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 (ton) 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 (tsample) 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 (ton - tsample): 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 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) Heat map showing log2 intensities of Nups and five randomly selected non-enriched proteins (“Other”) 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. Means per replica 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=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. Means per replica are indicated. Nr of cells analyzed (ton - tsample): 289 (20 min-4 hr), 215 (20min-8hr) from four biological replicas. See Figure 1–Source Data 1 for source data to EFGHIKL.

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=2hr following either a 20min (left) or 1hr induction pulse (right). Images represent sum projections of 3 z-slices (midplane +/- 0.1 µm). Scale bar = 2 µm