Figure 2.
Domain components are substantially cotransported in the retrograde direction following endocytosis.(A) Still images of domain components transported in the retrograde direction are shown. NF186 tagged with mKate2 (red) is compared with other components labeled with EGFP. Yellow arrows indicate vesicles with EGFP-tagged components that are cotransported with NF186-mKate2; white arrows indicate vesicles labeled with EGFP alone. Insets show higher power images of vesicles indicated by the arrows. Time of imaging (seconds) is indicated in each panel. (B) Kymographs of the retrogradely transported vesicles indicated by the arrows in the panels in A are shown. Trajectories of each vesicle are circled (yellow for cotransport, white for EGFP alone with the retrograde direction indicated by the dotted red arrows); vesicle positions are numbered in temporal sequence. (C) Quantification of vesicles that colabel for NF186 and the indicated EGFP+ component versus the total number of retrogradely transported NF186 vesicles (average ± SEM) is shown. The numbers of axons counted were NrCAM, n = 15; Navβ2, n = 18; Navβ1, n = 20; KCNQ3, n = 17; Caspr, n = 15; and Necl1, n = 16. NF186 is uniformly cotransported with NrCAM and substantially cotranported with all other components as shown. (D) Representative micrographs show biotinylated Avi-tagged NF186-EGFP labeled with streptavidin 568 and then live imaged. Upper rows show anterograde vesicles (white arrow, white circles); lower rows show retrograde vesicles (yellow arrow, yellow circle). Insets show higher power images of vesicles indicated by the arrows. Time of imaging (seconds) is indicated in each panel. Dotted arrows indicate the direction of transport (i.e., blue, anterograde and red, retrograde). The separation of red and green markers in the retrograde vesicles reflects ongoing transport during the change in the emission filters. (E) Avi-tagged NF186 EGFP transported in the retrograde direction colabeled with pHrodo Red Avidin, indicating it is an acidic compartment. In the kymograph, the vesicle trajectory is circled (yellow for cotransport) and numbered in temporal sequence; the dotted red arrow indicates the retrograde direction. Scale bars: 1 µm.

Domain components are substantially cotransported in the retrograde direction following endocytosis. (A) Still images of domain components transported in the retrograde direction are shown. NF186 tagged with mKate2 (red) is compared with other components labeled with EGFP. Yellow arrows indicate vesicles with EGFP-tagged components that are cotransported with NF186-mKate2; white arrows indicate vesicles labeled with EGFP alone. Insets show higher power images of vesicles indicated by the arrows. Time of imaging (seconds) is indicated in each panel. (B) Kymographs of the retrogradely transported vesicles indicated by the arrows in the panels in A are shown. Trajectories of each vesicle are circled (yellow for cotransport, white for EGFP alone with the retrograde direction indicated by the dotted red arrows); vesicle positions are numbered in temporal sequence. (C) Quantification of vesicles that colabel for NF186 and the indicated EGFP+ component versus the total number of retrogradely transported NF186 vesicles (average ± SEM) is shown. The numbers of axons counted were NrCAM, n = 15; Navβ2, n = 18; Navβ1, n = 20; KCNQ3, n = 17; Caspr, n = 15; and Necl1, n = 16. NF186 is uniformly cotransported with NrCAM and substantially cotranported with all other components as shown. (D) Representative micrographs show biotinylated Avi-tagged NF186-EGFP labeled with streptavidin 568 and then live imaged. Upper rows show anterograde vesicles (white arrow, white circles); lower rows show retrograde vesicles (yellow arrow, yellow circle). Insets show higher power images of vesicles indicated by the arrows. Time of imaging (seconds) is indicated in each panel. Dotted arrows indicate the direction of transport (i.e., blue, anterograde and red, retrograde). The separation of red and green markers in the retrograde vesicles reflects ongoing transport during the change in the emission filters. (E) Avi-tagged NF186 EGFP transported in the retrograde direction colabeled with pHrodo Red Avidin, indicating it is an acidic compartment. In the kymograph, the vesicle trajectory is circled (yellow for cotransport) and numbered in temporal sequence; the dotted red arrow indicates the retrograde direction. Scale bars: 1 µm.

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