Septins promote macropinosome maturation and traffic to the lysosome by facilitating membrane fusion

Lee Dolat; Elias T. Spiliotis

doi:10.1083/jcb.201603030

DOI: 10.1083/jcb.201603030 | Published August 22, 2016

Figure 1.
Septins localize to macropinosomes and their contact/fusion sites. (A) Confocal images of MDCKs stained for SEPT2 and F-actin with or without treatment with 0.1% Triton X-100 before fixation. (B) Bar graph shows the sum intensity (mean ± SEM) of SEPT2 per cell area (n = 15). AU, arbitrary units. (C and D) 3D-rendered confocal microscopy (C) and SIM (D) images of MDCK-PM-mCherry cells stained for endogenous SEPT2. Dashed lines outline the cell edge, and arrows point to SEPT2 at the contact sites of macropinocytic vacuoles. (E) MDCK-PM-mCherry cells were incubated with FITC-dextran and stained for SEPT2. 3D-rendered confocal images show SEPT2 at the periphery and in between (arrow) macropinosomes/endosomes. (F) MDCK-PM-mCherry cells were transfected with SEPT2-GFP and imaged live with wide-field deconvolution microscopy. Still frames show SEPT2 accumulation (arrows) at the site of fusion between two macropinosomes.
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.
Figure 3.
Septin depletion decreases macropinocytic fusion events and hinders macropinosome maturation and turnover. (A) Time-lapse frames from wide-field deconvolution microscopy show the dynamics of PM-mCherry–labeled macropinosomes in MDCK cells treated with control and SEPT2 shRNAs. Yellow arrow points to a cluster of macropinosomes that fuse into a single vacuole. (B) Control (n = 41) and SEPT2-depleted MDCK-PM-mCherry cells (n = 31) were imaged live every 5 s for 8 min. Bar graph shows percentage cells with a cluster of three or more macropinosomes formed from a lamellar ruffle. Error bars indicate the maximum and minimum values from three independent experiments. (C and D) Bar graphs show the lifetime (C) and fusion events (D) of nascent macropinosomes (n = 24–27). Error bars represent mean ± SEM. (E) Quantification of PM-mCherry fluorescence along the contacting (1) and noncontacting (2 and 3) segments of two macropinosomes from a SEPT2-depleted cell. AU, arbitrary units. (F and G) MDCKs were incubated with FITC-dextran for the indicated times. Bar graphshow the number of FITC-dextran–containing macropinosomes/endosomes (F) and their size (G) per cell (n = 18). Error bars represent mean ± SEM. (H) Still frames show the formation and detachment of PM-mCherry–labeled membrane tubules (arrows) in live MDCKs.
Figure 4.
Septins regulate the lysosomal delivery of fluid-phase cargo. (A) MDCKs were transfected for 48 h with control or SEPT2 shRNAs and GFP or shRNA-resistant SEPT2-YFP (rescue). After a 5-min pulse/15-min chase with TR-dextran, cells were stained for LAMP1. Images show maximum-intensity projections of confocal image stacks. (B) Bar graph shows percentage (mean ± SEM) of TR-dextran particles that colocalize with LAMP1 (n = 20 cells). (C) Plot shows percentage (mean ± SEM) of TR-dextran with LAMP1 in control and SEPT2-depleted MDCKs (n = 15), which were pulsed with TR-dextran for 5 min and chased for the indicated times. (D) Lysates from MDCKs that stably express SEPT2-YFP at low and high levels were analyzed by SDS-PAGE and blotted for SEPT2. (E) Plot shows percentage (mean ± SEM) of TR-dextran with LAMP1 in MDCK cells (n = 14) that express SEPT2-YFP at low and high levels. Cells were pulsed with TR-dextran for 5 min and chased for the indicated times.
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).

© 2016 Dolat and Spiliotis This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).