A tool to pulse-label yeast nuclear pore complexes in imaging and biochemical experiments
- European Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen, Netherlands
- Institute of Biochemistry, Department of Biology, ETH Zurich, Switzerland
Figures
Figure 1 with 1 supplement
Nanobody against Nup84 is a tool to pulse-label yeast nuclear pore complexes (NPCs) in imaging and biochemical experiments.
(A) Schematic of the experimental setup. At t0, VHH[Nup84]-mNG is expressed for the specified amount of time (ton) by the addition of 0.5% galactose to the medium. Expression is subsequently shut off by the addition of 1% glucose, and images are taken at time (tsample) to determine NPC labeling. (B) Colocalization of VHH[Nup84]-mNG with Pom34-mCh at t=2 hr following a 20 min induction pulse. Images represent summed projections of three z-slices (midplane ±0.5 µm). Scale bar = 5 µm. (C) Typical image of VHH[Nup84]-mNG in nup84Δ cells at t=2 hr following a 20 min induction pulse. Images represent a single z-slice. Scale bar = 5 µm. (D) Typical images of VHH[Nup84]-mNG at t=2 hr following either a 20 min or 1 hr induction pulse. Images represent sum projections of three z-slices (midplane ±0.1 µm). Scale bar = 5 µm. (E) Quantification of NE intensities in the midplane of cells at various time points following a 20 min (cyan) or 1 hr (purple) induction pulse. Data represents mean NE intensity ± standard deviation of three biological replicates. Numbers of cells analyzed (ton – tsample): 102 (20min-45 min), 139 (20min-1hr), 82 (20min-2 hr, N=2), 92 (20min-4 hr), 111 (1hr-45min), 186 (1hr-1hr), 153 (1hr-2hr), 193 (1hr-4hr). Mean NE intensity ± standard deviation of endogenously tagged Nup84-mNG is 726±148 (224 cells). (F) Volcano plot showing the log2 fold enrichment of proteins in affinity purifications using endogenously expressed Nup84-ZZ as a bait compared to mock-ZZ. Nups are colored in black, the Nup84 subcomplex is colored in magenta. (G) Volcano plot showing the log2 fold enrichment of proteins in affinity purifications using VHH[Nup84]-ZZ constitutively expressed from the Nup120 promoter as a bait compared to mock-ZZ. Nups are colored in black, the Nup84 subcomplex is colored in magenta. (H) Correlation between protein intensities in the affinity purifications with VHH[Nup84] from F and G. (I) Heatmap showing log2 intensities of Nups and five randomly selected non-enriched proteins (‘Other’) in VHH[Nup84]-ZZ affinity purifications at the various sampling time points following a 20 min VHH[Nup84] induction pulse. LFQ = label-free quantitation. (J) Typical image of the localization of VHH[Nup84]-mNG at t=4 hr and t=8 hr following a 20 min induction pulse. Each panel represents a sum projection of five consecutive z-slices (0.1 μm). Scale bar = 5 µm. (K) Quantification of the number of VHH[Nup84]-mNG foci per cell at t=4 hr and t=8 hr following a 20 min induction pulse. Each dot represents a cell, each color reflects a biological replicate. Means per replicate are indicated. Nr of cells analyzed (ton – tsample): 289 (20min-4hr), 215 (20min-8hr) from four biological replicas. (L) Intensities of detected VHH[Nup84]-mNG foci at t=4 hr and t=8 hr following a 20 min induction pulse. Intensities are normalized to median foci intensity at t=8 hr of the corresponding replica. Each dot represents a single focus, each color reflects a biological replicate. Means per replicate are indicated. Nr of cells analyzed (ton – tsample): 289 (20min-4hr), 215 (20min-8hr) from four biological replicas. See Figure 1—source data 1 for source data to EFGHIKL.
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Figure 1—source data 1
Numerical source for Figure 1.
- https://cdn.elifesciences.org/articles/108399/elife-108399-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Typical images of a cell expressing VHH[Nup84]-mNG (A) or endogenously tagged Nup84-mNG (B).
VHH[Nup84]-mNG expressing cells were imaged at t=2 hr following either a 20 min (left) or 1 hr induction pulse (right). Images represent sum projections of three z-slices (midplane ±0.1 µm). Scale bar = 2 µm.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Saccharomyces cerevisiae) | W303 MATa leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 | Open Biosystems | ||
| Strain, strain background (Saccharomyces cerevisiae) | yAV108 W303 leu2::pGAL1-VHH[Nup84]-mNeongreen::LEU2 | This study | yAV108 | VHH[Nup84]-mNG under galactose promoter integrated in the leu2 locus using pAV49 |
| Strain, strain background (Saccharomyces cerevisiae) | yAV115 W303 leu2::pGAL1-VHH[Nup84]-mNeongreen::LEU2 POM34-mCherry::URA3 | This study | yAV115 | C-terminal tagging of POM34-mCherry using pPP014 Rempel et al., 2019 |
| Strain, strain background (Saccharomyces cerevisiae) | yAV173 W303 NUP84-mNeongreen::URA3 | This study | yAV173 | C-terminal tagging of NUP84-mNeongreen using pAV54 Otto et al., 2024 |
| Strain, strain background (Saccharomyces cerevisiae) | yAV178 W303 leu2::pGAL1-VHH[Nup84]-mNeongreen::LEU2 nup84Δ::Hyg | This study | yAV178 | Deletion of nup84 using pFA6-HphNT1 |
| Strain, strain background (Saccharomyces cerevisiae) | yAV213 W303 lys2Δ::KanMX, leu2::pGAL1-VHH[Nup84]-S-TEV-ZZ::HIS3,LEU2 | This study | yAV213 | VHH[Nup84]-S-TEV-ZZ under galactose promoter integrated in the leu2 locus with HIS3 selection marker, using pKW803 to replace mNG with S-TEV-ZZ. Lys2 deletion by PCR amplification of lys2 deletion cassette from YKO collection |
| Strain, strain background (Saccharomyces cerevisiae) | KWY12171 W303 lys2Δ::KanMX his3::pNUP120-VHH[Nup84]-S-TEV-ZZ::HIS3 | This study | KWY12171 | Constitutively expressed VHH[Nup84]-S-TEV-ZZ in the his3 locus |
| Strain, strain background (Saccharomyces cerevisiae) | KWY12149 W303 lys2Δ::KanMX NUP84-S-TEV-ZZ::HIS3 | Onischenko et al., 2020 | KWY12149 | |
| Recombinant DNA reagent | pRS305 (plasmid) | Addgene Markus et al., 2009 | RRID:NCBITaxon_31828 | Integrative vector with LEU2 marker |
| Recombinant DNA reagent | pPP014 (plasmid) | Rempel et al., 2019 | pPP014 | PCR template for C-terminal mCherry tagging; URA3 selection |
| Recombinant DNA reagent | pFA6-HphNT1 (plasmid) | Janke et al., 2004 | pFA6-HphNT1 | Gene deletion cassette; hygromycin resistance |
| Recombinant DNA reagent | pKW803 (plasmid) | Onischenko et al., 2020 | pKW803 | |
| Recombinant DNA reagent | pAV49 (plasmid) | This study | pAV49 | pGAL1-VHH[Nup84]-mNeongreen-Cyc in pRS305 backbone |
| Recombinant DNA reagent | pAV54 (plasmid) | Otto et al., 2024 | pAV54 | PCR template for C-terminal mNeongreen tagging; URA3 selection |
| Chemical compound, drug | D-Glucose anhydrous | Fisher | G/0500/65 | |
| Chemical compound, drug | D(+)-Raffinose pentahydrate | Thermo Scientific | 195675000 | |
| Chemical compound, drug | D-Galactose | Acros Organics | 150610010 | |
| Chemical compound, drug | Minimal medium | Sigma-Aldrich | ||
| Chemical compound, drug | Protease inhibitor cocktail | Sigma-Aldrich | P8215 | |
| Chemical compound, drug | Dynabeads M-270 Epoxy | Invitrogen | 14301 | |
| Chemical compound, drug | Purified IgG protein from rabbit serum | Sigma-Aldrich | I5006 | |
| Chemical compound, drug | Coomassie Brilliant Blue R-250 | Bio-Rad | 161-0400 | |
| Chemical compound, drug | Sequencing grade porcine trypsin | Promega | V5113 | |
| Chemical compound, drug | L-Lysine:2HCl (13C6, 99%; 15N2, 99%) | Cambridge Isotope Laboratories | CNLM-291-H-0.25 | |
| Chemical compound, drug | Iodoacetamide | Sigma-Aldrich | I1149 | |
| Chemical compound, drug | Ammonium bicarbonate | Sigma-Aldrich | 9832 | |
| Chemical compound, drug | Formic acid 99–100% | VWR Chemicals | 20318.297 | |
| Chemical compound, drug | iRT Kit | Biognosis | Ki-3002-1 | |
| Chemical compound, drug | BioPureSPN MINI Columns Silica C18 | The Nest Group, Inc | HUM S18V | |
| Chemical compound, drug | BioPureSPN MACRO Columns Silica C18 | The Nest Group, Inc | HMM S18V | |
| Software, algorithm | ImageJ/Fiji | Schindelin et al., 2012 | RRID:SCR_002285 | |
| Software, algorithm | PunctaFinder | Terpstra et al., 2024 | ||
| Software, algorithm | R version 4.1.0 | R Development Core Team, 2024 | RRID:SCR_001905 | |
| Software, algorithm | RStudio v. 26.01.1 | Posit Software, PBC | RRID:SCR_000432 | |
| Software, algorithm | SoftWoRx | Cytiva | RRID:SCR_019157 | |
| Software, algorithm | Illustrator v. 30.2.1 | Adobe | RRID:SCR_010279 | |
| Software, algorithm | Jupyter Notebook v. 6.5.4 | Project Jupyter | RRID:SCR_018315 | |
| Software, algorithm | Python v. 3.11.4 | Python Software Foundation | RRID:SCR_008394 | |
| Other | screw-cap microtubes | Sarstedt Inc | 72.693.005 | See ‘APs and proteomic data acquisition and analysis’ in Materials and methods |
| Other | mini-BeadBeater-24, 230 V | BioSpec Products | 112011EUR | See ‘APs and proteomic data acquisition and analysis’ in Materials and methods |
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A tool to pulse-label yeast nuclear pore complexes in imaging and biochemical experiments
eLife 14:RP108399.
https://doi.org/10.7554/eLife.108399.3
