Figure 5. Septins are required for... | Rockefeller University Press

Figure 5.

$Figure 5. Septins are required for fusion of macropinosomes/endosomes with lysosomes and promote fusion directly in an in vitro reconstitution assay. (A) MDCKs were treated with control or SEPT2 siRNA, and two rounds of 5-min pulse/15-min chase were performed with TR- and FITC-dextran, successively. Cells were stained with anti-LAMP1 and imaged with confocal microscopy. Images show maximum-intensity projections of TR-dextran and LAMP1 and their areas of overlap (pseudo-colored in red masks), as well as FITC-dextran (green) and its overlay with the pseudo TR-dextran/LAMP1 channel. (B) Bar graph shows percentage TR-dextran/LAMP1 that contains FITC-dextran (n = 18 cells). (C and D) MDCKs were separately incubated with TR- or FITC-labeled dextran for 5 min, and postnuclear supernatants were mixed with canine kidney cytosol without or with ATP (3.3 mM) plus cytochalasin D (10 µM), nocodazole (10 µM), and control or anti-SEPT2 IgG. Images (C) show dextran-containing macropinosomes/endosomes after incubation in the indicated conditions. Bar graph (D) shows the fraction (mean ± SEM) of fused macropinosomes/endosomes with both TR- and FITC-labeled dextran (n = 15 images). n.s., not significant. (E) Bar graph shows the fraction (mean ± SEM) of fused endosomes after incubation of PNS with ATP and whole or SEPT2 immunodepleted (ΔSEPT2) cytosol (n = 15). (F) Gel shows equal volumes of whole and ΔSEPT2 cytosol blotted for SEPT2 and actin. (G) Bar graphs show the fraction (mean ± SEM) of fused endosomes after incubation of postnuclear supernatants with ATP and SEPT2 or SEPT2/6/7 (2.5 µM) in the presence/absence of 10 µM cytochalasin D (n = 15).$

Septins are required for fusion of macropinosomes/endosomes with lysosomes and promote fusion directly in an in vitro reconstitution assay. (A) MDCKs were treated with control or SEPT2 siRNA, and two rounds of 5-min pulse/15-min chase were performed with TR- and FITC-dextran, successively. Cells were stained with anti-LAMP1 and imaged with confocal microscopy. Images show maximum-intensity projections of TR-dextran and LAMP1 and their areas of overlap (pseudo-colored in red masks), as well as FITC-dextran (green) and its overlay with the pseudo TR-dextran/LAMP1 channel. (B) Bar graph shows percentage TR-dextran/LAMP1 that contains FITC-dextran (n = 18 cells). (C and D) MDCKs were separately incubated with TR- or FITC-labeled dextran for 5 min, and postnuclear supernatants were mixed with canine kidney cytosol without or with ATP (3.3 mM) plus cytochalasin D (10 µM), nocodazole (10 µM), and control or anti-SEPT2 IgG. Images (C) show dextran-containing macropinosomes/endosomes after incubation in the indicated conditions. Bar graph (D) shows the fraction (mean ± SEM) of fused macropinosomes/endosomes with both TR- and FITC-labeled dextran (n = 15 images). n.s., not significant. (E) Bar graph shows the fraction (mean ± SEM) of fused endosomes after incubation of PNS with ATP and whole or SEPT2 immunodepleted (ΔSEPT2) cytosol (n = 15). (F) Gel shows equal volumes of whole and ΔSEPT2 cytosol blotted for SEPT2 and actin. (G) Bar graphs show the fraction (mean ± SEM) of fused endosomes after incubation of postnuclear supernatants with ATP and SEPT2 or SEPT2/6/7 (2.5 µM) in the presence/absence of 10 µM cytochalasin D (n = 15).

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