Figure 1.
Figure 1. Colocalization of endocytosed NgCAM with markers for early and late endosomes. (A) NgCAM-expressing neurons were incubated for 20 min with anti-NgCAM antibodies, which were detected after permeabilization with an Alexa 488–goat anti–mouse antibody, whereas EEs were detected with a polyclonal anti-EEA1 antibody (red). Yellow arrowheads indicate examples of colocalizing puncta. (B) NgCAM-expressing neurons were incubated with anti-NgCAM antibody for 30 min at 16°C and washed, and internalized NgCAM antibody was chased at 37°C for 1 h. Surface NgCAM was detected before permeabilization with a cy5–goat anti–mouse secondary antibody (blue). After permeabilization, endocytosed NgCAM antibodies were detected with Alexa 568–goat anti–mouse antibody (red) and LEs/lysosomes were detected with a polyclonal anti-lgp120 antibody (green). A single confocal section is shown. The boxed regions shown in A and B are magnified sections of dendrites for easier comparison of colabeling. (C) Quantification of colocalization of EEA1 and NgCAM endocytosed for 20 min and of lgp120 and NgCAM endocytosed and chased for 60 min. For each puncta, the ratio of intensities of endocytosed NgCAM to either EEA1 (left) or lgp120 (right) was determined as described in Materials and methods. For EEA1 colocalization, 560 endosomes were scored. For lgp120 colocalization, 497 endosomes were scored. (D) Basic organization of endosomes, adapted from fibroblasts. Three distinct pathways are indicated by the arrows: degradative cargo follows the red arrows, somatodendritically recycling cargo follows the green arrows, and transcytosing cargo follows the blue arrows. Cargo enters via several pathways in small carriers vesicles (ECV) that fuse with existing EEs. EEs contain a vacuolar portion as well as tubular extensions. The tubular extensions accumulate recycling cargoes and bud off to transport recycling cargos back to the plasma membrane either directly or via the RE. Endosomal carrier vesicles (ECVs) can be spherical or elongated and serve as transport carriers between compartments. The vacuolar portions of the EE accumulate internal vesicles and mature into MVBs. MVBs are transport carriers that carry cargo to LE/lysosomes (LE/lys) for degradation (red). Some MVBs might not be predegradative but be capable of recycling. Cargo destined for either axons or dendrites are sorted in the RE and transported to their respective final destination from there.

Colocalization of endocytosed NgCAM with markers for early and late endosomes. (A) NgCAM-expressing neurons were incubated for 20 min with anti-NgCAM antibodies, which were detected after permeabilization with an Alexa 488–goat anti–mouse antibody, whereas EEs were detected with a polyclonal anti-EEA1 antibody (red). Yellow arrowheads indicate examples of colocalizing puncta. (B) NgCAM-expressing neurons were incubated with anti-NgCAM antibody for 30 min at 16°C and washed, and internalized NgCAM antibody was chased at 37°C for 1 h. Surface NgCAM was detected before permeabilization with a cy5–goat anti–mouse secondary antibody (blue). After permeabilization, endocytosed NgCAM antibodies were detected with Alexa 568–goat anti–mouse antibody (red) and LEs/lysosomes were detected with a polyclonal anti-lgp120 antibody (green). A single confocal section is shown. The boxed regions shown in A and B are magnified sections of dendrites for easier comparison of colabeling. (C) Quantification of colocalization of EEA1 and NgCAM endocytosed for 20 min and of lgp120 and NgCAM endocytosed and chased for 60 min. For each puncta, the ratio of intensities of endocytosed NgCAM to either EEA1 (left) or lgp120 (right) was determined as described in Materials and methods. For EEA1 colocalization, 560 endosomes were scored. For lgp120 colocalization, 497 endosomes were scored. (D) Basic organization of endosomes, adapted from fibroblasts. Three distinct pathways are indicated by the arrows: degradative cargo follows the red arrows, somatodendritically recycling cargo follows the green arrows, and transcytosing cargo follows the blue arrows. Cargo enters via several pathways in small carriers vesicles (ECV) that fuse with existing EEs. EEs contain a vacuolar portion as well as tubular extensions. The tubular extensions accumulate recycling cargoes and bud off to transport recycling cargos back to the plasma membrane either directly or via the RE. Endosomal carrier vesicles (ECVs) can be spherical or elongated and serve as transport carriers between compartments. The vacuolar portions of the EE accumulate internal vesicles and mature into MVBs. MVBs are transport carriers that carry cargo to LE/lysosomes (LE/lys) for degradation (red). Some MVBs might not be predegradative but be capable of recycling. Cargo destined for either axons or dendrites are sorted in the RE and transported to their respective final destination from there.

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