autolysosome  (New England Biolabs)

Journal: Developmental cell

Article Title: CENP-A modifications on Ser68 and Lys124 are dispensable for establishment, maintenance, and long-term function of human centromeres

doi: 10.1016/j.devcel.2016.12.014

Figure Lengend Snippet: Expression of CENP-A S68Q or CENP-A K124R (A) Schematic of the biallelic gene replacement approach used to replace the endogenous CENP-A gene with EGFP-AID-tagged CENP-A on one allele and CENP-A (wild type or mutant) tagged with SNAP-3xHA-P2A-NeoR on the other allele in DLD-1 TIR-1 cells via CRISPR/Cas9-mediated gene editing. (B) Schematic showing the indicated mutants of CENP-A tagged with SNAP-3xHA-P2A-NeoR, which replace the endogenous CENP-A gene on one allele, as indicated in Panel A. (C) Immunoblot of whole cell lysates from each of the indicated cell lines. Relevant cell lines were treated with 500 μM IAA for 24 hr to degrade EGFP-AID-tagged CENP-A. The blot was probed with anti-CENP-A and anti-tubulin antibodies. (D) Representative images showing localization of EGFP-AID-tagged CENP-A and CENP-A(wild type or mutant)-SNAP-3xHA at centromeres. Upon treatment with 500 μM IAA for 24 hr, the EGFP-AID-tagged CENP-A is no longer detected. (E) Quantification of the percentage of viable cells in the indicated cell lines upon treatment with 500 μM IAA for 8 d. Every 2 d, cells were collected and stained with Trypan Blue and counted on a hemocytometer to calculate the percentage of viable cells based on Trypan Blue uptake. Mean +/− SEM is shown for each time point. (F) Representative images of the indicated cell lines after 8 d of treatment with 500 μM IAA. The SNAP-3xHA-tagged CENP-A mutants are still present at endogenous centromeres. (G) Schematic for the quench-chase-pulse experiment in which the existing pool of CENP-A is quenched with SNAP-Cell Block, new CENP-A is synthesized, and newly loaded CENP-A is labeled with TMR- Star 24 hr later. (H) Quantification of the quench-chase-pulse experiment in which TMR- Star and total CENP-A signals are measured at centromeres in G1 cells (marked by a tubulin midbody remnant). Mean +/− SEM is shown. (I) Representative images showing that TMR- Star -labeled CENP-A is loaded at centromeres for each of the cell lines. The tubulin midbody remnant is shown between daughter G1 cells. Cells in which TMR- Star -labeled CENP-A is not detected at centromeres are shown in each representative image. Scale bar: 5 μm. Insets show magnification of the boxed region.

Article Snippet: 24 hr after block, the remaining coverslips were labeled with 2 μM SNAP-Cell TMR- Star for 30 min at 37°C, washed several times with warm growth medium, incubated in the growth medium supplemented with IAA for 2 hr, washed with warm growth medium again, and fixed in 4% formaldehyde (t=24 hr time point).

Techniques: Expressing, Mutagenesis, CRISPR, Staining, Blocking Assay, Synthesized, Labeling

Journal: The Journal of Neuroscience

Article Title: Densin-180 Controls the Trafficking and Signaling of L-Type Voltage-Gated Cav1.2 Ca2+ Channels at Excitatory Synapses

doi: 10.1523/JNEUROSCI.2583-16.2017

Figure Lengend Snippet: Densin accelerates the forward trafficking of Ca v 1.2 in transfected HEK293T cells. A , Cells expressing Ca v 1.2-HA-SNAP and eGFP (top) or GFP-densin (bottom) were processed for TMR-STAR labeling of newly synthesized channels (see Materials and Methods). After incubation for the indicated times to allow forward trafficking of channels, cells were subjected to labeling with HA antibodies to mark cell surface channels. Scale bars, 10 μm. B , Cell surface TMR-STAR fluorescence (arbitrary units, a.u.) was plotted against forward trafficking time. Results are representative of three independent experiments. C , Same as B but TMR-STAR signal was normalized to that at time = 60 min. Smooth line represents fit with a single exponential equation.

Article Snippet: The cells were then washed 3 times and incubated in complete medium for varying durations before incubating with TMR-STAR (2 μ m ; New England BioLabs catalog #S9105S) at 37°C for 25 min to label newly synthesized channels.

Techniques: Transfection, Expressing, Labeling, Synthesized, Incubation, Fluorescence

Journal: Nucleic Acids Research

Article Title: Phosphorylation of Drosophila CENP-A on serine 20 regulates protein turn-over and centromere-specific loading

doi: 10.1093/nar/gkz809

Figure Lengend Snippet: Mimicking the phosphorylated or non-phosphorylated state of S20 interferes with steady-state levels of CENP-A at the centromere. ( A ) TMR-staining of S2 cells expressing low levels of SNAP-tagged wt, S20A and S20D CENP-A in the absence ( top panels ) or presence ( bottom panels ) of RNAi against endogenous CENP-A (CENP-A endo ). Insets are 3× enlargement of the indicated nuclei. ( B ) Quantification of centromeric TMR signal intensities from ( A ) using Imaris v5.1. software. Three to five images were analyzed per experiment. Statistical significance of differences in cumulative centromere intensities per cell was determined by unpaired t -test with a significance threshold of P

Article Snippet: Fluorescence labelling of SNAP-CENP-A To determine steady-state levels of SNAP-tagged CENP-A wt and mutant proteins at centromeres, cells were incubated with 3 μM SNAP-Cell® TMR Star (NEB) for 30 min. Non-reacted TMR Star was washed out by incubating the cells in fresh medium for 10 min before removing the medium and repeating the washing steps three times.

Techniques: Staining, Expressing, Software

Journal: bioRxiv

Article Title: Two distinct HIRA-dependent pathways handle H3.3 de novo deposition and recycling during transcription

doi: 10.1101/2019.12.18.880716

Figure Lengend Snippet: The interaction domain between HIRA and ASF1 is required for old H3.3 recycling. a) Scheme depicting mutated HIRA-YFP transgenic proteins used for rescue experiments. Functional domains required for UBN1 interaction (WD40), ASF1 interaction (B-domain) and trimerization (C domain) are shown. Red indicates substituted amino acids. b) Top: Experimental strategy to rescue the effect of HIRA knockdown on new H3.3-deposition using HIRA-YFP transgenes. Bottom: Representative images for rescue experiment of impaired new H3.3 deposition with the different transgenic proteins. Cells were imaged for new H3.3-SNAP (TMR, red), as well as YFP (green), and DNA (DAPI, grey). YFP protein alone is undetectable in triton-extracted cells, but readily visible in all HIRA-YFP constructs. c) Quantification of total nuclear TMR signal from all conditions expressed as a percentage of new H3.3 gain relative to the siControl sample, indicating that HIRA trimerization and its interaction with UBN1 are required for new H3.3 deposition while ASF1 interaction can be partially bypassed. d) as in b), except the effect on old H3.3-SNAP loss is visualized. e) Quantification of total nuclear TMR signal from all conditions expressed as a percentage of total H3.3 in siControl sample, indicating that HIRA interaction with ASF1 is essential to recycle old H3.3, while its trimerization or interaction with UBN1 are dispensable. f) Summary table for all new and old H3.3 recue experiments results. For all samples, n > 200 cells were analysed. Plots show averages and standard errors for two (panel c) or three (panel e) biological replicates (* indicates p-value

Article Snippet: We incubated our cells in complete medium containing 2 μM of SNAP-Cell TMR-Star (New England Biolabs) during 20 min to label all pre-existing available SNAP-tag (Pulse).

Techniques: Transgenic Assay, Functional Assay, Construct

Journal: bioRxiv

Article Title: Two distinct HIRA-dependent pathways handle H3.3 de novo deposition and recycling during transcription

doi: 10.1101/2019.12.18.880716

Figure Lengend Snippet: Expression of HIRA-YFP mutant transgenic constructs a) Top: Experimental set-up to track new H3.3-SNAP (TMR, red) under siControl or SiHIRA. Bottom-left: representative wide field epifluorescence images of new H3.3-SNAP (red) and DAPI (grey) following 72h siRNA Control or HIRA. Bottom-right: quantification of average TMR signal for H3.3-SNAP (red) normalized to siControl. As expected, a YFP transgenic protein is not detected in the nucleus after permeabilization: this is used as a negative control for HIRA-YFP rescue experiments in Figure 6 . For all samples, n > 200 nuclei. b) as in a) but for old H3.3-SNAP. Plot shows 2h old H3.3-SNAP quantification under siControl or siHIRA normalized to 0h. Plots show average and standard error for two biological replicates. Scale bars represent 10 μm. c) HIRA-YFP wild type (WT) and mutants are present at comparable levels in the nucleus following transfection. Fluorescence quantification of YFP in HIRA knockdown cells expressing HIRA-YFP WT and HIRA-YFP mutants following 48h of siHIRA treatment in rescue experiments for new H3.3 deposition (left) and the old H3.3 loss (right). Plots show average and standard error for n > 200 cells from a single experiment.

Article Snippet: We incubated our cells in complete medium containing 2 μM of SNAP-Cell TMR-Star (New England Biolabs) during 20 min to label all pre-existing available SNAP-tag (Pulse).

Techniques: Expressing, Mutagenesis, Transgenic Assay, Construct, Negative Control, Transfection, Fluorescence

Journal: bioRxiv

Article Title: Two distinct HIRA-dependent pathways handle H3.3 de novo deposition and recycling during transcription

doi: 10.1101/2019.12.18.880716

Figure Lengend Snippet: Different partnerships for HIRA in new H3.3 deposition versus recycling. a) Top-right: experimental scheme to track new H3.3-SNAP in cells treated with siRNA for 72h. Left: representative wide field epifluorescence images of cells stained for new H3.3-SNAP after 0h (Background) or 2h of chase time (New H3.3) (TMR, red), and DNA (DAPI, grey), 72h after knockdown of HIRA, UBN1, CABIN1 or ASF1 isoforms a+b or using a control siRNA. Bottom-right: quantification of total nuclear TMR signal for each knockdown condition expressed as a percentage of H3.3 gain in 2h relative to the control condition. The results indicate that new H3.3 deposition requires HIRA, UBN1 and ASF1 but not CABIN1. b) as in a), except old H3.3-SNAP was tracked after 0h (Total H3.3), 1h or 2h (Old H3.3) of chase time, indicating that old H3.3 recycling requires HIRA and ASF1 but not UBN1 or CABIN1. For all samples, n > 200 cells were imaged per replicate. All plots show averages and standard errors for two (panel a) or three (panel b) biological replicates.

Article Snippet: We incubated our cells in complete medium containing 2 μM of SNAP-Cell TMR-Star (New England Biolabs) during 20 min to label all pre-existing available SNAP-tag (Pulse).

Techniques: Staining

Journal: bioRxiv

Article Title: Two distinct HIRA-dependent pathways handle H3.3 de novo deposition and recycling during transcription

doi: 10.1101/2019.12.18.880716

Figure Lengend Snippet: HIRA is required for both deposition of new H3.3 and retention of old H3.3 at transcriptionally active domains. a) Top-right: Experimental strategy to track old histones in cells treated with control or HIRA-targeting siRNA 72h prior to SNAP-tag labelling. Left: representative wide field epifluorescence images of cells stained for H3.1- or H3.3-SNAP following control (siControl) or HIRA-targeting (siHIRA) siRNA treatment, and after 0h and 2h of TMR chase time. Bottom-right: quantification of average nuclear TMR signal for H3.1 (purple) and H3.3 (green), in control (siControl, full lines) and HIRA knockdown (siHIRA, dashed lines) cells, expressed as a percentage of the average value at chase time 0h. The average percentage of loss at 2h chase time is indicated for each sample, indicating that H3.3 is more rapidly lost upon HIRA knockdown, while H3.1 loss is alleviated in these conditions. For each sample, n > 200 cells were analysed. Plots show the average and standard error of independent biological triplicates. b) Left: representative deconvolved epifluorescence images for cells stained for total H3.1- or H3.3-SNAP (TMR, red) or using a HIRA antibody (red), together with RNAPIIS7ph (green) and DNA (DAPI, white). Right: spatial relationship analysis plots showing enrichment of HIRA (orange) and H3.3 (green) but not H3.1 (purple) at RNAPIIS7ph foci. c) Top: Experimental strategy to track new H3.3 upon HIRA knockdown. Middle: representative deconvolved epifluorescence images of cells stained for new H3.3 (TMR, red), RNAPIIS7ph (green) and DNA (DAPI, grey). Bottom: spatial relationship analysis showing preferential depletion of new H3.3 at RNAPIIS7ph foci in the absence of HIRA. d) As in c), except old H3.3 was tracked, showing that old H3.3 is also preferentially depleted at RNAPIIS7ph foci in the absence of HIRA. For spatial relationship analysis, numbers are averages from n > 40 cells. Scale bars represent 10µm.

Article Snippet: We incubated our cells in complete medium containing 2 μM of SNAP-Cell TMR-Star (New England Biolabs) during 20 min to label all pre-existing available SNAP-tag (Pulse).

Techniques: Staining

Journal: bioRxiv

Article Title: Two distinct HIRA-dependent pathways handle H3.3 de novo deposition and recycling during transcription

doi: 10.1101/2019.12.18.880716

Figure Lengend Snippet: Loss of old H3.3 is dependent on transcriptional activity. Short term old H3.3 loss is dependent on transcriptional activity. a ) Top: Experimental set-up to track total and old H3.1/H3.3-SNAP (TMR, red) following different chase times (0h to 48h). Bottom-left: representative wide field epifluorescence images of H3.1- and H3.3-SNAP (red), and DAPI (grey). Right: quantification of average TMR signal for H3.1- and H3.3-SNAP, together with its best exponential fit (blue), the exponential fit over 0 to 2h (red) and the expected exponential decay for cell cycle-dependent histone dilution (yellow). b) Top: Experimental set-up to track total and old H3.1/H3.3-SNAP (TMR, red) in the presence or absence of the transcription inhibitor Flavopiridol (FLP) for 3 hours prior to TMR pulse and during the chase time. EU labelling (green) marks nascent RNA and is used to confirm the absence of transcription in Flavopiridol-treated cells at every chase time point. Bottom-left: representative wide field epifluorescence images of control and FLP-treated H3.1- or H3.3-SNAP cells after 0h and 2h of TMR chase time. Bottom-right: quantification of average TMR signal for H3.3-(green) and H3.1-SNAP (purple) of untreated (full lines) or FLP-treated (dashed lines) cells, expressed as a percentage of the average value at chase time 0h. For all samples, n > 200 nuclei per replicate were analysed. Numbers shown are the average and standard error for independent biological triplicates. Scale bars represent 10 μm.

Article Snippet: We incubated our cells in complete medium containing 2 μM of SNAP-Cell TMR-Star (New England Biolabs) during 20 min to label all pre-existing available SNAP-tag (Pulse).

Techniques: Activity Assay

Journal: bioRxiv

Article Title: Two distinct HIRA-dependent pathways handle H3.3 de novo deposition and recycling during transcription

doi: 10.1101/2019.12.18.880716

Figure Lengend Snippet: Transcription is required to reveal the HIRA dependent H3.3 recycling. Top: experimental strategy to track old H3.3 during steady state transcription and in cells exposed to FLP until transcription was fully arrested (as in Figure 1b ). Bottom-left: representative images of total (0h) or old (2h) H3.3-SNAP (TMR, red), in control conditions or during FLP treatment and 72h following knockdown using Control or HIRA-targeting siRNA. Bottom-right: quantification of TMR signal for control (full lines) and HIRA knockdown (dashed lines), untreated (green) and FLP-treated (blue) cells indicate that, in transcriptionally-arrested cells, absence of HIRA has no effect on old H3.3 dynamics. For all samples, n > 200 cells. Plots show averages and errors from independent biological triplicates. Scale bars represent 10µm.

Article Snippet: We incubated our cells in complete medium containing 2 μM of SNAP-Cell TMR-Star (New England Biolabs) during 20 min to label all pre-existing available SNAP-tag (Pulse).

Techniques:

Journal: bioRxiv

Article Title: Two distinct HIRA-dependent pathways handle H3.3 de novo deposition and recycling during transcription

doi: 10.1101/2019.12.18.880716

Figure Lengend Snippet: Effects of RNAPII-inhibiting drugs on H3.3. a) Representative images of cells treated with Flavopiridol (FLP) or Triptolide (TRP) for the indicated times and labelled with EU (green) to measure nascent transcript levels and DAPI (DNA, grey). b) Corresponding quantifications of total nuclear EU signal normalized to untreated cells (0h) allowed to distinguish between full signal in steady state transcription, to no signal after complete transcription shutdown (3h-5h). b) Left: representative epifluorescence images of cells stained for total H3.3-SNAP (TMR, red) EU (nascent RNA, green) and DAPI (grey) after 0h or 2h of FLP treatment. Right: quantification of average TMR signal (red), as a percentage of average values at 0h, showing an increase in total H3.3 upon transcriptional arrest. b) Same as in Figure 4 , excerpt Triptolide (TRP) was used to inhibit transcriptiopn. Left: representative images of total (0h) or old (2h) H3.3-SNAP (TMR, red), in control conditions or during TRP treatment and 72h following knockdown using Control or HIRA-targeting siRNA. Right: quantification of TMR signal for control (full lines) and HIRA knockdown (dashed lines), untreated (green) and TRP-treated (purple) cells. As for FLP treatment, the results indicate that transcriptional activity is necessary to reveal the effect of HIRA knockdown on H3.3 recycling. Control data is the same as in Figure 4 . All plots show average and standard error for n > 200 cells from two biological replicates. Standard t-test indicated statistical significance (*: p

Article Snippet: We incubated our cells in complete medium containing 2 μM of SNAP-Cell TMR-Star (New England Biolabs) during 20 min to label all pre-existing available SNAP-tag (Pulse).

Techniques: Staining, Activity Assay

Journal: bioRxiv

Article Title: Two distinct HIRA-dependent pathways handle H3.3 de novo deposition and recycling during transcription

doi: 10.1101/2019.12.18.880716

Figure Lengend Snippet: Transcription inhibited by Triptolide also prevents H3.3 loss. a) Top: Experimental set-up to track total or old H3.1/H3.3 using SNAP-tag labelling. A pulse using the fluorophore TMR (red) labels SNAP-tagged H3.3 or H3.1, cells are triton-extracted and fixed at different chase times to reveal total (0h) or old (1h, 2h) chromatin-bound histones. EdU labelling (green) marks nascent DNA allowing identification of cells in or outside S-phase, while DNA is stained with DAPI (grey). Bottom-left: representative wide field epifluorescence images of H3.1- or H3.3-SNAP after 0h, 1h and 2h of chase time. Bottom-right: quantification of average nuclear TMR signal for H3.1 (purple) and H3.3 (green), expressed as a percentage of the average value at chase time 0h. The average percentage of loss at 2h chase time is indicated for each sample. Cells were grouped as EdU positive (EdU+: cells in S-phase, full lines) or EdU negative (EdU-: cells outside of S-phase, dashed lines). b) Top: Experimental set-up to track total and old H3.1/H3.3-SNAP (TMR, red) in the presence or absence of the transcription inhibitor Triptolide (TRP), performed in parallel to Flavopiridol treatment in Figure 1b . EU labelling (green) marks nascent RNA and is used to confirm the absence of transcription in Triptolide-treated cells. Bottom-left: representative wide field epifluorescence images of control and TRP-treated H3.3- or H3.1-SNAP cells after 0h and 2h of TMR chase time. Bottom-right: quantification of average TMR signal for H3.3-(green) and H3.1-SNAP (purple) of untreated (full lines) or TRP-treated (dashed lines) cells, as a percentage of the average value at time 0h for each condition. For all samples, n > 200 nuclei. Plots show average and standard error for three biological replicates. Control data is the same as in Figure 1b . Scale bars represent 10 μm.

Article Snippet: We incubated our cells in complete medium containing 2 μM of SNAP-Cell TMR-Star (New England Biolabs) during 20 min to label all pre-existing available SNAP-tag (Pulse).

Techniques: Staining