Figure 3.
Microtubule-associated septins inhibit the motility of GFP-CD63–containing endosomes in cell-free in vitro assays. (A–D) Kymographs from time-lapse TIRF microscopy movies show motile and stationary GFP-CD63-containing endosomes (magenta) on uncoated taxol-stabilized microtubules (A; see also Video 4) and microtubules coated with recombinant mCherry-tagged SEPT2/6/7 (1.2 μM; B, see also Video 5), SEPT2/6/7/9 (100 nM; C, see also Video 6) and MAP4(654–1090) (100 nM; D) (green). The MAPs are shown in green. GFP-CD63 endosomes were isolated by differential centrifugation from subconfluent MDCK-GFP-CD63 cells. Blue arrow points to an immobilization event of GFP-CD63 at a microtubule domain with mCherry-SEPT2/6/7. Orange arrows point to stationary GFP-CD63 at microtubule-associated mCherry-SEPT2/6/7/9. Scale bars, 2 μm. (E and F) Quantification of the mean frequency (±SEM, error bars) of the landing (E) and processive (F) events of GFP-CD63 per μm of microtubule length per minute. Landing events were defined as the appearance of GFP-CD63 endosomes that persisted on microtubules for at least 2 s independently of motility status. Processive events were defined as any particles with diagonal trajectories in kymographs of time-lapse movies. Data were acquired from uncoated microtubules (n = 188, eight fields of view) and microtubules coated with mCherry-tagged SEPT2/6/7 (n = 149, five fields of view), SEPT2/6/7/9 (n = 205, seven fields of view) and MAP4(654–1090) (n = 218, six fields of view). Statistical analysis of pairwise comparisons was done with a Mann-Whitney test. * P < 0.05, *** P < 0.001, **** P < 0.0001. (G and H) Quantification of the mean frequency (±SEM, error bars) of dissociation (G) and stationary (H) events of GFP-CD63 per μm of microtubule length per minute. Dissociation events were defined as the dissociation of microtubule-bound GFP-CD63 endosomes, which were on microtubules for at least 2 s independently of motility status. Stationary events were defined as any GFP-CD63 endosomes that did not exhibit processive movement for >5 s regardless of motility status before or after the stationary phase. Data were acquired from uncoated microtubules (n = 188, eight fields of view) and microtubules coated with mCherry-tagged SEPT2/6/7 (n = 149, five fields of view), SEPT2/67/9 (n = 205, seven fields of view) and MAP4(654–1090) (n = 218, six fields of view). Statistical analysis of pairwise comparisons between them was done with a Mann-Whitney test and comparisons were with uncoated microtubules or as indicated by the horizontal bars. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Microtubule-associated septins inhibit the motility of GFP-CD63–containing endosomes in cell-free in vitro assays. (A–D) Kymographs from time-lapse TIRF microscopy movies show motile and stationary GFP-CD63-containing endosomes (magenta) on uncoated taxol-stabilized microtubules (A; see also Video 4) and microtubules coated with recombinant mCherry-tagged SEPT2/6/7 (1.2 μM; B, see also Video 5), SEPT2/6/7/9 (100 nM; C, see also Video 6) and MAP4(654–1090) (100 nM; D) (green). The MAPs are shown in green. GFP-CD63 endosomes were isolated by differential centrifugation from subconfluent MDCK-GFP-CD63 cells. Blue arrow points to an immobilization event of GFP-CD63 at a microtubule domain with mCherry-SEPT2/6/7. Orange arrows point to stationary GFP-CD63 at microtubule-associated mCherry-SEPT2/6/7/9. Scale bars, 2 μm. (E and F) Quantification of the mean frequency (±SEM, error bars) of the landing (E) and processive (F) events of GFP-CD63 per μm of microtubule length per minute. Landing events were defined as the appearance of GFP-CD63 endosomes that persisted on microtubules for at least 2 s independently of motility status. Processive events were defined as any particles with diagonal trajectories in kymographs of time-lapse movies. Data were acquired from uncoated microtubules (n = 188, eight fields of view) and microtubules coated with mCherry-tagged SEPT2/6/7 (n = 149, five fields of view), SEPT2/6/7/9 (n = 205, seven fields of view) and MAP4(654–1090) (n = 218, six fields of view). Statistical analysis of pairwise comparisons was done with a Mann-Whitney test. * P < 0.05, *** P < 0.001, **** P < 0.0001. (G and H) Quantification of the mean frequency (±SEM, error bars) of dissociation (G) and stationary (H) events of GFP-CD63 per μm of microtubule length per minute. Dissociation events were defined as the dissociation of microtubule-bound GFP-CD63 endosomes, which were on microtubules for at least 2 s independently of motility status. Stationary events were defined as any GFP-CD63 endosomes that did not exhibit processive movement for >5 s regardless of motility status before or after the stationary phase. Data were acquired from uncoated microtubules (n = 188, eight fields of view) and microtubules coated with mCherry-tagged SEPT2/6/7 (n = 149, five fields of view), SEPT2/67/9 (n = 205, seven fields of view) and MAP4(654–1090) (n = 218, six fields of view). Statistical analysis of pairwise comparisons between them was done with a Mann-Whitney test and comparisons were with uncoated microtubules or as indicated by the horizontal bars. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

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