Figure 9

RabGDI triggers membrane insertion of Robo1. Robo1 expressed in COS cells was mainly found in the perinuclear area, the endoplasmic reticulum, and the Golgi apparatus (A and B). Co-transfection of RabGDI resulted in redistribution of Robo1 to vesicular structures in the periphery of the cells (C and D). Inserts show RabGDI expression. Note that the staining intensity in the perinuclear area was markedly decreased in the presence of RabGDI. To demonstrate surface expression of Robo1 in the presence of RabGDI commissural neurons dissected from HH21/22 chicken embryos expressing either HA-Robo1-myc alone (E-G and K-M) or in combination with RabGDI (H-J and N-P) were grown in culture. Growth cones shown in (E-J) and (K-P) were taken from independent experiments. Surface expression of Robo1 was visualized by staining the N-terminal HA-tag before fixation and permeabilization (E,H,K,N). The myc staining after fixation and permeabilization revealed total Robo1 levels (F,I,L,O). The overlay of both stainings is shown in G,J,M and P, respectively. In the presence of RabGDI 27.8% (p = 0.0051) more Robo1 could be detected on the surface of commissural axons. Bar: 10 μm.