Figure 2.
Temporal and spatial distributions of Aux1 and GAK. (A and B) Maximum-intensity projections from a thin 2-µm optical section of a gene-edited cell expressing EGFP-Aux1 (A) or EGFP-GAK (B) recorded in 3D by lattice light-sheet microscopy. Scale bars, 10 µm. (C) Representative plot from 3D automated trackings of AP2-TagRFP and EGFP-Aux1 (872 traces from six cells) or AP2-TagRFP and EGFP-GAK (755 traces from six cells) in the double-edited cells imaged by lattice light-sheet microscopy. Distribution (fit with a single Gaussian) of the interval between maximum fluorescent signals for AP2 and Aux1 or AP2 and GAK (2.4 ± 0.1 and 3.2 ± 0.1 s, mean ± SE, respectively). (D) Distribution of GAK in cells gene-edited for EGFP-GAK+/+ and stably expressing AP1-TagRFP recorded in 3D by lattice light-sheet microscopy. Left: Maximum-intensity projections in X-Y, X-Z, and Y-Z of EGFP-GAK and AP1-TagRFP from a 3D rendered cell. Right: Representative plot of EGFP-GAK recruitment to a single AP1-positive carrier. Scale bar, 10 µm. (E) Snapshot from a representative TIRF microscopy time series showing the transient burst of EGFP-Aux1 and TagRFP-GAK+/+ on the plasma membrane of a cell double-edited for EGFP-Aux1+/+ and TagRFP-GAK+/+. Time series with single molecule detection sensitivity for EGFP and TagRFP acquired for 120 s at 1-s intervals using 100-ms exposures. Kymograph (bottom panel) shifted laterally by 5 pixels. Scale bar, 5 µm. (F) Representative plot (left) for a single endocytic event showing sequential recruitment of EGFP-Aux1 and TagRFP-GAK (recruitment peaks highlighted by arrows) imaged at 0.5-s intervals by TIRF microscopy. The traces (right) are averaged relative fluorescence intensity (mean ± SE) of EGFP-Aux1 and TagRFP-GAK for the cohort of EGFP-Aux1 bursts with residence times of 3–12 s (1,516 traces from eight cells). (G) The relative time differences (<0, 0, and >0 s) between peaks of EGFP-Aux1 and TagRFP-GAK recruitment (mean ± SD, n = 8 cells). (H) Distribution of interval between peaks of EGFP-Aux1 and TagRFP-GAK recruitment. The mean interval and SD from eight cells was 1.34 ± 0.26 s. (I) Averaged relative fluorescence intensity (mean ± SE) for bursts of duration of 3–12 s of transiently expressed mCherry-Aux1 and gene-edited EGFP-GAK (1,859 traces from eight cells). (J) Scatter plot of maximum (max.) fluorescence intensities of EGFP-Aux1 and TagRFP-GAK (750 traces from eight cells double-edited for EGFP-Aux1+/+ and TagRFP-GAK+/+, Pearson correlation coefficient r = 0.189).

Temporal and spatial distributions of Aux1 and GAK. (A and B) Maximum-intensity projections from a thin 2-µm optical section of a gene-edited cell expressing EGFP-Aux1 (A) or EGFP-GAK (B) recorded in 3D by lattice light-sheet microscopy. Scale bars, 10 µm. (C) Representative plot from 3D automated trackings of AP2-TagRFP and EGFP-Aux1 (872 traces from six cells) or AP2-TagRFP and EGFP-GAK (755 traces from six cells) in the double-edited cells imaged by lattice light-sheet microscopy. Distribution (fit with a single Gaussian) of the interval between maximum fluorescent signals for AP2 and Aux1 or AP2 and GAK (2.4 ± 0.1 and 3.2 ± 0.1 s, mean ± SE, respectively). (D) Distribution of GAK in cells gene-edited for EGFP-GAK+/+ and stably expressing AP1-TagRFP recorded in 3D by lattice light-sheet microscopy. Left: Maximum-intensity projections in X-Y, X-Z, and Y-Z of EGFP-GAK and AP1-TagRFP from a 3D rendered cell. Right: Representative plot of EGFP-GAK recruitment to a single AP1-positive carrier. Scale bar, 10 µm. (E) Snapshot from a representative TIRF microscopy time series showing the transient burst of EGFP-Aux1 and TagRFP-GAK+/+ on the plasma membrane of a cell double-edited for EGFP-Aux1+/+ and TagRFP-GAK+/+. Time series with single molecule detection sensitivity for EGFP and TagRFP acquired for 120 s at 1-s intervals using 100-ms exposures. Kymograph (bottom panel) shifted laterally by 5 pixels. Scale bar, 5 µm. (F) Representative plot (left) for a single endocytic event showing sequential recruitment of EGFP-Aux1 and TagRFP-GAK (recruitment peaks highlighted by arrows) imaged at 0.5-s intervals by TIRF microscopy. The traces (right) are averaged relative fluorescence intensity (mean ± SE) of EGFP-Aux1 and TagRFP-GAK for the cohort of EGFP-Aux1 bursts with residence times of 3–12 s (1,516 traces from eight cells). (G) The relative time differences (<0, 0, and >0 s) between peaks of EGFP-Aux1 and TagRFP-GAK recruitment (mean ± SD, n = 8 cells). (H) Distribution of interval between peaks of EGFP-Aux1 and TagRFP-GAK recruitment. The mean interval and SD from eight cells was 1.34 ± 0.26 s. (I) Averaged relative fluorescence intensity (mean ± SE) for bursts of duration of 3–12 s of transiently expressed mCherry-Aux1 and gene-edited EGFP-GAK (1,859 traces from eight cells). (J) Scatter plot of maximum (max.) fluorescence intensities of EGFP-Aux1 and TagRFP-GAK (750 traces from eight cells double-edited for EGFP-Aux1+/+ and TagRFP-GAK+/+, Pearson correlation coefficient r = 0.189).

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal