Figure 6.

Rab5 endosomes move into developing axons and Clstn-1 knockdown inhibits transport of early endosomes from the cell body into axons. A, B, Time-lapse images of PA-GFP-Rab5c-labeled endosomes in TagRFP-CAAX-labeled RB cells in control (A) and Clstn KD (B) RB neurons. Photoactivation was in the cell body (yellow circle) and spread of photoactivated Rab5c vesicles into axons was measured as the farthest vesicle from the cell body (open arrowheads). C, Quantification of Rab5 vesicle movement into central axons. Vesicles advance more slowly in Clstn-1 knockdown compared with wild-type. p = 0.0003 (two-way ANOVA). D, Rab5 vesicles move more slowly into peripheral axons in Clstn-1 knockdown embryos compared with wild-type. p = 0.03 (two-way ANOVA). E, Time-lapse images of peripheral axon initiation in neuron in which PA-GFP-Rab5 was photoactivated in the cell body. GFP-Rab5 accumulation is present at peripheral axon initiation site (arrowhead) and fills forming growth cone. F, Time-lapse images of peripheral growth cone turning. Growth cone is initially growing up, but little Rab5 enters the upper protrusion, which retracts. Growth cone redirects to area of large Rab5 accumulation. The accumulation moves into lower branch, which extends. *Axon shaft. G, Time-lapse images of bifurcating peripheral growth cone. Rab5 moves into both branches (arrowheads) of bifurcating growth cone. Scale bars: A, B, E, 20 μm; F, G, 5 μm. Time is minutes after photoactivation.