Figure 8.
Pak1 removal from the plasma membrane is associated with endocytic patch internalization. (A) Fim1-mCherry and punctate Pak1-mEGFP localization at cell ends. Middle slice shows a single frame of a cell end expressing Pak1-mEGFP and Fim1-mCherry. Arrowhead marks overlapping Fim1-mCherry and Pak1-mEGFP puncta (scale bar = 2 µm). Bull’s eye view shows 3D reconstructed image of the same cell end. A dashed circle marks the outline of the cell end (scale bar = 6 µm). Center panel shows a whole cell with a white dashed outline (scale bar = 10 µm). Red box indicates the region shown as kymographs in the right panels. Yellow circles mark Pak1-mEGFP loss from the membrane while red arrows mark the onset of Fim1-mCherry internalization at the membrane (scale bar = 800 nm). Kymograph brightness is adjusted for ease of visibility for changes in Pak1 intensity. Representative images of the cell end in the left panel are deconvolved, clarified, and denoised with Nikon NIS elements. (B) Super-resolution images of cells expressing Pak1-mEGFP and Fim1-mCherry using Airyscan microscopy. Top row shows a maximum intensity projection. The dotted ROI indicates the cell that is presented in 3-D below). The bottom row shows 3D view of Pak1-mEGFP and Fim1-mCherry localization in the indicated cell. Magenta outline indicates the location of the cell (scale bar = 10 µm). Arrows indicate examples of Pak1-mEGFP at Fim1-mCherry patches. Airyscan images are processed and presented in 3D using Zeiss Zen Blue Automatic processing and 3D deconvolution. (C) Kymograph of Pak1-mEGFP and Fim1-mCherry as captured in a 3-min timelapse movie with 1-s intervals. Red box marks the region quantified in C (scale bar = 800 nm; frame rates = 1 s per frame [SPF]). (D) Quantification of Pak1-mEGFP and Fim1-mCherry intensities at the membrane over time. Red dashed lines indicate the peak and subsequent drop of Fim1-mCherry intensity. (E) Quantification of the time taken for dissipation of Pak1-mEGFP relative to Fim1-mCherry internalization (n ≥ 6 endocytic events per kymograph, N = 21 kymographs from 3 replicates).

Pak1 removal from the plasma membrane is associated with endocytic patch internalization. (A) Fim1-mCherry and punctate Pak1-mEGFP localization at cell ends. Middle slice shows a single frame of a cell end expressing Pak1-mEGFP and Fim1-mCherry. Arrowhead marks overlapping Fim1-mCherry and Pak1-mEGFP puncta (scale bar = 2 µm). Bull’s eye view shows 3D reconstructed image of the same cell end. A dashed circle marks the outline of the cell end (scale bar = 6 µm). Center panel shows a whole cell with a white dashed outline (scale bar = 10 µm). Red box indicates the region shown as kymographs in the right panels. Yellow circles mark Pak1-mEGFP loss from the membrane while red arrows mark the onset of Fim1-mCherry internalization at the membrane (scale bar = 800 nm). Kymograph brightness is adjusted for ease of visibility for changes in Pak1 intensity. Representative images of the cell end in the left panel are deconvolved, clarified, and denoised with Nikon NIS elements. (B) Super-resolution images of cells expressing Pak1-mEGFP and Fim1-mCherry using Airyscan microscopy. Top row shows a maximum intensity projection. The dotted ROI indicates the cell that is presented in 3-D below). The bottom row shows 3D view of Pak1-mEGFP and Fim1-mCherry localization in the indicated cell. Magenta outline indicates the location of the cell (scale bar = 10 µm). Arrows indicate examples of Pak1-mEGFP at Fim1-mCherry patches. Airyscan images are processed and presented in 3D using Zeiss Zen Blue Automatic processing and 3D deconvolution. (C) Kymograph of Pak1-mEGFP and Fim1-mCherry as captured in a 3-min timelapse movie with 1-s intervals. Red box marks the region quantified in C (scale bar = 800 nm; frame rates = 1 s per frame [SPF]). (D) Quantification of Pak1-mEGFP and Fim1-mCherry intensities at the membrane over time. Red dashed lines indicate the peak and subsequent drop of Fim1-mCherry intensity. (E) Quantification of the time taken for dissipation of Pak1-mEGFP relative to Fim1-mCherry internalization (n ≥ 6 endocytic events per kymograph, N = 21 kymographs from 3 replicates).

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