Figure 4.
The AIS of AcD neurons serves as an efficient barrier, preventing entry of dendritic cargo into the axon. (A) Representative images of nonAcD (left) and AcD (right) neurons expressing a presynaptic vesicle marker EGFP-Rab3A, the AIS is live-labeled with anti-NF-CF640R (NF). White dashed line indicates the analyzed area; white arrowhead indicates the AIS; red, yellow, and cyan arrowheads indicate the stem dendrite, AcD, and regular dendrite of AcD neuron, respectively. Scale bar is 15 µm. Related to Video 6. (B) Representative kymographs showing trajectories of EGFP-Rab3A vesicles entering the AIS of nonAcD neuron shown in A, corresponding to area indicated by white dashed line. Scale is 10 s (vertical) and 2 µm (horizontal). (C) Representative kymographs showing trajectories of EGFP-Rab3A vesicles moving in the stem dendrite (Stem D), AIS and regular dendrite (Regular D) of the AcD neuron shown in A, corresponding to area indicated by white dashed line. Scale is 10 s (vertical) and 2 µm (horizontal). (D) Representative images of nonAcD (left) and AcD (right) neurons labelled with anti-NF-CF640R (NF) for AIS and Transferrin-Alexa568 for TfRs as dendritic cargo. White dashed line indicates analyzed area; white arrowhead indicates the AIS; cyan arrowhead indicates regular dendrite of nonAcD neuron; yellow and red arrowhead indicates the AcD and stem dendrite of AcD neuron, respectively. Scale bar is 15 µm. Relate to Video 7. (E) Representative kymographs showing trajectories of TfR vesicles entering and moving in the AIS, axon and regular dendrite (Regular D) of nonAcD neuron shown in D, corresponding to the area indicated by white dashed line. Scale is 5 s (vertical) and 2 µm (horizontal). (F) Representative kymographs showing trajectories of TfR vesicles moving in the AIS, axon, stem dendrite (Stem D) and AcD of the AcD neuron shown in D, corresponding to the area indicated by white dashed line. Scale is 5 s (vertical) and 2 µm (horizontal). (G and H) Motility of TfR vesicles in the AIS of nonAcD neuron, the AIS, stem dendrite (Stem D) and AcD of AcD neuron, and the regular dendrite (Regular D) of both nonAcD and AcD neurons. (G) Percentage of mobile and stationary TfR vesicles. (H) Number of mobile TfR vesicles per cell. Mean ± SEM, five independent cultures, AIS (nonAcD) n = 25 cells, AIS (AcD) n = 23 cells, Stem D n = 23 cells, AcD n = 19 cells, Regular D n = 56 cells. (I) Percentage of TfR vesicles running towards anterograde and retrograde directions in the AIS of nonAcD neuron, the AIS, stem dendrite (Stem D) and AcD of AcD neuron, and the regular dendrite (Regular D) of both nonAcD and AcD neurons. Mean ± SEM, five independent cultures, AIS (nonAcD) n = 25 cells, AIS (AcD) n = 23 cells, Stem D n = 23 cells, AcD n = 19 cells, Regular D n = 56 cells. (J) Percentage of time a mobile TfR vesicle is running, pausing or passively moving during anterograde transport. Mean ± SEM, five independent cultures, AIS (nonAcD) n = 25 cells, AIS (AcD) n = 23 cells, Stem D n = 23 cells, AcD n = 19 cells, Regular D n = 56 cells. One-way ANOVA with Tukey’s multiple comparisons test, no significance (n.s.) P > 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (K and L) Motility of EGFP-Rab3A vesicles in the AIS of nonAcD neuron, the AIS and stem dendrite (Stem D) of AcD neuron, and the regular dendrite (Regular D) of both nonAcD and AcD neurons. (K) Percentage of mobile and stationary EGFP-Rab3A vesicles. (L) Number of mobile EGFP-Rab3A vesicles per cell. Mean ± SEM, seven independent cultures, AIS (nonAcD): n = 42 cells, AIS (AcD) n = 42 cells, Stem D: n = 28 cells, Regular D: n = 27 cells. (M) Percentage of EGFP-Rab3A vesicles running towards anterograde and retrograde directions in the AIS of nonAcD neuron, the AIS and stem dendrite (Stem D) of AcD neuron, and the regular dendrite (Regular D) of both nonAcD and AcD neurons. Mean ± SEM, seven independent cultures, AIS (nonAcD): n = 42 cells, AIS (AcD) n = 42 cells, Stem D: n = 28 cells, Regular D: n = 27 cells. (N) Percentage of time a mobile Rab3A vesicle that is running, pausing or passively moving during anterograde transport. Mean ± SEM, seven independent cultures, AIS (nonAcD) n = 42 cells, AIS (AcD) n = 42 cells, Stem D n = 28 cells, Regular D n = 27 cells. (O–Q) Average length (O), duration (P) and velocity (Q) of EGFP-Rab3A vesicles running towards anterograde and retrograde direction within the AIS of nonAcD neuron, the AIS and stem dendrite (Stem D) of AcD neuron, and the regular dendrite (Regular D) of both nonAcD and AcD neurons. Mean ± SEM, seven independent cultures, Anterograde: AIS (nonAcD) n = 42 cells, AIS (AcD) n = 41 cells, Stem D n = 28 cells, Regular D n = 26 cells, Retrograde: AIS (nonAcD) n = 25 cells, AIS (AcD) n = 37 cells, Stem D n = 17 cells, Regular D n = 20 cells. More detailed statistical information see Data S1.

The AIS of AcD neurons serves as an efficient barrier, preventing entry of dendritic cargo into the axon. (A) Representative images of nonAcD (left) and AcD (right) neurons expressing a presynaptic vesicle marker EGFP-Rab3A, the AIS is live-labeled with anti-NF-CF640R (NF). White dashed line indicates the analyzed area; white arrowhead indicates the AIS; red, yellow, and cyan arrowheads indicate the stem dendrite, AcD, and regular dendrite of AcD neuron, respectively. Scale bar is 15 µm. Related to Video 6. (B) Representative kymographs showing trajectories of EGFP-Rab3A vesicles entering the AIS of nonAcD neuron shown in A, corresponding to area indicated by white dashed line. Scale is 10 s (vertical) and 2 µm (horizontal). (C) Representative kymographs showing trajectories of EGFP-Rab3A vesicles moving in the stem dendrite (Stem D), AIS and regular dendrite (Regular D) of the AcD neuron shown in A, corresponding to area indicated by white dashed line. Scale is 10 s (vertical) and 2 µm (horizontal). (D) Representative images of nonAcD (left) and AcD (right) neurons labelled with anti-NF-CF640R (NF) for AIS and Transferrin-Alexa568 for TfRs as dendritic cargo. White dashed line indicates analyzed area; white arrowhead indicates the AIS; cyan arrowhead indicates regular dendrite of nonAcD neuron; yellow and red arrowhead indicates the AcD and stem dendrite of AcD neuron, respectively. Scale bar is 15 µm. Relate to Video 7. (E) Representative kymographs showing trajectories of TfR vesicles entering and moving in the AIS, axon and regular dendrite (Regular D) of nonAcD neuron shown in D, corresponding to the area indicated by white dashed line. Scale is 5 s (vertical) and 2 µm (horizontal). (F) Representative kymographs showing trajectories of TfR vesicles moving in the AIS, axon, stem dendrite (Stem D) and AcD of the AcD neuron shown in D, corresponding to the area indicated by white dashed line. Scale is 5 s (vertical) and 2 µm (horizontal). (G and H) Motility of TfR vesicles in the AIS of nonAcD neuron, the AIS, stem dendrite (Stem D) and AcD of AcD neuron, and the regular dendrite (Regular D) of both nonAcD and AcD neurons. (G) Percentage of mobile and stationary TfR vesicles. (H) Number of mobile TfR vesicles per cell. Mean ± SEM, five independent cultures, AIS (nonAcD) n = 25 cells, AIS (AcD) n = 23 cells, Stem D n = 23 cells, AcD n = 19 cells, Regular D n = 56 cells. (I) Percentage of TfR vesicles running towards anterograde and retrograde directions in the AIS of nonAcD neuron, the AIS, stem dendrite (Stem D) and AcD of AcD neuron, and the regular dendrite (Regular D) of both nonAcD and AcD neurons. Mean ± SEM, five independent cultures, AIS (nonAcD) n = 25 cells, AIS (AcD) n = 23 cells, Stem D n = 23 cells, AcD n = 19 cells, Regular D n = 56 cells. (J) Percentage of time a mobile TfR vesicle is running, pausing or passively moving during anterograde transport. Mean ± SEM, five independent cultures, AIS (nonAcD) n = 25 cells, AIS (AcD) n = 23 cells, Stem D n = 23 cells, AcD n = 19 cells, Regular D n = 56 cells. One-way ANOVA with Tukey’s multiple comparisons test, no significance (n.s.) P > 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (K and L) Motility of EGFP-Rab3A vesicles in the AIS of nonAcD neuron, the AIS and stem dendrite (Stem D) of AcD neuron, and the regular dendrite (Regular D) of both nonAcD and AcD neurons. (K) Percentage of mobile and stationary EGFP-Rab3A vesicles. (L) Number of mobile EGFP-Rab3A vesicles per cell. Mean ± SEM, seven independent cultures, AIS (nonAcD): n = 42 cells, AIS (AcD) n = 42 cells, Stem D: n = 28 cells, Regular D: n = 27 cells. (M) Percentage of EGFP-Rab3A vesicles running towards anterograde and retrograde directions in the AIS of nonAcD neuron, the AIS and stem dendrite (Stem D) of AcD neuron, and the regular dendrite (Regular D) of both nonAcD and AcD neurons. Mean ± SEM, seven independent cultures, AIS (nonAcD): n = 42 cells, AIS (AcD) n = 42 cells, Stem D: n = 28 cells, Regular D: n = 27 cells. (N) Percentage of time a mobile Rab3A vesicle that is running, pausing or passively moving during anterograde transport. Mean ± SEM, seven independent cultures, AIS (nonAcD) n = 42 cells, AIS (AcD) n = 42 cells, Stem D n = 28 cells, Regular D n = 27 cells. (O–Q) Average length (O), duration (P) and velocity (Q) of EGFP-Rab3A vesicles running towards anterograde and retrograde direction within the AIS of nonAcD neuron, the AIS and stem dendrite (Stem D) of AcD neuron, and the regular dendrite (Regular D) of both nonAcD and AcD neurons. Mean ± SEM, seven independent cultures, Anterograde: AIS (nonAcD) n = 42 cells, AIS (AcD) n = 41 cells, Stem D n = 28 cells, Regular D n = 26 cells, Retrograde: AIS (nonAcD) n = 25 cells, AIS (AcD) n = 37 cells, Stem D n = 17 cells, Regular D n = 20 cells. More detailed statistical information see Data S1.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal