Figure 2.
Facilitated transport of MBP-GFP cargoes into FG-Nup49 droplets. Epifluorescence microscopy of whole microfluidic device/chip. (A) Interaction between the IBB-MBP-GFP cargo and FG-Nup49 droplets in the presence of Importin β. (B and C) Two control experiments: the IBB-MBP-GFP cargo without Importin β and the M9-MBP-GFP cargo in the presence of Importin β. The rows of the figures are organized as follows. Top row: Confocal images of the respective cargo construct in permeabilized cells assays with a functional nuclear transport machinery. Second and third rows: Snapshot of the droplet channel (FG-Nup49), followed by a corresponding snapshot of the cargo channel. Fourth row: Two zooms corresponding to the white boxes in images above. Fifth row: Ratiometric maximum projection of an entire image series (see Fig. S2 for details on ratiometric analysis). Bottom row: Single-droplet analysis of the fluorescent cargo signals within the FG-Nup49 droplets while they travel along the device during each experiment. Briefly, droplets were segmented from the ratiometric video, and the intensity was estimated and plotted in relation to the droplet centroid position along the device. Each dot on the plots represents a single droplet. All rows: The FG-Nup49 droplets channel is displayed in red, the GFP cargo channel is in grayscale, and the ratiometric projections of the experiment (last row) show a colorimetric interaction of the cargo with the droplets. The more yellow/white the color, the more the cargo enriched inside the FG-Nup droplet. Scale bar is 50 µm. See corresponding Videos 1, 2, and 3 and details in Materials and methods for the ratiometric analysis. Scale bar in the zoom is 20 µm. Shown are exemplarily datasets from three technical replicates. In each dataset, >10,000 droplets are analyzed.

Facilitated transport of MBP-GFP cargoes into FG-Nup49 droplets. Epifluorescence microscopy of whole microfluidic device/chip. (A) Interaction between the IBB-MBP-GFP cargo and FG-Nup49 droplets in the presence of Importin β. (B and C) Two control experiments: the IBB-MBP-GFP cargo without Importin β and the M9-MBP-GFP cargo in the presence of Importin β. The rows of the figures are organized as follows. Top row: Confocal images of the respective cargo construct in permeabilized cells assays with a functional nuclear transport machinery. Second and third rows: Snapshot of the droplet channel (FG-Nup49), followed by a corresponding snapshot of the cargo channel. Fourth row: Two zooms corresponding to the white boxes in images above. Fifth row: Ratiometric maximum projection of an entire image series (see Fig. S2 for details on ratiometric analysis). Bottom row: Single-droplet analysis of the fluorescent cargo signals within the FG-Nup49 droplets while they travel along the device during each experiment. Briefly, droplets were segmented from the ratiometric video, and the intensity was estimated and plotted in relation to the droplet centroid position along the device. Each dot on the plots represents a single droplet. All rows: The FG-Nup49 droplets channel is displayed in red, the GFP cargo channel is in grayscale, and the ratiometric projections of the experiment (last row) show a colorimetric interaction of the cargo with the droplets. The more yellow/white the color, the more the cargo enriched inside the FG-Nup droplet. Scale bar is 50 µm. See corresponding Videos 1, 2, and 3 and details in Materials and methods for the ratiometric analysis. Scale bar in the zoom is 20 µm. Shown are exemplarily datasets from three technical replicates. In each dataset, >10,000 droplets are analyzed.

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