Figure 2.
Membrane-associated SEPT9 recruits dynein and induces retrograde dynein-driven transport.(A) Images show COS-7 cells that were transfected with GFP-SEPT9-FRB and PEX-RFP-FKBP after 45 min of treatment with rapalog or control carrier. Bar graph shows percentage of cells (n = 37–42; χ2 test) with dispersed, partially clustered, and clustered peroxisomes. Scale bar, 20 µm. (B) COS-7 cells were transfected with MitoTagRFP or SEPT9-MitoTagRFP and stained with DAPI. Bar graph shows perinuclear and peripheral mitochondria as percentage (mean ± SEM; two-way ANOVA) of total mitochondria per cell (n = 20–44). Scale bar, 20 µm. (C) COS-7 cells were transfected with Mito-TagRFP or SEPT9-MitoTagRFP, treated with nocodazole (33 µM) or DMSO for 3 h, and stained with anti-GM130 and DAPI. Quantification shows the amount of perinuclear and peripheral mitochondria as percentage (mean ± SEM; two-way ANOVA) of total mitochondria per cell (n = 53–54). Scale bar, 10 µm. (D) Images show DAPI-stained COS-7 cells that were transfected with SEPT9-MitoTagRFP and GFP (control) or GFP-p50 (dynamitin). Bar graph shows quantification of perinuclear and peripheral mitochondria as percentage (mean ± SEM; two-way ANOVA) of total per cell (n = 55–70). Scale bars, 20 µm. (E) COS-7 cells were transfected with MitoTagRFP or SEPT9-MitoTagRFP and stained with anti-DIC after 3-h treatment with DMSO or nocodazole (33 µM). Plot shows DIC fluorescence intensity (mean ± SEM) on MitoTagRFP-labeled mitochondria per cell (n = 9–16; Kruskal–Wallis one-way ANOVA). Scale bars, 20 µm. (F) Lysates of HEK-293 cells expressing mCherry or mCherry-SEPT9 were incubated with mRFP trap beads, and immunoprecipitates were blotted with antibodies against mCherry (top), DIC, and p150GLUED. A.U., arbitrary units. (G) Coomassie-stained gel (top) shows recombinant SEPT9 and SEPT2/6/7, which were used in pull-down assays with dynein–dynactin purified from HEK-293 cells. Western blots (bottom) were performed with antibodies against DHC and p150GLUED. *, P < 0.05; *** P < 0.001.

Membrane-associated SEPT9 recruits dynein and induces retrograde dynein-driven transport. (A) Images show COS-7 cells that were transfected with GFP-SEPT9-FRB and PEX-RFP-FKBP after 45 min of treatment with rapalog or control carrier. Bar graph shows percentage of cells (n = 37–42; χ2 test) with dispersed, partially clustered, and clustered peroxisomes. Scale bar, 20 µm. (B) COS-7 cells were transfected with MitoTagRFP or SEPT9-MitoTagRFP and stained with DAPI. Bar graph shows perinuclear and peripheral mitochondria as percentage (mean ± SEM; two-way ANOVA) of total mitochondria per cell (n = 20–44). Scale bar, 20 µm. (C) COS-7 cells were transfected with Mito-TagRFP or SEPT9-MitoTagRFP, treated with nocodazole (33 µM) or DMSO for 3 h, and stained with anti-GM130 and DAPI. Quantification shows the amount of perinuclear and peripheral mitochondria as percentage (mean ± SEM; two-way ANOVA) of total mitochondria per cell (n = 53–54). Scale bar, 10 µm. (D) Images show DAPI-stained COS-7 cells that were transfected with SEPT9-MitoTagRFP and GFP (control) or GFP-p50 (dynamitin). Bar graph shows quantification of perinuclear and peripheral mitochondria as percentage (mean ± SEM; two-way ANOVA) of total per cell (n = 55–70). Scale bars, 20 µm. (E) COS-7 cells were transfected with MitoTagRFP or SEPT9-MitoTagRFP and stained with anti-DIC after 3-h treatment with DMSO or nocodazole (33 µM). Plot shows DIC fluorescence intensity (mean ± SEM) on MitoTagRFP-labeled mitochondria per cell (n = 9–16; Kruskal–Wallis one-way ANOVA). Scale bars, 20 µm. (F) Lysates of HEK-293 cells expressing mCherry or mCherry-SEPT9 were incubated with mRFP trap beads, and immunoprecipitates were blotted with antibodies against mCherry (top), DIC, and p150GLUED. A.U., arbitrary units. (G) Coomassie-stained gel (top) shows recombinant SEPT9 and SEPT2/6/7, which were used in pull-down assays with dynein–dynactin purified from HEK-293 cells. Western blots (bottom) were performed with antibodies against DHC and p150GLUED. *, P < 0.05; *** P < 0.001.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal