Figure 2. Septins associate... | Rockefeller University Press

Figure 2.

$Figure 2. Septins associate preferentially with maturing macropinosomes in a PI(3,5)P2-dependent manner. (A) MDCKs were pulsed with TR-dextran for 5 min, chased for the indicated times, stained for SEPT2, and imaged with confocal microscopy. Bar graph shows the percentage (mean ± SEM) of dextran-containing macropinosomes/endosomes with SEPT2 (n = 12 cells). (B) Maximum-intensity projections of confocal stacks and SIM images of MDCKs transfected with ML1Nx2-GFP and stained for SEPT2. Insets show a vacuole (arrow) in higher magnification. (C) Maximum-intensity projections of confocal images of MDCK-PM-mCherry cells stained for SEPT2 after 2-h treatment with DMSO or YM201636 (800 nM). (D) Bar graph shows the sum intensity (mean ± SEM) of peripheral SEPT2 per square micrometer (n = 20 cells). AU, arbitrary units. (E) Bar graph shows the fraction (mean ± SEM) of TR-dextran puncta with SEPT2 (n = 20 cells) in MDCKs incubated with TR-dextran for 10 min after DMSO or YM201636 treatment. (F) Recombinant SEPT2/6/7 was mixed with liposomes of the indicated phosphoinositides and centrifuged on a sucrose gradient. Coomassie-stained gels show equal volumes from the top (liposome-bound SEPT2/6/7; B) and bottom (unbound SEPT2/6/7; U) fractions. (G) Bar graphs show the fraction of bound and unbound SEPT2/6/7 from three independent experiments. Error bars represent the maximum and minimum values from the three independent experiments.$

Septins associate preferentially with maturing macropinosomes in a PI(3,5)P2-dependent manner. (A) MDCKs were pulsed with TR-dextran for 5 min, chased for the indicated times, stained for SEPT2, and imaged with confocal microscopy. Bar graph shows the percentage (mean ± SEM) of dextran-containing macropinosomes/endosomes with SEPT2 (n = 12 cells). (B) Maximum-intensity projections of confocal stacks and SIM images of MDCKs transfected with ML1Nx2-GFP and stained for SEPT2. Insets show a vacuole (arrow) in higher magnification. (C) Maximum-intensity projections of confocal images of MDCK-PM-mCherry cells stained for SEPT2 after 2-h treatment with DMSO or YM201636 (800 nM). (D) Bar graph shows the sum intensity (mean ± SEM) of peripheral SEPT2 per square micrometer (n = 20 cells). AU, arbitrary units. (E) Bar graph shows the fraction (mean ± SEM) of TR-dextran puncta with SEPT2 (n = 20 cells) in MDCKs incubated with TR-dextran for 10 min after DMSO or YM201636 treatment. (F) Recombinant SEPT2/6/7 was mixed with liposomes of the indicated phosphoinositides and centrifuged on a sucrose gradient. Coomassie-stained gels show equal volumes from the top (liposome-bound SEPT2/6/7; B) and bottom (unbound SEPT2/6/7; U) fractions. (G) Bar graphs show the fraction of bound and unbound SEPT2/6/7 from three independent experiments. Error bars represent the maximum and minimum values from the three independent experiments.

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