Figure S3.
Trafficking of LAMP1 and NPY at the AIS of nonAcD and AcD neurons. (A) Representative images of nonAcD and AcD neurons co-transfected with NPY-EGFP (left) to visualize dense core vesicles as additional axonal cargo, and Tag-RFP (right) to visualize neuronal morphology. The AIS is live-labeled with anti-NF-CF640R (NF). White arrowhead on the left panel and black arrowhead on the right panel indicates the AIS. Scale bar is 15 µm. Related to Video 8. (B and C) Representative kymographs showing trajectories of NPY-EGFP vesicles entering the AIS of nonAcD (B) and AcD (C) neuron shown in A. Scale is 10 s (vertical) and 2 µm (horizontal). (D) Representative images of nonAcD and AcD neurons transfected with LAMP1-mCherry to visualize lysosomes as neutral cargo. The AIS is live-labelled with anti-NF-CF640R (NF). White arrowhead indicates the AIS. Scale bar is 15 µm. Related to Video 9. (E and F) Representative kymographs showing trajectories of LAMP1-mCherry vesicles entering the AIS of nonAcD (E) and AcD (F) neuron shown in D. Scale is 10 s (vertical) and 2 µm (horizontal). (G) Velocity of NPY-EGFP vesicles running towards anterograde and retrograde directions in the AIS of nonAcD and AcD neurons. Mean ± SEM, four independent cultures, nonAcD neurons: n = 12 cells, AcD neurons: n = 12 cells. (H) Velocity of LAMP1-mCherry vesicles running towards anterograde and retrograde directions in the AIS of nonAcD and AcD neurons. Mean ± SEM, four independent cultures, nonAcD neurons: n = 20 cells, AcD neurons: n = 17 cells. (I and J) Run length (I) and run duration (J) of NPY-EGFP vesicles running towards anterograde and retrograde directions in the AIS of nonAcD and AcD neurons. Mean ± SEM, four independent cultures, nonAcD neurons: n = 12 cells, AcD neurons: n = 12 cells. (K and L) Number of pause and total pausing duration of NPY-EGFP vesicles travelling within the AIS of nonAcD and AcD neurons (regardless of directions). Mean ± SEM, four independent cultures, nonAcD neurons: n = 12 cells, AcD neurons: n = 12 cells. (M and N) Run length (M) and run duration (N) of LAMP1-mCherry vesicles running towards anterograde and retrograde directions in the AIS of nonAcD and AcD neurons. Mean ± SEM, four independent cultures, nonAcD neurons: n = 20 cells, AcD neurons: n = 17 cells. (O and P) Number of pause (O) and total pausing duration (P) of LAMP1-mCherry vesicles travelling within the AIS of nonAcD and AcD neurons (regardless of directions). Mean ± SEM, four independent cultures, nonAcD neurons: n = 20 cells, AcD neurons: n = 17 cells. Mann–Whitney test (two-sided): not significant (n.s.) P > 0.05, *P < 0.05.

Trafficking of LAMP1 and NPY at the AIS of nonAcD and AcD neurons. (A) Representative images of nonAcD and AcD neurons co-transfected with NPY-EGFP (left) to visualize dense core vesicles as additional axonal cargo, and Tag-RFP (right) to visualize neuronal morphology. The AIS is live-labeled with anti-NF-CF640R (NF). White arrowhead on the left panel and black arrowhead on the right panel indicates the AIS. Scale bar is 15 µm. Related to Video 8. (B and C) Representative kymographs showing trajectories of NPY-EGFP vesicles entering the AIS of nonAcD (B) and AcD (C) neuron shown in A. Scale is 10 s (vertical) and 2 µm (horizontal). (D) Representative images of nonAcD and AcD neurons transfected with LAMP1-mCherry to visualize lysosomes as neutral cargo. The AIS is live-labelled with anti-NF-CF640R (NF). White arrowhead indicates the AIS. Scale bar is 15 µm. Related to Video 9. (E and F) Representative kymographs showing trajectories of LAMP1-mCherry vesicles entering the AIS of nonAcD (E) and AcD (F) neuron shown in D. Scale is 10 s (vertical) and 2 µm (horizontal). (G) Velocity of NPY-EGFP vesicles running towards anterograde and retrograde directions in the AIS of nonAcD and AcD neurons. Mean ± SEM, four independent cultures, nonAcD neurons: n = 12 cells, AcD neurons: n = 12 cells. (H) Velocity of LAMP1-mCherry vesicles running towards anterograde and retrograde directions in the AIS of nonAcD and AcD neurons. Mean ± SEM, four independent cultures, nonAcD neurons: n = 20 cells, AcD neurons: n = 17 cells. (I and J) Run length (I) and run duration (J) of NPY-EGFP vesicles running towards anterograde and retrograde directions in the AIS of nonAcD and AcD neurons. Mean ± SEM, four independent cultures, nonAcD neurons: n = 12 cells, AcD neurons: n = 12 cells. (K and L) Number of pause and total pausing duration of NPY-EGFP vesicles travelling within the AIS of nonAcD and AcD neurons (regardless of directions). Mean ± SEM, four independent cultures, nonAcD neurons: n = 12 cells, AcD neurons: n = 12 cells. (M and N) Run length (M) and run duration (N) of LAMP1-mCherry vesicles running towards anterograde and retrograde directions in the AIS of nonAcD and AcD neurons. Mean ± SEM, four independent cultures, nonAcD neurons: n = 20 cells, AcD neurons: n = 17 cells. (O and P) Number of pause (O) and total pausing duration (P) of LAMP1-mCherry vesicles travelling within the AIS of nonAcD and AcD neurons (regardless of directions). Mean ± SEM, four independent cultures, nonAcD neurons: n = 20 cells, AcD neurons: n = 17 cells. Mann–Whitney test (two-sided): not significant (n.s.) P > 0.05, *P < 0.05.

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