistical significance: quadruple dots P
    Figure Legend Snippet: dCENP-A abundance at the centromere is affected by phosphorylation. a Stably transfected cells with SNAP-tagged wildtype (WT) or S77D mutant dCENP-A were visualized by staining with TMR Star. Boxes indicate the 2.5 times enlarged inset. b Quantification of centromeric signal intensities of wildtype ( n = 196) and S77A CENP-A ( n = 182 centromeres). Representative images ( a ) and quantification ( b ) of one out of N = 3 independent experiments are shown. c SNAP-tagged wildtype and S77A-mutant dCENP-A staining by TMR Star. d Quantification of centromeric signal intensities of wildtype ( n = 110) and S77D CENP-A ( n = 112). Representative images ( c ) and quantification ( d ) of one out of N = 5 independent experiments. Values are normalized relative to the wildtype mean (set to 100%). Data are represented as scatter plots with mean and 95% CI. Statistical significance: quadruple dots P

    Techniques Used: Stable Transfection, Transfection, Mutagenesis, Staining

    Depletion of human SPT6 leads to the loss of CENP-A maintenance. a Experimental setup. HeLa cells expressing SNAP-tagged CENP-A were treated with TMR-star to detect previously incorporated CENP-A and siRNA-treated to deplete proteins indicated in ( b , c ). Cells were then synchronized in S phase by a thymidine block and released. Cells were allowed transit through G1 phase and were collected at the next G1/S boundary by re-addition of thymine. b Cells were treated with indicated siRNAs for 48 h and extracts were processed for immunoblotting and probed with indicated antibodies. CC CENP-C, M Marker. N = 3 independent experiments. c Representative images of siRNA-treated cells, 48 h after TMR pulse labeling and mRNA depletion. Cells were counter stained with DAPI and anti-CENP-B antibodies to label DNA and centromeres, respectively. Scale bar represents 10 μm. d Quantification of experiments shown in ( a , c ). Mean and ± SEM of N = 3 independent experiments is shown normalized to median control siRNA (ctrl). Images are quantified with CrAQ2. n Crtl = 3989, n CC = 4097, n Spt6_1 = 3635, n Spt6_1 = 3559 centromeres. Statistical significance: triple dots P CC = 0.0001, single dots P 1 = 0,0374, single dots P 2 = 0.019. (One-way ANOVA, Dunnett’s multiple comparisons test). Source data are provided as a Source Data file.
    Figure Legend Snippet: Depletion of human SPT6 leads to the loss of CENP-A maintenance. a Experimental setup. HeLa cells expressing SNAP-tagged CENP-A were treated with TMR-star to detect previously incorporated CENP-A and siRNA-treated to deplete proteins indicated in ( b , c ). Cells were then synchronized in S phase by a thymidine block and released. Cells were allowed transit through G1 phase and were collected at the next G1/S boundary by re-addition of thymine. b Cells were treated with indicated siRNAs for 48 h and extracts were processed for immunoblotting and probed with indicated antibodies. CC CENP-C, M Marker. N = 3 independent experiments. c Representative images of siRNA-treated cells, 48 h after TMR pulse labeling and mRNA depletion. Cells were counter stained with DAPI and anti-CENP-B antibodies to label DNA and centromeres, respectively. Scale bar represents 10 μm. d Quantification of experiments shown in ( a , c ). Mean and ± SEM of N = 3 independent experiments is shown normalized to median control siRNA (ctrl). Images are quantified with CrAQ2. n Crtl = 3989, n CC = 4097, n Spt6_1 = 3635, n Spt6_1 = 3559 centromeres. Statistical significance: triple dots P CC = 0.0001, single dots P 1 = 0,0374, single dots P 2 = 0.019. (One-way ANOVA, Dunnett’s multiple comparisons test). Source data are provided as a Source Data file.

    Techniques Used: Expressing, Blocking Assay, Marker, Labeling, Staining

    8) Product Images from "Epigenetic engineering shows that a human centromere resists silencing mediated by H3K27me3/K9me3"

    Article Title: Epigenetic engineering shows that a human centromere resists silencing mediated by H3K27me3/K9me3

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E15-08-0605

    Retention of PRC1 at HAC chromatin during mitosis is insufficient to affect CENP-A assembly. (A) PRC1 relocates away from chromatin during mitosis. Microscopy images of 1C7-EZH2 cells, after 4 d of EZH2 tethering (green), were analyzed for PRC1 signal at the HAC (as detected by its RING1A subunit) both at interphase and at various stages of mitosis. Antibodies against H3K27me3 (red) and RING1A (blue) were used for detection. Note that H3K27me3 signal is also shown as being reduced during mitosis, but the anti-H3K27me3 antibody used suffers from lack of epitope accessibility due to occlusion by the adjacent H3S28p modification present during mitosis (Hiroshi Kimura, personal communication). (B, B′) Inhibition of Aurora B activity leads to retention of PRC1 during mitosis at the EZH2-tethered HAC. Microscopy images of mitotic 1C7-EZH2 cells after 1 d of EZH2 tethering (green) in the presence or absence of Aurora B inhibitor ZM447439. (B) Confirmation of the inhibition of Aurora B kinase activity; antibodies against α-tubulin (red) and H3S10 phosphorylation (gray) were used. (B′) PRC1 is retained at the HAC during mitosis upon inhibition of Aurora B; antibodies against RING1A (red) were used to detect the PRC1 complex. Scale bar, 5 μm. (C) Experimental outline of procedure to analyze CENP-A assembly on EZH2-tethered HACs with or without PRC1 retention during mitosis (by inhibiting Aurora B activity). 1C7-EZH2 cells were grown in the absence of doxycycline, to allow for EZH2 tethering, for a total of 1 or 4 d before fixation. At 1 d before fixation, a plasmid expressing CENP-A-SNAP was transfected into cells, and ∼9 h before fixation, a quench-chase-pulse assay was performed to specifically label newly synthesized CENP-A-SNAP with TMR- Star . During the quench-chase-pulse period, cells were grown in parallel in medium supplemented either with dimethyl sulfoxide (DMSO) or ZM447439 (to inhibit Aurora B). G1 cells (those that loaded newly synthesized CENP-A onto centromeres during the assay timeline) were analyzed for the levels of CENP-A:SNAP∼TMR on the HAC. (D) PRC1 retention during mitosis at EZH2-tethered HACs has no visible effect on CENP-A assembly in the subsequent G1 phase. Quantification of C. Two independent experiments; ≥23 cells for each condition. Mann–Whitney U statistical test was used to evaluate significance of differences observed. A reduction in CENP-A assembly could be noted between controls of days 1 and 4; this loss of total CENP-A assembly is likely due to reduction in total centromere proteins (and consequently the assembly recruitment platform), in line with results in Figure 3A .
    Figure Legend Snippet: Retention of PRC1 at HAC chromatin during mitosis is insufficient to affect CENP-A assembly. (A) PRC1 relocates away from chromatin during mitosis. Microscopy images of 1C7-EZH2 cells, after 4 d of EZH2 tethering (green), were analyzed for PRC1 signal at the HAC (as detected by its RING1A subunit) both at interphase and at various stages of mitosis. Antibodies against H3K27me3 (red) and RING1A (blue) were used for detection. Note that H3K27me3 signal is also shown as being reduced during mitosis, but the anti-H3K27me3 antibody used suffers from lack of epitope accessibility due to occlusion by the adjacent H3S28p modification present during mitosis (Hiroshi Kimura, personal communication). (B, B′) Inhibition of Aurora B activity leads to retention of PRC1 during mitosis at the EZH2-tethered HAC. Microscopy images of mitotic 1C7-EZH2 cells after 1 d of EZH2 tethering (green) in the presence or absence of Aurora B inhibitor ZM447439. (B) Confirmation of the inhibition of Aurora B kinase activity; antibodies against α-tubulin (red) and H3S10 phosphorylation (gray) were used. (B′) PRC1 is retained at the HAC during mitosis upon inhibition of Aurora B; antibodies against RING1A (red) were used to detect the PRC1 complex. Scale bar, 5 μm. (C) Experimental outline of procedure to analyze CENP-A assembly on EZH2-tethered HACs with or without PRC1 retention during mitosis (by inhibiting Aurora B activity). 1C7-EZH2 cells were grown in the absence of doxycycline, to allow for EZH2 tethering, for a total of 1 or 4 d before fixation. At 1 d before fixation, a plasmid expressing CENP-A-SNAP was transfected into cells, and ∼9 h before fixation, a quench-chase-pulse assay was performed to specifically label newly synthesized CENP-A-SNAP with TMR- Star . During the quench-chase-pulse period, cells were grown in parallel in medium supplemented either with dimethyl sulfoxide (DMSO) or ZM447439 (to inhibit Aurora B). G1 cells (those that loaded newly synthesized CENP-A onto centromeres during the assay timeline) were analyzed for the levels of CENP-A:SNAP∼TMR on the HAC. (D) PRC1 retention during mitosis at EZH2-tethered HACs has no visible effect on CENP-A assembly in the subsequent G1 phase. Quantification of C. Two independent experiments; ≥23 cells for each condition. Mann–Whitney U statistical test was used to evaluate significance of differences observed. A reduction in CENP-A assembly could be noted between controls of days 1 and 4; this loss of total CENP-A assembly is likely due to reduction in total centromere proteins (and consequently the assembly recruitment platform), in line with results in Figure 3A .

    Techniques Used: HAC Assay, Microscopy, Modification, Inhibition, Activity Assay, Plasmid Preparation, Expressing, Transfection, Synthesized, MANN-WHITNEY

    9) Product Images from "Snap-, CLIP- and Halo-Tag Labelling of Budding Yeast Cells"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0078745

    Labelling of SNAP-, CLIP- and Halo-tagged proteins in chemically fixed and living budding yeast cells. (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.
    Figure Legend Snippet: Labelling of SNAP-, CLIP- and Halo-tagged proteins in chemically fixed and living budding yeast cells. (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.

    Techniques Used: Cross-linking Immunoprecipitation, Expressing, Electroporation, Construct, Plasmid Preparation

    Live cell super-resolution microscopy and multi-colour microscopy using self-labelling proteins. (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.
    Figure Legend Snippet: Live cell super-resolution microscopy and multi-colour microscopy using self-labelling proteins. (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.

    Techniques Used: Microscopy, Expressing, Cross-linking Immunoprecipitation, Electroporation, Epifluorescence Microscopy

    10) Product Images from "Optimization of fluorophores for chemical tagging and immunohistochemistry of Drosophila neurons"

    Article Title: Optimization of fluorophores for chemical tagging and immunohistochemistry of Drosophila neurons

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0200759

    Detail comparison of SS02565 neuronal membrane labeling. All samples show the same region of projections crossing from the left optic lobe to the central brain. Only the neuronal membrane channel is shown, and is labeled via antibodies in (A-B) and CLIP-tag in (C-D). (A) SS02565 was crossed to w;; 5XUAS-IVS-myr :: smFLAG in VK00005 , pJFRC51-3XUAS-IVS-Syt :: smHA in su(Hw)attP1 and brains were labeled with pure IHC, including rat anti-FLAG and ATTO 647N goat anti-rat antibodies over a period of 13 days. (B) SS02565 was crossed to w; brp-SNAP; 5XUAS-IVS-myr :: smFLAG in VK00005 , pJFRC51-3XUAS-IVS-Syt :: smHA in su(Hw)attP1 and brains were labeled with hybrid IHC, including rat anti-FLAG and ATTO 647N goat anti-rat antibodies over a period of 6 days. (C) SS02565 was crossed to w; brp-SNAP; UAS-myr :: 4xCLIPf in VK00005 , UAS-Syt :: Halo7 in VK0027 and brains were labeled for 15 minutes with pure chemical tags, including TMR CLIP-tag ligand. (D) SS02565 was crossed to w; brp-SNAP; UAS-myr :: 4xCLIPf in VK00005 , UAS-Syt :: Halo7 in VK0027 and brains were labeled for 15 minutes with pure chemical tags, including JF 549 CLIP-tag ligand.
    Figure Legend Snippet: Detail comparison of SS02565 neuronal membrane labeling. All samples show the same region of projections crossing from the left optic lobe to the central brain. Only the neuronal membrane channel is shown, and is labeled via antibodies in (A-B) and CLIP-tag in (C-D). (A) SS02565 was crossed to w;; 5XUAS-IVS-myr :: smFLAG in VK00005 , pJFRC51-3XUAS-IVS-Syt :: smHA in su(Hw)attP1 and brains were labeled with pure IHC, including rat anti-FLAG and ATTO 647N goat anti-rat antibodies over a period of 13 days. (B) SS02565 was crossed to w; brp-SNAP; 5XUAS-IVS-myr :: smFLAG in VK00005 , pJFRC51-3XUAS-IVS-Syt :: smHA in su(Hw)attP1 and brains were labeled with hybrid IHC, including rat anti-FLAG and ATTO 647N goat anti-rat antibodies over a period of 6 days. (C) SS02565 was crossed to w; brp-SNAP; UAS-myr :: 4xCLIPf in VK00005 , UAS-Syt :: Halo7 in VK0027 and brains were labeled for 15 minutes with pure chemical tags, including TMR CLIP-tag ligand. (D) SS02565 was crossed to w; brp-SNAP; UAS-myr :: 4xCLIPf in VK00005 , UAS-Syt :: Halo7 in VK0027 and brains were labeled for 15 minutes with pure chemical tags, including JF 549 CLIP-tag ligand.

    Techniques Used: Labeling, Cross-linking Immunoprecipitation, Immunohistochemistry

    Comparison of Polarity IHC and chemical tag labeling methods. All samples show the Drosophila left optic lobe imaged at 63X. Each image is independently scaled for optimal intensity. (A). Polarity pure IHC : Split GAL4 SS02565 was crossed to w;; 5XUAS-IVS-myr :: smFLAG in VK00005 , pJFRC51-3XUAS-IVS-Syt :: smHA in su(Hw)attP1 and was labeled over a period of 13 days with nc82 mouse anti-Brp/Cy2 anti-mouse, rabbit anti-HA/Cy3 anti-rabbit, and rat anti-FLAG/Cy5 anti-rat. (B). Polarity hybrid IHC : SS02565 was crossed to w; brp-SNAP; 5XUAS-IVS-myr :: smFLAG in VK00005 , pJFRC51-3XUAS-IVS-Syt :: smHA in su(Hw)attP1 and labeled with Cy2 SNAP-tag ligand for 15 minutes, followed by rabbit anti-HA/Cy3 anti-rabbit, and rat anti-FLAG/Cy5 anti-rat over 6 days. Arrowheads indicate bleed-through of Cy5 into Cy2 channel. (C). Polarity pure chemical tag : SS02565 was crossed to w; brp-SNAP; UAS-myr :: 4xCLIPf in VK00005 , UAS-Syt :: Halo7 in VK0027 and labeled for 15 minutes with Cy2 SNAP-tag ligand, TMR CLIP-tag ligand, and ATTO 647N HaloTag ligand. (D). Polarity ATTO 647N pure IHC : As in (A) but with ATTO 647N instead of Cy5. (E). Polarity ATTO 647N hybrid IHC : As in (B) but with ATTO 647N instead of Cy5.
    Figure Legend Snippet: Comparison of Polarity IHC and chemical tag labeling methods. All samples show the Drosophila left optic lobe imaged at 63X. Each image is independently scaled for optimal intensity. (A). Polarity pure IHC : Split GAL4 SS02565 was crossed to w;; 5XUAS-IVS-myr :: smFLAG in VK00005 , pJFRC51-3XUAS-IVS-Syt :: smHA in su(Hw)attP1 and was labeled over a period of 13 days with nc82 mouse anti-Brp/Cy2 anti-mouse, rabbit anti-HA/Cy3 anti-rabbit, and rat anti-FLAG/Cy5 anti-rat. (B). Polarity hybrid IHC : SS02565 was crossed to w; brp-SNAP; 5XUAS-IVS-myr :: smFLAG in VK00005 , pJFRC51-3XUAS-IVS-Syt :: smHA in su(Hw)attP1 and labeled with Cy2 SNAP-tag ligand for 15 minutes, followed by rabbit anti-HA/Cy3 anti-rabbit, and rat anti-FLAG/Cy5 anti-rat over 6 days. Arrowheads indicate bleed-through of Cy5 into Cy2 channel. (C). Polarity pure chemical tag : SS02565 was crossed to w; brp-SNAP; UAS-myr :: 4xCLIPf in VK00005 , UAS-Syt :: Halo7 in VK0027 and labeled for 15 minutes with Cy2 SNAP-tag ligand, TMR CLIP-tag ligand, and ATTO 647N HaloTag ligand. (D). Polarity ATTO 647N pure IHC : As in (A) but with ATTO 647N instead of Cy5. (E). Polarity ATTO 647N hybrid IHC : As in (B) but with ATTO 647N instead of Cy5.

    Techniques Used: Immunohistochemistry, Labeling, Cross-linking Immunoprecipitation

    11) Product Images from "Light-induced cell damage in live-cell super-resolution microscopy"

    Article Title: Light-induced cell damage in live-cell super-resolution microscopy

    Journal: Scientific Reports

    doi: 10.1038/srep15348

    Dependence of cell survival on irradiation intensity at 514 nm for 240 s of differently modified U2OS cells. ( a ) Wildtype cells irradiated at 21 °C, ( b ) cells stably transfected with CLIP-H2B irradiated at 21 °C, ( c ) cells stably transfected with CLIP-H2B and labeled with TMR, irradiated at 21 °C and ( d ) cells stably transfected with CLIP-H2B irradiated at 37 °C. Red dots are masked data points and were not considered for fitting. Error bars are given as one standard deviation. For each data point 20–50 cells were irradiated ( Table 1 ).
    Figure Legend Snippet: Dependence of cell survival on irradiation intensity at 514 nm for 240 s of differently modified U2OS cells. ( a ) Wildtype cells irradiated at 21 °C, ( b ) cells stably transfected with CLIP-H2B irradiated at 21 °C, ( c ) cells stably transfected with CLIP-H2B and labeled with TMR, irradiated at 21 °C and ( d ) cells stably transfected with CLIP-H2B irradiated at 37 °C. Red dots are masked data points and were not considered for fitting. Error bars are given as one standard deviation. For each data point 20–50 cells were irradiated ( Table 1 ).

    Techniques Used: Irradiation, Modification, Stable Transfection, Transfection, Cross-linking Immunoprecipitation, Labeling, Standard Deviation

    12) Product Images from "CAS-LiveFISH: Simple and versatile imaging of genomic loci in live mammalian cells and early pre-implantation embryos"

    Article Title: CAS-LiveFISH: Simple and versatile imaging of genomic loci in live mammalian cells and early pre-implantation embryos

    Journal: bioRxiv

    doi: 10.1101/2020.08.25.265306

    Pools of pre-assembled RNPs enable visualizing the MYC locus in live HeLa cells. ( A ) Organization of the extended MYC locus though proximity ligation (ChIA-PET) and ChIP-Seq assays. HeLa Pol II ChIA-PET data (GSM832461) ( Li et al., 2012 ),as well as ChIP-Seq data for p300 (GSM935500), H3K27ac (GSM733684) and Pol2-Ser2Ph (GSM935383), are visualized using the WashU Epigenome Browser ( Zhou et al., 2011 ). ( B ) Visualization of the HPV integration site using Alexa 647-labeled dCas9 RNPs, assembled with a pool of 36 distinct gRNAs. The right cell contains doublets of closely-spaced puncta, indicative of replicated loci in S/G2. ( C ) Distribution of number of discernible Alexa 647-HPV spots per cell. ( D ) Two-color imaging of HPV integration site (Alexa 647) and MYC (TMR) using co-delivery of preassembled RNPs in live HeLa cells. ( E ) 2D scatter plot of relative positions and statistics (histogram and box plot) of 2D distances between Alexa 647-HPV and TMR-MYC in live HeLa cells. 2D distance is 177±155 nm (mean±S.D.; n =472 measurements from > 30 loci).
    Figure Legend Snippet: Pools of pre-assembled RNPs enable visualizing the MYC locus in live HeLa cells. ( A ) Organization of the extended MYC locus though proximity ligation (ChIA-PET) and ChIP-Seq assays. HeLa Pol II ChIA-PET data (GSM832461) ( Li et al., 2012 ),as well as ChIP-Seq data for p300 (GSM935500), H3K27ac (GSM733684) and Pol2-Ser2Ph (GSM935383), are visualized using the WashU Epigenome Browser ( Zhou et al., 2011 ). ( B ) Visualization of the HPV integration site using Alexa 647-labeled dCas9 RNPs, assembled with a pool of 36 distinct gRNAs. The right cell contains doublets of closely-spaced puncta, indicative of replicated loci in S/G2. ( C ) Distribution of number of discernible Alexa 647-HPV spots per cell. ( D ) Two-color imaging of HPV integration site (Alexa 647) and MYC (TMR) using co-delivery of preassembled RNPs in live HeLa cells. ( E ) 2D scatter plot of relative positions and statistics (histogram and box plot) of 2D distances between Alexa 647-HPV and TMR-MYC in live HeLa cells. 2D distance is 177±155 nm (mean±S.D.; n =472 measurements from > 30 loci).

    Techniques Used: Ligation, ChIA Pet Assay, Chromatin Immunoprecipitation, Labeling, Imaging

    Imaging genomic loci in live cells with CAS-LiveFISH. ( A ) Schematic of cell delivery of in vitro transcribed gRNAs. ( B, C ) Visualization of telomeres in U-2 OS cells stably expressing dCas9-EGFP, by electroporation ( B ) or microinjection ( C ) of an in vitro transcribed gRNA (sgTelomere). A non-targeting control (sgGal4) shows no discernible nuclear puncta ( B ), demonstrating locus-specific tagging. In ( B ), cells are imaged 20hrs after electroporation. Co-injection of a BFP-LifeAct-expressing plasmid ( B ) or Alexa 647-Benzylguanine ( C ) results in simultaneous visualization of actin filaments and RNA Polymerase II (SNAP-Pol II), respectively. ( D ) Schematic of recombinant fluorescent dCas9:gRNA RNP assembly and cell delivery. ( E, F, G ) Visualization of genomic loci in U-2 OS ( E ) and HeLa ( F, G ) cells using pre-assembled dCas9-gRNA RNPs. ( F ) Co-delivery of TMR-RNPs targeting telomeres and Alexa 647-RNPs targeting a-satellite sequences shows distinct, non-overlapping nuclear puncta. ( G ) Co-injection of telomere-targeting Atto 488- or TMR-RNPs and non-targeting control Alexa 647-RNPs (sgGal4) shows no discernible nuclear puncta in the Alexa 647 channel, demonstrating locus-specific tagging and minimal cross-talk between colors.
    Figure Legend Snippet: Imaging genomic loci in live cells with CAS-LiveFISH. ( A ) Schematic of cell delivery of in vitro transcribed gRNAs. ( B, C ) Visualization of telomeres in U-2 OS cells stably expressing dCas9-EGFP, by electroporation ( B ) or microinjection ( C ) of an in vitro transcribed gRNA (sgTelomere). A non-targeting control (sgGal4) shows no discernible nuclear puncta ( B ), demonstrating locus-specific tagging. In ( B ), cells are imaged 20hrs after electroporation. Co-injection of a BFP-LifeAct-expressing plasmid ( B ) or Alexa 647-Benzylguanine ( C ) results in simultaneous visualization of actin filaments and RNA Polymerase II (SNAP-Pol II), respectively. ( D ) Schematic of recombinant fluorescent dCas9:gRNA RNP assembly and cell delivery. ( E, F, G ) Visualization of genomic loci in U-2 OS ( E ) and HeLa ( F, G ) cells using pre-assembled dCas9-gRNA RNPs. ( F ) Co-delivery of TMR-RNPs targeting telomeres and Alexa 647-RNPs targeting a-satellite sequences shows distinct, non-overlapping nuclear puncta. ( G ) Co-injection of telomere-targeting Atto 488- or TMR-RNPs and non-targeting control Alexa 647-RNPs (sgGal4) shows no discernible nuclear puncta in the Alexa 647 channel, demonstrating locus-specific tagging and minimal cross-talk between colors.

    Techniques Used: Imaging, In Vitro, Stable Transfection, Expressing, Electroporation, Injection, Plasmid Preparation, Recombinant

    13) Product Images from "A Gateway-Based System for Fast Evaluation of Protein-Protein Interactions in Bacteria"

    Article Title: A Gateway-Based System for Fast Evaluation of Protein-Protein Interactions in Bacteria

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0123646

    Subcellular localization of chemotaxis proteins CheY and CheZ. Two representative S . Typhimurium cells co-expressing fusions of CheY and CheZ to Halo- and SNAP-Tag from Expression clone pWRG602 are shown. Fusion proteins were labeled with Atto655-coupled HaloTag ligand and the SNAP-tag ligand TMR-Star. Subcellular localization of protein clusters was visualized using super-resolution microscopy (dSTORM). Colocalization of the labeled fusion proteins was calculated in separate images using the Matlab-based software Slimfast with a distance threshold of 100 nm. The insets show autofluorescence of the bacteria after excitation at 488 nm which was used to determine their shapes (dashed white lines). Scale bars = 1 μm.
    Figure Legend Snippet: Subcellular localization of chemotaxis proteins CheY and CheZ. Two representative S . Typhimurium cells co-expressing fusions of CheY and CheZ to Halo- and SNAP-Tag from Expression clone pWRG602 are shown. Fusion proteins were labeled with Atto655-coupled HaloTag ligand and the SNAP-tag ligand TMR-Star. Subcellular localization of protein clusters was visualized using super-resolution microscopy (dSTORM). Colocalization of the labeled fusion proteins was calculated in separate images using the Matlab-based software Slimfast with a distance threshold of 100 nm. The insets show autofluorescence of the bacteria after excitation at 488 nm which was used to determine their shapes (dashed white lines). Scale bars = 1 μm.

    Techniques Used: Chemotaxis Assay, Expressing, Labeling, Microscopy, Software

    Related Articles

    Staining:

    Article Title: Snap-, CLIP- and Halo-Tag Labelling of Budding Yeast Cells
    Article Snippet: .. Fluorescent CLIP-, SNAP- and Halo-tag ligands 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. .. Alternatively, CLIP-, SNAP- and Halo-tag ligands were assembled from commercially available building blocks.

    Article Title: Light-induced cell damage in live-cell super-resolution microscopy
    Article Snippet: .. Cells were stained with 0.2 μM CLIP-Cell™ TMR-Star (New England Biolabs, cat. S9219S) for 30 minutes at 37 °C. ..

    Incubation:

    Article Title: Aurora A and Aurora B jointly coordinate chromosome segregation and anaphase microtubule dynamics
    Article Snippet: .. Within the last hour of 1NMPP1 incubation, cells were pulse labeled with 5 µM TMR-Star (New England Biolabs, Inc.) for 30 min and then washed twice with warm medium containing 10 µM 1NMPP1 and doxycyclin/4-hydroxytamoxifen if required. ..

    Article Title: Ran-GTP is non-essential to activate NuMA for spindle pole focusing, but dynamically polarizes HURP to control mitotic spindle length
    Article Snippet: .. To visualize SNAP-tagged HURP in , cells were incubated with 0.1 μM TMR-Star (New England BioLabs) for > 2 hr, and TMR-Star were removed before observation. ..

    Cross-linking Immunoprecipitation:

    Article Title: Snap-, CLIP- and Halo-Tag Labelling of Budding Yeast Cells
    Article Snippet: .. Fluorescent CLIP-, SNAP- and Halo-tag ligands 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. .. Alternatively, CLIP-, SNAP- and Halo-tag ligands were assembled from commercially available building blocks.

    Article Title: Light-induced cell damage in live-cell super-resolution microscopy
    Article Snippet: .. Cells were stained with 0.2 μM CLIP-Cell™ TMR-Star (New England Biolabs, cat. S9219S) for 30 minutes at 37 °C. ..

    Article Title: Optimization of fluorophores for chemical tagging and immunohistochemistry of Drosophila neurons
    Article Snippet: .. In addition to the novel chemical tag ligands described here, we used CLIP-Cell TMR-Star (S9219S, New England Biolabs, Ipswich, MA) and JF549 SNAP-tag ligand [ ]. .. Hybrid IHC & chemical tag The hybrid IHC/chemical tag protocol combined Cy2 SNAP-tag ligand labeling of the brp-SNAP reference with antibody labeling of specific neurons.

    Article Title: CAS-LiveFISH: Simple and versatile imaging of genomic loci in live mammalian cells and early pre-implantation embryos
    Article Snippet: .. For CLIP tag labelling, CLIP-Cell TMR star substrate or CLIP-Surface 488 substrate (NEB S9219 and S9232) was reacted with dCas9-CLIP proteins at 37°C for 60min. .. The excess fluorescent SNAP or CLIP substrate was removed with three washes using 10 K molecular weight (MWCO) spin concentrators (Millipore).

    Labeling:

    Article Title: Aurora A and Aurora B jointly coordinate chromosome segregation and anaphase microtubule dynamics
    Article Snippet: .. Within the last hour of 1NMPP1 incubation, cells were pulse labeled with 5 µM TMR-Star (New England Biolabs, Inc.) for 30 min and then washed twice with warm medium containing 10 µM 1NMPP1 and doxycyclin/4-hydroxytamoxifen if required. ..

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    New England Biolabs clip cell tmr star
    <t>CLIP-Cas9</t> activity and internalization in keratinocytes. ( a ) Schematic of CLIP-Cas9::sgRNA electroporation strategy. ( b ) Indel spectrum determined by TIDE of primary keratinocytes electroporated with CLIP-Cas9::sgRNA targeting the Atat1 gene. The inset show T7 endonuclease 1 assay performed on genomic DNA from electroporated keratinocytes. t.e. = total efficiency. ( c ) Quantification (% cells) and representative images ( d ) of <t>TMR</t> positive cells upon 2 hours treatment with 2 μM of ligand cross-linked Cas9 (#1 no sgRNA; #2 with sgRNA; #3 with sgRNA+ protamine; #4 with sgRNA + ppTG21). Nuclei were stained with Hoechst. Scale bars, 20 μm. The horizontal lines mark the geometric mean and the error bars mark the standard error.
    Clip Cell Tmr Star, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Labelling specificity probed by single molecule localization. ( A ) MvP818 [p3725], the invC deletion strain expressing invC ::HaloTag, was treated with 20 nM <t>HTL-TMR</t> or 150 mM HTL-Atto655. MvP818 [p3723], the invC deletion strain expressing invC ::SNAP-tag, was treated with 30 nM TMR-Star. Labeling reactions were performed for 45 min at 37 °C. For the shape of the bacteria, the maximum intensity projection with bilinear interpolation of all 500 acquired frames is shown (left panel). After localization distinct patches of InvC are visible for each condition. The white dots in the localized images show the localized molecules of all 500 acquired frames (right panel). Clusters are indicated with red arrows. ( B ) Salmonella WT cells were treated with 20 nM HTL-TMR, 150 nM HTL-Atto655 or 30 nM TMR-Star for 45 min at 37 °C. 500 frames were acquired as described before with 5 mW laser power at the focal plane. The autofluorescence of bacteria upon excitation with a 488 nm laser is shown (left panel). SRM images rendered after localization of individual emitters (right panel) confirmed labelling specificity in all cases. Scale bars, 1 μm.
    Tmr Star, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    CLIP-Cas9 activity and internalization in keratinocytes. ( a ) Schematic of CLIP-Cas9::sgRNA electroporation strategy. ( b ) Indel spectrum determined by TIDE of primary keratinocytes electroporated with CLIP-Cas9::sgRNA targeting the Atat1 gene. The inset show T7 endonuclease 1 assay performed on genomic DNA from electroporated keratinocytes. t.e. = total efficiency. ( c ) Quantification (% cells) and representative images ( d ) of TMR positive cells upon 2 hours treatment with 2 μM of ligand cross-linked Cas9 (#1 no sgRNA; #2 with sgRNA; #3 with sgRNA+ protamine; #4 with sgRNA + ppTG21). Nuclei were stained with Hoechst. Scale bars, 20 μm. The horizontal lines mark the geometric mean and the error bars mark the standard error.

    Journal: Scientific Reports

    Article Title: A ligand-based system for receptor-specific delivery of proteins

    doi: 10.1038/s41598-019-55797-1

    Figure Lengend Snippet: CLIP-Cas9 activity and internalization in keratinocytes. ( a ) Schematic of CLIP-Cas9::sgRNA electroporation strategy. ( b ) Indel spectrum determined by TIDE of primary keratinocytes electroporated with CLIP-Cas9::sgRNA targeting the Atat1 gene. The inset show T7 endonuclease 1 assay performed on genomic DNA from electroporated keratinocytes. t.e. = total efficiency. ( c ) Quantification (% cells) and representative images ( d ) of TMR positive cells upon 2 hours treatment with 2 μM of ligand cross-linked Cas9 (#1 no sgRNA; #2 with sgRNA; #3 with sgRNA+ protamine; #4 with sgRNA + ppTG21). Nuclei were stained with Hoechst. Scale bars, 20 μm. The horizontal lines mark the geometric mean and the error bars mark the standard error.

    Article Snippet: SDS-PAGE and western blotting To assess the coupling reaction, CLIP-Cre or CLIP-Cas9 were coupled with an excess of BC-Surface488 or BCTMR at a 1:1.5 molar ratio (NEB #S9232S; #S9219S) for 1 hour at 37 °C in PBS (pH 7.4).

    Techniques: Cross-linking Immunoprecipitation, Activity Assay, Electroporation, Staining

    Binding of SNAP-tagged ligands to keratinocytes and selective cross-linking to CLIP-tagged enzymes. ( a ) Schematic representation of one keratinocyte expressing the receptors of interest and the ligands used. ( b ) Quantification of labelled IL-31 K138A SNAP-BG 549 ( c ), NGF R121W SNAP-BG 549 ( d ) and SNAP-BG 549 (green bars) binding to primary keratinocytes. Nuclear localization was observed after 2 hours treatment. The nuclei were stained with Hoechst. Scale bars, 20 μm. The insets represent corresponding brightfield images. ( e ) 3D structures showing selective cross-linking of SNAP-tagged ligands (NGF-SNAP) and CLIP-tagged proteins (CLIP-Cre) through a BG-TMR-PEG-BC linker (PDB ID codes: 1BET, 1KBU, 3KZY). ( f ) Schematic representation of S-CROSS optimized chemical reaction. ( g ) Representative Coomassie gel showing cross-linking complexes (red asterisks). First lane (#1) is IL-31 SNAP::CLIP CRE, second lane (#2) is NGF SNAP::CLIP CRE and third lane (#3) is SNAP::CLIP-CRE. ( h ) Quantification of cross-linking from Coomassie gel ( g ).

    Journal: Scientific Reports

    Article Title: A ligand-based system for receptor-specific delivery of proteins

    doi: 10.1038/s41598-019-55797-1

    Figure Lengend Snippet: Binding of SNAP-tagged ligands to keratinocytes and selective cross-linking to CLIP-tagged enzymes. ( a ) Schematic representation of one keratinocyte expressing the receptors of interest and the ligands used. ( b ) Quantification of labelled IL-31 K138A SNAP-BG 549 ( c ), NGF R121W SNAP-BG 549 ( d ) and SNAP-BG 549 (green bars) binding to primary keratinocytes. Nuclear localization was observed after 2 hours treatment. The nuclei were stained with Hoechst. Scale bars, 20 μm. The insets represent corresponding brightfield images. ( e ) 3D structures showing selective cross-linking of SNAP-tagged ligands (NGF-SNAP) and CLIP-tagged proteins (CLIP-Cre) through a BG-TMR-PEG-BC linker (PDB ID codes: 1BET, 1KBU, 3KZY). ( f ) Schematic representation of S-CROSS optimized chemical reaction. ( g ) Representative Coomassie gel showing cross-linking complexes (red asterisks). First lane (#1) is IL-31 SNAP::CLIP CRE, second lane (#2) is NGF SNAP::CLIP CRE and third lane (#3) is SNAP::CLIP-CRE. ( h ) Quantification of cross-linking from Coomassie gel ( g ).

    Article Snippet: SDS-PAGE and western blotting To assess the coupling reaction, CLIP-Cre or CLIP-Cas9 were coupled with an excess of BC-Surface488 or BCTMR at a 1:1.5 molar ratio (NEB #S9232S; #S9219S) for 1 hour at 37 °C in PBS (pH 7.4).

    Techniques: Binding Assay, Cross-linking Immunoprecipitation, Expressing, Staining

    Labelling specificity probed by single molecule localization. ( A ) MvP818 [p3725], the invC deletion strain expressing invC ::HaloTag, was treated with 20 nM HTL-TMR or 150 mM HTL-Atto655. MvP818 [p3723], the invC deletion strain expressing invC ::SNAP-tag, was treated with 30 nM TMR-Star. Labeling reactions were performed for 45 min at 37 °C. For the shape of the bacteria, the maximum intensity projection with bilinear interpolation of all 500 acquired frames is shown (left panel). After localization distinct patches of InvC are visible for each condition. The white dots in the localized images show the localized molecules of all 500 acquired frames (right panel). Clusters are indicated with red arrows. ( B ) Salmonella WT cells were treated with 20 nM HTL-TMR, 150 nM HTL-Atto655 or 30 nM TMR-Star for 45 min at 37 °C. 500 frames were acquired as described before with 5 mW laser power at the focal plane. The autofluorescence of bacteria upon excitation with a 488 nm laser is shown (left panel). SRM images rendered after localization of individual emitters (right panel) confirmed labelling specificity in all cases. Scale bars, 1 μm.

    Journal: Scientific Reports

    Article Title: Single molecule super-resolution imaging of proteins in living Salmonella enterica using self-labelling enzymes

    doi: 10.1038/srep31601

    Figure Lengend Snippet: Labelling specificity probed by single molecule localization. ( A ) MvP818 [p3725], the invC deletion strain expressing invC ::HaloTag, was treated with 20 nM HTL-TMR or 150 mM HTL-Atto655. MvP818 [p3723], the invC deletion strain expressing invC ::SNAP-tag, was treated with 30 nM TMR-Star. Labeling reactions were performed for 45 min at 37 °C. For the shape of the bacteria, the maximum intensity projection with bilinear interpolation of all 500 acquired frames is shown (left panel). After localization distinct patches of InvC are visible for each condition. The white dots in the localized images show the localized molecules of all 500 acquired frames (right panel). Clusters are indicated with red arrows. ( B ) Salmonella WT cells were treated with 20 nM HTL-TMR, 150 nM HTL-Atto655 or 30 nM TMR-Star for 45 min at 37 °C. 500 frames were acquired as described before with 5 mW laser power at the focal plane. The autofluorescence of bacteria upon excitation with a 488 nm laser is shown (left panel). SRM images rendered after localization of individual emitters (right panel) confirmed labelling specificity in all cases. Scale bars, 1 μm.

    Article Snippet: After 2 h 45 min of subculture, fluorescent ligands were added, i.e. 20 nM HTL-TMR (Promega) or 150 nM HTL-Atto655 (self-synthesized) for HaloTag fusions, and 30 nM TMR-Star (NEB) for SNAP-tag fusions.

    Techniques: Expressing, Labeling

    Effect of enzyme tags and ligands on functionality of protein secretion systems in Salmonella . Invasion of polarized epithelial cell line MDCK by various Salmonella strains was quantified. Salmonella WT, mutant strains, and strains expressing HaloTag or HaloTag and SNAP-tag fusions as indicated were used to infect MDCK cells at an multiplicity of infection (MOI) of 5. Non-internalized bacteria were removed by washing and remaining bacteria killed by addition of gentamicin for 1 h. Subsequently, the cells were lysed and serial dilutions were plated onto agar plates for colony-forming units (CFU) determination. Invasion is expressed as percentage of the inoculum that was internalized by host ells. After adding the inoculum, infection was synchronized by centrifugation ( A ), or no centrifugation was performed ( B,C ). Mutant strains siiF, spaS and fliM served as SPI4-T1SS-defective, SPI1-T3SS-defective and non-motile control strains, respectively ( C ). ( D,E ) Prior to invasion, the cells were mock-treated (−) or 20 nM HTL-TMR ( D , +, hatched bars) or 30 nM TMR-Star was added ( E , +, hatched bars). Cells were infected without centrifugation. Statistical significance as determined by Student’s t test is indicated as ns, not significant; * P

    Journal: Scientific Reports

    Article Title: Single molecule super-resolution imaging of proteins in living Salmonella enterica using self-labelling enzymes

    doi: 10.1038/srep31601

    Figure Lengend Snippet: Effect of enzyme tags and ligands on functionality of protein secretion systems in Salmonella . Invasion of polarized epithelial cell line MDCK by various Salmonella strains was quantified. Salmonella WT, mutant strains, and strains expressing HaloTag or HaloTag and SNAP-tag fusions as indicated were used to infect MDCK cells at an multiplicity of infection (MOI) of 5. Non-internalized bacteria were removed by washing and remaining bacteria killed by addition of gentamicin for 1 h. Subsequently, the cells were lysed and serial dilutions were plated onto agar plates for colony-forming units (CFU) determination. Invasion is expressed as percentage of the inoculum that was internalized by host ells. After adding the inoculum, infection was synchronized by centrifugation ( A ), or no centrifugation was performed ( B,C ). Mutant strains siiF, spaS and fliM served as SPI4-T1SS-defective, SPI1-T3SS-defective and non-motile control strains, respectively ( C ). ( D,E ) Prior to invasion, the cells were mock-treated (−) or 20 nM HTL-TMR ( D , +, hatched bars) or 30 nM TMR-Star was added ( E , +, hatched bars). Cells were infected without centrifugation. Statistical significance as determined by Student’s t test is indicated as ns, not significant; * P

    Article Snippet: After 2 h 45 min of subculture, fluorescent ligands were added, i.e. 20 nM HTL-TMR (Promega) or 150 nM HTL-Atto655 (self-synthesized) for HaloTag fusions, and 30 nM TMR-Star (NEB) for SNAP-tag fusions.

    Techniques: Mutagenesis, Expressing, Infection, Centrifugation

    SMT of TMR-labelled SiiF, SpaS, FliN and HilA. ( A ) SiiF-HaloTag, HilA-HaloTag, SpaS-HaloTag, or FliN-HaloTag were labelled with TMR-Star and imaged as described for Fig. 4 . Selected frames from series of 500 frames are shown (frame rate: 15 frames per second) and frame numbers are indicated. Inserts in frame 2 show localized images. Each trajectory has a different colour. Using pooled trajectories for proteins in at least 20 bacteria recorded under the same conditions, the diffusion coefficient D was calculated using the Jaqaman algorithm 5 20 61 . ( B ) Values for the diffusion coefficients. Scale bars, 0.5 μm (SiiF-HaloTag), 1 μm (SpaS-HaloTag, FliN-HaloTag, HilA-HaloTag). The sequences of 500 frames are shown in Movie S1 for (SiiF-HaloTag), Movie S2 (SpaS-HaloTag), Movie S3 (FliN-HaloTag), and Movie S4 (HilA-HaloTag).

    Journal: Scientific Reports

    Article Title: Single molecule super-resolution imaging of proteins in living Salmonella enterica using self-labelling enzymes

    doi: 10.1038/srep31601

    Figure Lengend Snippet: SMT of TMR-labelled SiiF, SpaS, FliN and HilA. ( A ) SiiF-HaloTag, HilA-HaloTag, SpaS-HaloTag, or FliN-HaloTag were labelled with TMR-Star and imaged as described for Fig. 4 . Selected frames from series of 500 frames are shown (frame rate: 15 frames per second) and frame numbers are indicated. Inserts in frame 2 show localized images. Each trajectory has a different colour. Using pooled trajectories for proteins in at least 20 bacteria recorded under the same conditions, the diffusion coefficient D was calculated using the Jaqaman algorithm 5 20 61 . ( B ) Values for the diffusion coefficients. Scale bars, 0.5 μm (SiiF-HaloTag), 1 μm (SpaS-HaloTag, FliN-HaloTag, HilA-HaloTag). The sequences of 500 frames are shown in Movie S1 for (SiiF-HaloTag), Movie S2 (SpaS-HaloTag), Movie S3 (FliN-HaloTag), and Movie S4 (HilA-HaloTag).

    Article Snippet: After 2 h 45 min of subculture, fluorescent ligands were added, i.e. 20 nM HTL-TMR (Promega) or 150 nM HTL-Atto655 (self-synthesized) for HaloTag fusions, and 30 nM TMR-Star (NEB) for SNAP-tag fusions.

    Techniques: Diffusion-based Assay

    Dual colour dSTORM of Salmonella using fusion proteins to HaloTag and SNAP-tag. Flagellar motor subunit FliN and SPI1-T3SS subunit SpaS were visualized within one cell. ( A ) FliN-SNAP-tag and SpaS-HaloTag were stained with 30 nM TMR-Star (green) and 150 nM HTL-Atto655 (red), respectively. ( B ) SpaS-SNAP-tag and FliN-HaloTag were stained with 30 nM TMR-Star (green) and 150 nM HTL-Atto655 (red), respectively. After staining, cells were washed and fixed with 3% PFA and immobilized on glass slides. For dSTORM imaging, cells were incubated in a buffer containing 100 mM β-Mercaptoethylamine, 4.5 mg × ml −1 D-Glucose, 40 μg × ml −1 Catalase and 0.5 mg × ml −1 Glucose-Oxidase and maximum laser power was used for excitation. SR images were rendered from single emitter localizations obtained within 500 frames. SR images of the TMR channel (i), of the Atto655 channel (ii) and merged images of both channels (iii) are shown. For the shape of the bacteria, the maximum intensity projection with bilinear interpolation of all 500 acquired frames is shown in the lower corner in (iii). Scale bar, 0.5 μm.

    Journal: Scientific Reports

    Article Title: Single molecule super-resolution imaging of proteins in living Salmonella enterica using self-labelling enzymes

    doi: 10.1038/srep31601

    Figure Lengend Snippet: Dual colour dSTORM of Salmonella using fusion proteins to HaloTag and SNAP-tag. Flagellar motor subunit FliN and SPI1-T3SS subunit SpaS were visualized within one cell. ( A ) FliN-SNAP-tag and SpaS-HaloTag were stained with 30 nM TMR-Star (green) and 150 nM HTL-Atto655 (red), respectively. ( B ) SpaS-SNAP-tag and FliN-HaloTag were stained with 30 nM TMR-Star (green) and 150 nM HTL-Atto655 (red), respectively. After staining, cells were washed and fixed with 3% PFA and immobilized on glass slides. For dSTORM imaging, cells were incubated in a buffer containing 100 mM β-Mercaptoethylamine, 4.5 mg × ml −1 D-Glucose, 40 μg × ml −1 Catalase and 0.5 mg × ml −1 Glucose-Oxidase and maximum laser power was used for excitation. SR images were rendered from single emitter localizations obtained within 500 frames. SR images of the TMR channel (i), of the Atto655 channel (ii) and merged images of both channels (iii) are shown. For the shape of the bacteria, the maximum intensity projection with bilinear interpolation of all 500 acquired frames is shown in the lower corner in (iii). Scale bar, 0.5 μm.

    Article Snippet: After 2 h 45 min of subculture, fluorescent ligands were added, i.e. 20 nM HTL-TMR (Promega) or 150 nM HTL-Atto655 (self-synthesized) for HaloTag fusions, and 30 nM TMR-Star (NEB) for SNAP-tag fusions.

    Techniques: Staining, Imaging, Incubation

    Labelling of distinct subunits of the SPI4-T1SS. Salmonella expressing siiF ::HaloTag and siiC ::SNAP-tag was subcultured for 2 h 45 min in LB and incubation was continued for 45 min at 37 °C cells with labelling by 30 nM TMR-Star (red), and 20 nM HTL-SiR (green). Subsequently, cells were applied to agarose pads and 750 images were recorded with excitation with 561 nm and 642 nm lasers at 15% power at the focal plane. After applying threshold settings as described in Fig. S7 , localization using modulated MTT was performed. DIC images were recorded in order to outline the bacterial cell body of three representative cells. Cluster of individual SiiF and SiiC subunits, as well as complexes containing both subunits are observed. Of the events analysed, 21.4% and 42.8% represented cluster of only SiiF or only SiiC, respectively, and co-cluster of SiiC and SiiF were observed for 35.8% of the events. Scale bar, 1 μm.

    Journal: Scientific Reports

    Article Title: Single molecule super-resolution imaging of proteins in living Salmonella enterica using self-labelling enzymes

    doi: 10.1038/srep31601

    Figure Lengend Snippet: Labelling of distinct subunits of the SPI4-T1SS. Salmonella expressing siiF ::HaloTag and siiC ::SNAP-tag was subcultured for 2 h 45 min in LB and incubation was continued for 45 min at 37 °C cells with labelling by 30 nM TMR-Star (red), and 20 nM HTL-SiR (green). Subsequently, cells were applied to agarose pads and 750 images were recorded with excitation with 561 nm and 642 nm lasers at 15% power at the focal plane. After applying threshold settings as described in Fig. S7 , localization using modulated MTT was performed. DIC images were recorded in order to outline the bacterial cell body of three representative cells. Cluster of individual SiiF and SiiC subunits, as well as complexes containing both subunits are observed. Of the events analysed, 21.4% and 42.8% represented cluster of only SiiF or only SiiC, respectively, and co-cluster of SiiC and SiiF were observed for 35.8% of the events. Scale bar, 1 μm.

    Article Snippet: After 2 h 45 min of subculture, fluorescent ligands were added, i.e. 20 nM HTL-TMR (Promega) or 150 nM HTL-Atto655 (self-synthesized) for HaloTag fusions, and 30 nM TMR-Star (NEB) for SNAP-tag fusions.

    Techniques: Expressing, Incubation, MTT Assay

    Detail comparison of SS02565 neuronal membrane labeling. All samples show the same region of projections crossing from the left optic lobe to the central brain. Only the neuronal membrane channel is shown, and is labeled via antibodies in (A-B) and CLIP-tag in (C-D). (A) SS02565 was crossed to w;; 5XUAS-IVS-myr :: smFLAG in VK00005 , pJFRC51-3XUAS-IVS-Syt :: smHA in su(Hw)attP1 and brains were labeled with pure IHC, including rat anti-FLAG and ATTO 647N goat anti-rat antibodies over a period of 13 days. (B) SS02565 was crossed to w; brp-SNAP; 5XUAS-IVS-myr :: smFLAG in VK00005 , pJFRC51-3XUAS-IVS-Syt :: smHA in su(Hw)attP1 and brains were labeled with hybrid IHC, including rat anti-FLAG and ATTO 647N goat anti-rat antibodies over a period of 6 days. (C) SS02565 was crossed to w; brp-SNAP; UAS-myr :: 4xCLIPf in VK00005 , UAS-Syt :: Halo7 in VK0027 and brains were labeled for 15 minutes with pure chemical tags, including TMR CLIP-tag ligand. (D) SS02565 was crossed to w; brp-SNAP; UAS-myr :: 4xCLIPf in VK00005 , UAS-Syt :: Halo7 in VK0027 and brains were labeled for 15 minutes with pure chemical tags, including JF 549 CLIP-tag ligand.

    Journal: PLoS ONE

    Article Title: Optimization of fluorophores for chemical tagging and immunohistochemistry of Drosophila neurons

    doi: 10.1371/journal.pone.0200759

    Figure Lengend Snippet: Detail comparison of SS02565 neuronal membrane labeling. All samples show the same region of projections crossing from the left optic lobe to the central brain. Only the neuronal membrane channel is shown, and is labeled via antibodies in (A-B) and CLIP-tag in (C-D). (A) SS02565 was crossed to w;; 5XUAS-IVS-myr :: smFLAG in VK00005 , pJFRC51-3XUAS-IVS-Syt :: smHA in su(Hw)attP1 and brains were labeled with pure IHC, including rat anti-FLAG and ATTO 647N goat anti-rat antibodies over a period of 13 days. (B) SS02565 was crossed to w; brp-SNAP; 5XUAS-IVS-myr :: smFLAG in VK00005 , pJFRC51-3XUAS-IVS-Syt :: smHA in su(Hw)attP1 and brains were labeled with hybrid IHC, including rat anti-FLAG and ATTO 647N goat anti-rat antibodies over a period of 6 days. (C) SS02565 was crossed to w; brp-SNAP; UAS-myr :: 4xCLIPf in VK00005 , UAS-Syt :: Halo7 in VK0027 and brains were labeled for 15 minutes with pure chemical tags, including TMR CLIP-tag ligand. (D) SS02565 was crossed to w; brp-SNAP; UAS-myr :: 4xCLIPf in VK00005 , UAS-Syt :: Halo7 in VK0027 and brains were labeled for 15 minutes with pure chemical tags, including JF 549 CLIP-tag ligand.

    Article Snippet: In addition to the novel chemical tag ligands described here, we used CLIP-Cell TMR-Star (S9219S, New England Biolabs, Ipswich, MA) and JF549 SNAP-tag ligand [ ].

    Techniques: Labeling, Cross-linking Immunoprecipitation, Immunohistochemistry

    Comparison of Polarity IHC and chemical tag labeling methods. All samples show the Drosophila left optic lobe imaged at 63X. Each image is independently scaled for optimal intensity. (A). Polarity pure IHC : Split GAL4 SS02565 was crossed to w;; 5XUAS-IVS-myr :: smFLAG in VK00005 , pJFRC51-3XUAS-IVS-Syt :: smHA in su(Hw)attP1 and was labeled over a period of 13 days with nc82 mouse anti-Brp/Cy2 anti-mouse, rabbit anti-HA/Cy3 anti-rabbit, and rat anti-FLAG/Cy5 anti-rat. (B). Polarity hybrid IHC : SS02565 was crossed to w; brp-SNAP; 5XUAS-IVS-myr :: smFLAG in VK00005 , pJFRC51-3XUAS-IVS-Syt :: smHA in su(Hw)attP1 and labeled with Cy2 SNAP-tag ligand for 15 minutes, followed by rabbit anti-HA/Cy3 anti-rabbit, and rat anti-FLAG/Cy5 anti-rat over 6 days. Arrowheads indicate bleed-through of Cy5 into Cy2 channel. (C). Polarity pure chemical tag : SS02565 was crossed to w; brp-SNAP; UAS-myr :: 4xCLIPf in VK00005 , UAS-Syt :: Halo7 in VK0027 and labeled for 15 minutes with Cy2 SNAP-tag ligand, TMR CLIP-tag ligand, and ATTO 647N HaloTag ligand. (D). Polarity ATTO 647N pure IHC : As in (A) but with ATTO 647N instead of Cy5. (E). Polarity ATTO 647N hybrid IHC : As in (B) but with ATTO 647N instead of Cy5.

    Journal: PLoS ONE

    Article Title: Optimization of fluorophores for chemical tagging and immunohistochemistry of Drosophila neurons

    doi: 10.1371/journal.pone.0200759

    Figure Lengend Snippet: Comparison of Polarity IHC and chemical tag labeling methods. All samples show the Drosophila left optic lobe imaged at 63X. Each image is independently scaled for optimal intensity. (A). Polarity pure IHC : Split GAL4 SS02565 was crossed to w;; 5XUAS-IVS-myr :: smFLAG in VK00005 , pJFRC51-3XUAS-IVS-Syt :: smHA in su(Hw)attP1 and was labeled over a period of 13 days with nc82 mouse anti-Brp/Cy2 anti-mouse, rabbit anti-HA/Cy3 anti-rabbit, and rat anti-FLAG/Cy5 anti-rat. (B). Polarity hybrid IHC : SS02565 was crossed to w; brp-SNAP; 5XUAS-IVS-myr :: smFLAG in VK00005 , pJFRC51-3XUAS-IVS-Syt :: smHA in su(Hw)attP1 and labeled with Cy2 SNAP-tag ligand for 15 minutes, followed by rabbit anti-HA/Cy3 anti-rabbit, and rat anti-FLAG/Cy5 anti-rat over 6 days. Arrowheads indicate bleed-through of Cy5 into Cy2 channel. (C). Polarity pure chemical tag : SS02565 was crossed to w; brp-SNAP; UAS-myr :: 4xCLIPf in VK00005 , UAS-Syt :: Halo7 in VK0027 and labeled for 15 minutes with Cy2 SNAP-tag ligand, TMR CLIP-tag ligand, and ATTO 647N HaloTag ligand. (D). Polarity ATTO 647N pure IHC : As in (A) but with ATTO 647N instead of Cy5. (E). Polarity ATTO 647N hybrid IHC : As in (B) but with ATTO 647N instead of Cy5.

    Article Snippet: In addition to the novel chemical tag ligands described here, we used CLIP-Cell TMR-Star (S9219S, New England Biolabs, Ipswich, MA) and JF549 SNAP-tag ligand [ ].

    Techniques: Immunohistochemistry, Labeling, Cross-linking Immunoprecipitation

    HURP dynamically accumulates on metaphase k-fibers in an importin-β-dependent manner. (A) Live fluorescent images of HURP-SNAP visualized with TMR-star (magenta) and SiR-tubulin (TUB) in control (top) and importin-β-depleted cells (bottom). Fluorescent signals were bleached in the indicated box region at t = 0, and the fluorescence recoveries were monitored for 120 sec. (B) A graph showing fluorescence recovery after photobleaching. An average of 7 samples was plotted. Bars indicate SDs. (C) Schematic diagram of the metaphase degradation assay. Following release from RO-3336-mediated G2 arrest, proTAME and Apcin were added to arrest cells in metaphase. Auxin (IAA) was added (indicated by the red line) to induce RCC1 degradation during metaphase. (D) Live fluorescent images of SiR-DNA, importin-β-mAC, HURP-mCh, and SiR-700-tubulin (TUB). IAA was added at t = 0. Arrows indicate a cell showing a reduction of importin-β signal during metaphase. (E) Enlarged images from (D) showing a re-localization of HURP-mCh from k-fibers (t = 0) to the spindle (t =90). Scale bars = 10 μm.

    Journal: bioRxiv

    Article Title: Ran-GTP is non-essential to activate NuMA for spindle pole focusing, but dynamically polarizes HURP to control mitotic spindle length

    doi: 10.1101/473538

    Figure Lengend Snippet: HURP dynamically accumulates on metaphase k-fibers in an importin-β-dependent manner. (A) Live fluorescent images of HURP-SNAP visualized with TMR-star (magenta) and SiR-tubulin (TUB) in control (top) and importin-β-depleted cells (bottom). Fluorescent signals were bleached in the indicated box region at t = 0, and the fluorescence recoveries were monitored for 120 sec. (B) A graph showing fluorescence recovery after photobleaching. An average of 7 samples was plotted. Bars indicate SDs. (C) Schematic diagram of the metaphase degradation assay. Following release from RO-3336-mediated G2 arrest, proTAME and Apcin were added to arrest cells in metaphase. Auxin (IAA) was added (indicated by the red line) to induce RCC1 degradation during metaphase. (D) Live fluorescent images of SiR-DNA, importin-β-mAC, HURP-mCh, and SiR-700-tubulin (TUB). IAA was added at t = 0. Arrows indicate a cell showing a reduction of importin-β signal during metaphase. (E) Enlarged images from (D) showing a re-localization of HURP-mCh from k-fibers (t = 0) to the spindle (t =90). Scale bars = 10 μm.

    Article Snippet: To visualize SNAP-tagged HURP in , cells were incubated with 0.1 μM TMR-Star (New England BioLabs) for > 2 hr, and TMR-Star were removed before observation.

    Techniques: Fluorescence, Degradation Assay