Figure 2.
CD63-containing endosomes are less motile on septin-coated microtubules than EEA1-positive endosomes. (A and B) Still frames from time-lapse super-resolution spinning disk confocal microscopy of MDCK cells that express mCherry-SEPT9_i1 and GFP-CD63 show examples of septin-bound endosomes that are immotile (A, orange arrows) and shift between septin filaments (B, orange arrows). See also Videos 1 and 2. Scale bars, 1 μm. (C and D) Still frames from time-lapse super-resolution spinning disk confocal microscopy of MDCK cells that express mCherry-SEPT9_i1 and GFP-CD63 show examples of septin-bound endosomes, which transiently merge and disengage in a touch-and-go-like fashion (C, orange arrows and blue arrows), and a GFP-CD63 endosome that undergoes processive movement (D, orange arrow; see also Video 3). A kymograph illustrates the septin-bound track of the processive GFP-CD63 endosome. Scale bars, 1 µm. (E) Quantification of the mean percentage (±SEM, error bars) of motile GFP-CD63 endosomes with overlap with mCherry-SEPT9_i1 (SEPT9-bound) and without overlap (unbound) in subconfluent MDCK cells (n = 6). Statistical analysis of pairwise comparisons was performed with an unpaired t test with Welch’s correction. ** P < 0.01. (F) Quantification of the mean percentage (±SEM, error bars) of motile septin-bound GFP-CD63 endosomes that undergo bidirectional, unidirectional, and diffusive movements on septin-coated microtubules (n = 30). Data were statistically analyzed with a Mann-Whitney test. *** P < 0.001; **** P < 0.0001. (G) Quantification of the mean percentage (±SEM, error bars) of motile GFP-EEA1 endosomes with overlap with mCherry-SEPT9_i1 (SEPT9-bound) and without overlap (unbound) in subconfluent MDCK cells (n = 6). Data were statistically analyzed with a Mann-Whitney test. n.s., not significant.

CD63-containing endosomes are less motile on septin-coated microtubules than EEA1-positive endosomes. (A and B) Still frames from time-lapse super-resolution spinning disk confocal microscopy of MDCK cells that express mCherry-SEPT9_i1 and GFP-CD63 show examples of septin-bound endosomes that are immotile (A, orange arrows) and shift between septin filaments (B, orange arrows). See also Videos 1 and 2. Scale bars, 1 μm. (C and D) Still frames from time-lapse super-resolution spinning disk confocal microscopy of MDCK cells that express mCherry-SEPT9_i1 and GFP-CD63 show examples of septin-bound endosomes, which transiently merge and disengage in a touch-and-go-like fashion (C, orange arrows and blue arrows), and a GFP-CD63 endosome that undergoes processive movement (D, orange arrow; see also Video 3). A kymograph illustrates the septin-bound track of the processive GFP-CD63 endosome. Scale bars, 1 µm. (E) Quantification of the mean percentage (±SEM, error bars) of motile GFP-CD63 endosomes with overlap with mCherry-SEPT9_i1 (SEPT9-bound) and without overlap (unbound) in subconfluent MDCK cells (n = 6). Statistical analysis of pairwise comparisons was performed with an unpaired t test with Welch’s correction. ** P < 0.01. (F) Quantification of the mean percentage (±SEM, error bars) of motile septin-bound GFP-CD63 endosomes that undergo bidirectional, unidirectional, and diffusive movements on septin-coated microtubules (n = 30). Data were statistically analyzed with a Mann-Whitney test. *** P < 0.001; **** P < 0.0001. (G) Quantification of the mean percentage (±SEM, error bars) of motile GFP-EEA1 endosomes with overlap with mCherry-SEPT9_i1 (SEPT9-bound) and without overlap (unbound) in subconfluent MDCK cells (n = 6). Data were statistically analyzed with a Mann-Whitney test. n.s., not significant.

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