Figure 5.
Figure 5. Dual live imaging of internalized Tf and NgCAM. (A) Neurons were transfected with NgCAM and incubated with anti-NgCAM antibodies (red) and FITC-Tf (green) at 16°C, washed and chased at 37°C in the continued presence of Tf for 15 (A, left) or 60 min (A, right), and then fixed. Surface NgCAM (blue) was detected with a cy5–anti–mouse secondary antibody before permeabilization. Yellow arrowheads indicate overlapping puncta. At 60 min of chase, the overlap is frequently not precise, resulting in a traffic light appearance. Single confocal sections are shown. The traffic light pattern is most abundant at late chase times. (B and C) Neurons (DIV12) expressing NgCAM were loaded with Texas red–Tf and Alexa 488–anti-NgCAM antibodies for 1 h and then washed and imaged live every 3 s. Tf (red) and endocytosed NgCAM (green) can be easily detected in somatodendritic endosomes in live neurons. See Video 3 (available). (B) Examples of endosome behavior is shown in four consecutive frames 3 s apart: stationary endosomes containing only NgCAM (green), only Tf (red), or both NgCAM and Tf (yellow) are indicated with arrowheads. Motile endosomal carriers are indicated by arrows (red, Tf only; green, NgCAM only). The four compartments marked with symbols were traced over time and their trajectories plotted in the adjacent graph. For the moving compartments, the arrow points at the first frame of the sequence. A proximal dendrite is shown. See Video 4. (C) Consecutive frames of a traffic light endosome are shown. The endosome appeared initially yellow but in subsequent frames appears as one slightly elongated carrier (or two tethered carriers) in which Tf (red) is laterally segregated away from endocytosed NgCAM (green). The field shown is from the soma of the cell. See Video 5. (D) Labeled endosomal puncta were categorized as Tf only, NgCAM only, or both NgCAM and Tf. All labeled structures in each panel were subdivided by shape (round or elongated) and size (large, medium, or small) and by motility (stationary or moving). Structures containing both Tf and NgCAM rarely moved, whereas structures containing Tf only moved occasionally. Structures containing NgCAM only were the most motile. n = 365 endosomes.

Dual live imaging of internalized Tf and NgCAM. (A) Neurons were transfected with NgCAM and incubated with anti-NgCAM antibodies (red) and FITC-Tf (green) at 16°C, washed and chased at 37°C in the continued presence of Tf for 15 (A, left) or 60 min (A, right), and then fixed. Surface NgCAM (blue) was detected with a cy5–anti–mouse secondary antibody before permeabilization. Yellow arrowheads indicate overlapping puncta. At 60 min of chase, the overlap is frequently not precise, resulting in a traffic light appearance. Single confocal sections are shown. The traffic light pattern is most abundant at late chase times. (B and C) Neurons (DIV12) expressing NgCAM were loaded with Texas red–Tf and Alexa 488–anti-NgCAM antibodies for 1 h and then washed and imaged live every 3 s. Tf (red) and endocytosed NgCAM (green) can be easily detected in somatodendritic endosomes in live neurons. See Video 3 . (B) Examples of endosome behavior is shown in four consecutive frames 3 s apart: stationary endosomes containing only NgCAM (green), only Tf (red), or both NgCAM and Tf (yellow) are indicated with arrowheads. Motile endosomal carriers are indicated by arrows (red, Tf only; green, NgCAM only). The four compartments marked with symbols were traced over time and their trajectories plotted in the adjacent graph. For the moving compartments, the arrow points at the first frame of the sequence. A proximal dendrite is shown. See Video 4. (C) Consecutive frames of a traffic light endosome are shown. The endosome appeared initially yellow but in subsequent frames appears as one slightly elongated carrier (or two tethered carriers) in which Tf (red) is laterally segregated away from endocytosed NgCAM (green). The field shown is from the soma of the cell. See Video 5. (D) Labeled endosomal puncta were categorized as Tf only, NgCAM only, or both NgCAM and Tf. All labeled structures in each panel were subdivided by shape (round or elongated) and size (large, medium, or small) and by motility (stationary or moving). Structures containing both Tf and NgCAM rarely moved, whereas structures containing Tf only moved occasionally. Structures containing NgCAM only were the most motile. n = 365 endosomes.

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