Figure 6.
Septins restrict the intracellular motility of GFP-CD63 endosomes and promote their maturation from EEA1- to LBPA-positive MVBs/LEs. (A) Images show representative trajectories of motile GFP-CD63 endosomes with the indicated velocity ranges in the midzones of subconfluent MDCK cells, which were transfected with plasmids expressing mCherry (lower left insets) and control or SEPT7 shRNAs. Images show the cumulative trajectories of a 5-min acquisition by time-lapse super-resolution spinning disk confocal microscopy. Frames with thicker lines highlight the reduction in the abundance of trajectories with low velocities (0.2–0.3 μm/s) and an increase in the abundance of trajectories with higher velocities (>0.5 μm/s) in cells transfected with SEPT7 shRNAs. Scale bars, 10 μm. See also Videos 7 and 8. (B) Distribution of the velocity of GFP-CD63 in MDCK cells transfected with control (n = 2,640 endosomes from six cells) and SEPT7 shRNAs (n = 2,312 endosomes from six cells) fitted to a Gaussian non-linear regression line of best fit. (C) Distribution of displacements of GFP-CD63 in MDCK cells transfected with control (n = 2,640 endosomes from six cells) and SEPT7 shRNAs (n = 2,312 endosomes from six cells) fitted to a Gaussian non-linear regression line of best fit. (D and E) Maximum intensity projections of super-resolution confocal microscopy sections of subconfluent MDCK-GFP-CD63 cells (inverted forest green), which were stained for endogenous EEA1 (D; inverted magenta) or LBPA (E; inverted magenta) after transfection with plasmids that express mCherry (inverted blue) and control or SEPT7 shRNAs. Outlined regions (dashed rectangles) are shown in higher magnification (upper right insets). Lower left insets show the fluorescence of mCherry, which is coexpressed with shRNAs. Scale bars, 10 and 5 μm (insets). (F) Mean percentage (±SEM, error bars) of total GFP-CD63 endosomes that overlap with EEA1 in MDCK-GFP-CD63 cells (n = 8) transfected with control or SEPT7 shRNAs. Data were statistically analyzed with an unpaired t test with Welch’s correction. n.s., not significant. (G) Mean percentage (±SEM, error bars) of total GFP-CD63 endosomes that overlap with LBPA in MDCK-GFP-CD63 cells (n = 8) transfected with control or SEPT7 shRNAs. Data were statistically analyzed with an unpaired t test with Welch’s correction. **** P < 0.0001.

Septins restrict the intracellular motility of GFP-CD63 endosomes and promote their maturation from EEA1- to LBPA-positive MVBs/LEs. (A) Images show representative trajectories of motile GFP-CD63 endosomes with the indicated velocity ranges in the midzones of subconfluent MDCK cells, which were transfected with plasmids expressing mCherry (lower left insets) and control or SEPT7 shRNAs. Images show the cumulative trajectories of a 5-min acquisition by time-lapse super-resolution spinning disk confocal microscopy. Frames with thicker lines highlight the reduction in the abundance of trajectories with low velocities (0.2–0.3 μm/s) and an increase in the abundance of trajectories with higher velocities (>0.5 μm/s) in cells transfected with SEPT7 shRNAs. Scale bars, 10 μm. See also Videos 7 and 8. (B) Distribution of the velocity of GFP-CD63 in MDCK cells transfected with control (n = 2,640 endosomes from six cells) and SEPT7 shRNAs (n = 2,312 endosomes from six cells) fitted to a Gaussian non-linear regression line of best fit. (C) Distribution of displacements of GFP-CD63 in MDCK cells transfected with control (n = 2,640 endosomes from six cells) and SEPT7 shRNAs (n = 2,312 endosomes from six cells) fitted to a Gaussian non-linear regression line of best fit. (D and E) Maximum intensity projections of super-resolution confocal microscopy sections of subconfluent MDCK-GFP-CD63 cells (inverted forest green), which were stained for endogenous EEA1 (D; inverted magenta) or LBPA (E; inverted magenta) after transfection with plasmids that express mCherry (inverted blue) and control or SEPT7 shRNAs. Outlined regions (dashed rectangles) are shown in higher magnification (upper right insets). Lower left insets show the fluorescence of mCherry, which is coexpressed with shRNAs. Scale bars, 10 and 5 μm (insets). (F) Mean percentage (±SEM, error bars) of total GFP-CD63 endosomes that overlap with EEA1 in MDCK-GFP-CD63 cells (n = 8) transfected with control or SEPT7 shRNAs. Data were statistically analyzed with an unpaired t test with Welch’s correction. n.s., not significant. (G) Mean percentage (±SEM, error bars) of total GFP-CD63 endosomes that overlap with LBPA in MDCK-GFP-CD63 cells (n = 8) transfected with control or SEPT7 shRNAs. Data were statistically analyzed with an unpaired t test with Welch’s correction. **** P < 0.0001.

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