A tool to pulse-label yeast Nuclear Pore Complexes in imaging and biochemical experiments

Annemiek C Veldsink
Jonas S Fischer
Sophie Hell
Karsten Weis
Liesbeth M Veenhoff author has email address

European Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen, Groningen, Netherlands
Institute of Biochemistry, Department of Biology, ETH Zurich, Zurich, Switzerland

https://doi.org/10.7554/eLife.108399.1

Open access
Copyright information

Figures and data

Nanobody against Nup84 is a tool to pulse-label yeast NPCs in imaging and biochemical experiments
A) Schematic of the experimental set-up. At t₀, VHH[Nup84]-mNG is expressed for the specified amount of time (t_on) by addition of 0.5% galactose to the medium. Expression is subsequently shut off by addition of 1% glucose and images are taken at time (t_sample) to determine NPC labeling.
B) Colocalization of VHH[Nup84]-mNG with Pom34-mCh at t=2hr following a 20min induction pulse. Images represent summed projections of 3 z-slices (midplane +/- 0.5 μm). Scale bar = 5 μm.
C) Typical image of VHH[Nup84]-mNG in nup84Δ cells at t=2hr following a 20min induction pulse. Images represent a single z-slice. Scale bar = 5 μm
D) Typical images of VHH[Nup84]-mNG at t=2hr following either a 20min or 1hr induction pulse. Images represent sum projections of 3 z-slices (midplane +/- 0.1 μm). Scale bar = 5 μm.
E) Quantification of NE intensities in the midplane of cells at various timepoints following a 20min (cyan) or 1hr (purple) induction pulse. Data represents mean NE intensity +/- standard deviation of three biological replicas. Numbers of cells analyzed (t_on - t_sample): 102 (20min-45min), 139 (20min-1hr), 82 (20min-2hr, N=2), 92 (20min-4hr), 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 promotor 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) Heat map showing log2 intensities of Nups and NTRs in VHH[Nup84]-ZZ affinity purifications at the various sampling timepoints following a 20 min VHH[Nup84] induction pulse. LFQ = label free quantitation.
J) Typical image of the localization of VHH[Nup84]-mNG at t=4hr and t=8hr following a 20-minute induction pulse. Each panel represents a sum projection of 5 consecutive z-slices (0.1 μm). Scale bar = 5 μm.
K) Quantification of the number of VHH[Nup84]-mNG foci per cell at t=4hr and t=8hr following a 20- minute induction pulse. Each dot represents a cell, each color reflects a biological replica. Boxplots represent median and first and third quartiles, whiskers extend to 1.5x IQR. Nr of cells analyzed (t_on - t_sample): 289 (20min-4hr), 215 (20min-8hr) from four biological replicas.
L) Intensities of detected VHH[Nup84]-mNG foci at t=4hr and t=8hr following a 20-minute induction pulse. Intensities are normalized to median foci intensity at t=8hr of the corresponding replica. Each dot represents a single focus, each color reflects a biological replica. Boxplots represent median and first and third quartiles, whiskers extend to 1.5x IQR. Nr of cells analyzed (t_on - t_sample): 289 (20 min-4 hr), 215 (20min-8hr) from four biological replicas.

Strains and plasmids

Sign up for email alerts