Figure 6.

Dynamic colocalization of EHD1 with EEA1-positive early endosomes. A–D, Neurons were cotransfected with Cherry–EHD1 and GFP–EEA1. Time lapse was performed for 10 min duration (A, B) and 1 min duration (C, D). First frame of time lapse of part of dendrite and kymograph are shown. The time dimension is shown on the y-axis, and the retrograde direction is indicated above. A, General view of dynamic colocalization of Cherry–EHD1 (cyan) and GFP–EEA1 (red). Arrows indicate stationary vesicles and arrowheads motile compartments. Scale bar, 6 μm. B, Example of stationary (arrow) and motile (arrowhead) EHD1–EEA1-positive vesicles. C, Example of motile EHD1-positive vesicle (arrowhead) and stationary EHD1–EEA1-positive vesicles (arrows). D, Example of motile EHD1–EEA1-positive vesicle that is losing EEA1 during the time-lapse (arrowhead) and stationary EHD1–EEA1-positive vesicles (arrows). E, Example of presumed fusion of EEA1-positive vesicle with EHD1-positive vesicle (image is magnified 2× for comparing with images A–D). F, Quantification of colocalization of EHD1-positive stationary and motile vesicles with EEA1-positive stationary and motile vesicles (n = 357). EHD1 alone are shown in white, and EHD1 + EEA1 are shown in black. G, Quantification of colocalization of all EEA1-positive stationary and motile vesicles with EHD1-positive stationary and motile vesicles. EEA1 alone are shown in white, and EEA1 + EHD1 are shown in black. H–J, Fluorescence intensity changes in EHD1–EEA1-positive stationary vesicles. Examples of line traces of single vesicles EHD1 (cyan) and EEA1 (red) fluorescence intensity over 10 min time lapse (10 s interval between frames). H, Example of a line trace of fluorescence intensity of vesicle in which neither EHD1 nor EEA1 changed their intensity. I, Example of a vesicle in which EEA1 fluorescence intensity diminished while EHD1 remained constant. J, Example of vesicle in which EHD1 intensity diminished while EEA1 intensity was constant. AU, Arbitrary units.