Figure S5.
Dextran and EGFR’s interaction with dynein. (a) HILO microscopy image (left) from a 10-s-long time-lapse video of mDHC-GFP (green) and dextran-A647 (magenta) and the corresponding kymograph (right). The yellow arrowhead points to a dextran vesicle with no dynein fluorescence and the white arrowhead points to a dextran vesicle with associated dynein intensity. The kymograph shows that a dextran vesicle with dynein bound to it (white arrowhead) made a short run toward the MT minus end. The dextran vesicle without visible dynein intensity (yellow arrowhead) remained stationary. (b) Displacement versus time plot of dextran vesicles without associated dynein intensity. (c) Displacement versus time plot of dextran vesicles with visible dynein intensity. Comparing b and c, while 28% of dextran vesicles with visible dynein intensity moved toward the MT minus ends (n = 12/44 vesicles), only 10% of those without associated dynein intensity moved to the minus end of MTs (n = 11/114 dextran vesicles; n = 2 independent experiments with >25 cells). In other words, of all the minus end–directed dextran vesicles observed in these experiments (all the teal-colored traces in b and c), ∼52% (n = 12/23 vesicles) had associated dynein intensity. Fisher’s exact test yielded a significant difference in dynein-associated and non-dynein-associated endosome behavior (P < 0.005). (d) Box plots comparing the intensity of dynein on dextran vesicles and intensity of single molecules of dynein (measured from binding events) showed that there were one to two dynein molecules per dextran vesicle (n = 44 dextran vesicles with dynein intensity, n = 115 single molecules of dynein from n = 2 independent experiments with >25 cells). (e) Analysis of b–d was done in cells expressing mDHC-GFP and treated with 25 nM siRNA against endogenous hDHC. Quantification of western blot confirmed that the RNAi successfully knocked down levels of DHC by 85%. (f) HILO microscopy image (left) from a time-lapse video of mDHC-GFP (green) and dextran-A647 (magenta), and the kymograph corresponding to the region marked with the white square (right). The yellow arrowheads point to a short run of the dextran vesicle toward the MT minus end upon binding of a dynein molecule; the portion of the kymograph with the run is enlarged and shown on the bottom right. A longer run in the same cell has been depicted in Fig. 6 a. (g) HILO microscopy image (left) from a time-lapse video of mDHC-GFP (green) and dextran-A647 (magenta) in cells with RNAi-mediated depletion of hDHC, and the kymograph and plots corresponding to the region marked with the white square (right). The yellow arrowheads point to a short run of a previously plus end–directed dextran vesicle toward the MT minus end upon binding of a dynein molecule. (h) Immunofluorescence images of EGFR (left, green), DHC (middle, magenta), and their merge (right) were obtained using Airyscan confocal microscopy. The inset (marked with a white box) is depicted to the right of the images. EGFR and anti-DHC channel insets are depicted as intensity maps and the white arrowheads point to EGFR punctae, some of which colocalize with anti-DHC. (i) Fluorescence images of anti-EGFR (left, green), mDHC-GFP (middle, magenta), and their merge (right) obtained using Airyscan confocal microscopy. The inset (marked with a white box) is depicted to the right of the images. EGFR and mDHC-GFP channel insets are depicted as intensity maps and the white arrowheads point to EGFR punctae, some of which colocalize with mDHC-GFP. (j) Plot of the probability of co-occurrence of EGFR with anti-DHC and mDHC-GFP 53 ± 18% of EGFR containing endosomes colocalized with DHC (human/mouse), and 33 ± 18% EGFR vesicles colocalized with mDHC-GFP (n = 76 cells across n = 3 independent experiments). This indicates that we are likely missing ∼40% ([53-33]/55) of the runs by these cargo (and presumably other cargo moved by dynein) in our imaging, which could explain the fewer runs we see in our imaging. Error bars represent SD. In a, f, and g, “N” marks the location/direction of the nucleus. Source data are available for this figure: SourceData FS5.

Dextran and EGFR’s interaction with dynein. (a) HILO microscopy image (left) from a 10-s-long time-lapse video of mDHC-GFP (green) and dextran-A647 (magenta) and the corresponding kymograph (right). The yellow arrowhead points to a dextran vesicle with no dynein fluorescence and the white arrowhead points to a dextran vesicle with associated dynein intensity. The kymograph shows that a dextran vesicle with dynein bound to it (white arrowhead) made a short run toward the MT minus end. The dextran vesicle without visible dynein intensity (yellow arrowhead) remained stationary. (b) Displacement versus time plot of dextran vesicles without associated dynein intensity. (c) Displacement versus time plot of dextran vesicles with visible dynein intensity. Comparing b and c, while 28% of dextran vesicles with visible dynein intensity moved toward the MT minus ends (n = 12/44 vesicles), only 10% of those without associated dynein intensity moved to the minus end of MTs (n = 11/114 dextran vesicles; n = 2 independent experiments with >25 cells). In other words, of all the minus end–directed dextran vesicles observed in these experiments (all the teal-colored traces in b and c), ∼52% (n = 12/23 vesicles) had associated dynein intensity. Fisher’s exact test yielded a significant difference in dynein-associated and non-dynein-associated endosome behavior (P < 0.005). (d) Box plots comparing the intensity of dynein on dextran vesicles and intensity of single molecules of dynein (measured from binding events) showed that there were one to two dynein molecules per dextran vesicle (n = 44 dextran vesicles with dynein intensity, n = 115 single molecules of dynein from n = 2 independent experiments with >25 cells). (e) Analysis of b–d was done in cells expressing mDHC-GFP and treated with 25 nM siRNA against endogenous hDHC. Quantification of western blot confirmed that the RNAi successfully knocked down levels of DHC by 85%. (f) HILO microscopy image (left) from a time-lapse video of mDHC-GFP (green) and dextran-A647 (magenta), and the kymograph corresponding to the region marked with the white square (right). The yellow arrowheads point to a short run of the dextran vesicle toward the MT minus end upon binding of a dynein molecule; the portion of the kymograph with the run is enlarged and shown on the bottom right. A longer run in the same cell has been depicted in Fig. 6 a. (g) HILO microscopy image (left) from a time-lapse video of mDHC-GFP (green) and dextran-A647 (magenta) in cells with RNAi-mediated depletion of hDHC, and the kymograph and plots corresponding to the region marked with the white square (right). The yellow arrowheads point to a short run of a previously plus end–directed dextran vesicle toward the MT minus end upon binding of a dynein molecule. (h) Immunofluorescence images of EGFR (left, green), DHC (middle, magenta), and their merge (right) were obtained using Airyscan confocal microscopy. The inset (marked with a white box) is depicted to the right of the images. EGFR and anti-DHC channel insets are depicted as intensity maps and the white arrowheads point to EGFR punctae, some of which colocalize with anti-DHC. (i) Fluorescence images of anti-EGFR (left, green), mDHC-GFP (middle, magenta), and their merge (right) obtained using Airyscan confocal microscopy. The inset (marked with a white box) is depicted to the right of the images. EGFR and mDHC-GFP channel insets are depicted as intensity maps and the white arrowheads point to EGFR punctae, some of which colocalize with mDHC-GFP. (j) Plot of the probability of co-occurrence of EGFR with anti-DHC and mDHC-GFP 53 ± 18% of EGFR containing endosomes colocalized with DHC (human/mouse), and 33 ± 18% EGFR vesicles colocalized with mDHC-GFP (n = 76 cells across n = 3 independent experiments). This indicates that we are likely missing ∼40% ([53-33]/55) of the runs by these cargo (and presumably other cargo moved by dynein) in our imaging, which could explain the fewer runs we see in our imaging. Error bars represent SD. In a, f, and g, “N” marks the location/direction of the nucleus. Source data are available for this figure: SourceData FS5.

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