Figure 1.
Septins associate with lysosomes and promote lysosome positioning in the perinuclear cytoplasm.(A and B) Confocal images of COS-7 cells stained for SEPT9 (inverted magenta) and LAMTOR4 (inverted green; A) or EEA1 (inverted green; B). Scale bars, 20 µm. Insets show lysosomes from perinuclear regions in higher magnification (scale bars, 3 µm). Arrowheads point to SEPT9 puncta that localize on LAMTOR4-stained lysosomes. (C) Bar graph shows percentage (mean ± SEM; Mann–Whitney U test) of LAMTOR4 and EEA1 organelles with SEPT9 puncta per cell (n = 6 cells). (D) Super-resolution structured illumination microscopy images show SEPT9 puncta on the delimiting membrane of LAMP2-mCherry–labeled compartments in BSC-1 cells. (E and F) Spinning-disk confocal microscopy image (E) and still frames from time-lapse imaging (F) of COS-7 cells transfected with SEPT9-mCherry and LAMP1-mGFP. Insets show higher-magnification LAMP1-mGFP compartments with SEPT9-mCherry puncta (arrows; scale bars, 1 µm), and arrowheads point to retrograde cotraffic toward the nucleus. Scale bar, 5 µm. (G) Color-coded trajectory of the SEPT9-positive lysosome moving retrogradely in F. Arrowheads point to the early (blue), medial (green), and late (red) stages of movement. (H) COS-7 cells were transfected with mCherry or SEPT9-mCherry and stained with anti-LAMP1. Scale bars, 20 µm. (I) Plot shows the ratio (mean ± SEM) of perinuclear to peripheral LAMP1 fluorescence intensity per cell (n = 70–79; Mann–Whitney U test). (J) Images of LAMP1-stained COS-7 cells after transfection with plasmids coexpressing scramble control or SEPT9-targeting shRNAs and GFP or SEPT9-GFP. Arrows point to lysosomes (LAMP1) with SEPT9-GFP. Scale bars, 20 µm. (K) Plot shows the ratio (mean ± SEM; Mann–Whitney U test) of perinuclear to peripheral LAMP1 fluorescence intensity per cell (n = 45–47). (L) Images show lysosome (LAMP-1) distribution in COS-7 cells transfected with mCherry and GFP, SEPT9-mCherry and GFP-Rab7-DN, or SEPT9-mCherry and GFP. Scale bars, 20 µm. (M) Quantification shows the ratio (mean ± SEM; Kruskal–Wallis one-way ANOVA) of perinuclear to peripheral LAMP1 fluorescence intensity per cell (n = 45–51). (N) Embryonic (E18) hippocampal neurons (DIV4) were transfected with LAMP1-mGFP and mCherry or rat SEPT9-mCherry, and axons were imaged live by TIRF microscopy. Kymographs show stationary or diffusive (blue), retrogradely (magenta), or anterogradely (green) moving particles with LAMP1-mGFP. Scale bar, 5 µm. (O) Bar graph shows the ratio (mean ± SEM; unpaired t test) of retrogradely to anterogradely moving LAMP-1-mGFP particles/2 min per axon (n = 18–19). ns, nonsignificant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Septins associate with lysosomes and promote lysosome positioning in the perinuclear cytoplasm. (A and B) Confocal images of COS-7 cells stained for SEPT9 (inverted magenta) and LAMTOR4 (inverted green; A) or EEA1 (inverted green; B). Scale bars, 20 µm. Insets show lysosomes from perinuclear regions in higher magnification (scale bars, 3 µm). Arrowheads point to SEPT9 puncta that localize on LAMTOR4-stained lysosomes. (C) Bar graph shows percentage (mean ± SEM; Mann–Whitney U test) of LAMTOR4 and EEA1 organelles with SEPT9 puncta per cell (n = 6 cells). (D) Super-resolution structured illumination microscopy images show SEPT9 puncta on the delimiting membrane of LAMP2-mCherry–labeled compartments in BSC-1 cells. (E and F) Spinning-disk confocal microscopy image (E) and still frames from time-lapse imaging (F) of COS-7 cells transfected with SEPT9-mCherry and LAMP1-mGFP. Insets show higher-magnification LAMP1-mGFP compartments with SEPT9-mCherry puncta (arrows; scale bars, 1 µm), and arrowheads point to retrograde cotraffic toward the nucleus. Scale bar, 5 µm. (G) Color-coded trajectory of the SEPT9-positive lysosome moving retrogradely in F. Arrowheads point to the early (blue), medial (green), and late (red) stages of movement. (H) COS-7 cells were transfected with mCherry or SEPT9-mCherry and stained with anti-LAMP1. Scale bars, 20 µm. (I) Plot shows the ratio (mean ± SEM) of perinuclear to peripheral LAMP1 fluorescence intensity per cell (n = 70–79; Mann–Whitney U test). (J) Images of LAMP1-stained COS-7 cells after transfection with plasmids coexpressing scramble control or SEPT9-targeting shRNAs and GFP or SEPT9-GFP. Arrows point to lysosomes (LAMP1) with SEPT9-GFP. Scale bars, 20 µm. (K) Plot shows the ratio (mean ± SEM; Mann–Whitney U test) of perinuclear to peripheral LAMP1 fluorescence intensity per cell (n = 45–47). (L) Images show lysosome (LAMP-1) distribution in COS-7 cells transfected with mCherry and GFP, SEPT9-mCherry and GFP-Rab7-DN, or SEPT9-mCherry and GFP. Scale bars, 20 µm. (M) Quantification shows the ratio (mean ± SEM; Kruskal–Wallis one-way ANOVA) of perinuclear to peripheral LAMP1 fluorescence intensity per cell (n = 45–51). (N) Embryonic (E18) hippocampal neurons (DIV4) were transfected with LAMP1-mGFP and mCherry or rat SEPT9-mCherry, and axons were imaged live by TIRF microscopy. Kymographs show stationary or diffusive (blue), retrogradely (magenta), or anterogradely (green) moving particles with LAMP1-mGFP. Scale bar, 5 µm. (O) Bar graph shows the ratio (mean ± SEM; unpaired t test) of retrogradely to anterogradely moving LAMP-1-mGFP particles/2 min per axon (n = 18–19). ns, nonsignificant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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