Fig. 4. Time-lapse microscopy of retinal growth cone responses to soluble EphB-ECDs or Fc protein applied by micropipette. Time in minutes relative to beginning of reagent application (0) is indicated in top left of panels. (A) Retinal axon elongation and growth cone behavior before and after application of soluble Fc protein. The growth cone was unaffected and continued towards the micropipette tip (outline in upper right). The 4th panel shows the fluorescent marker gradient after expulsion of reagent from the pipette. (B) Growth cone collapse triggered by EphB1-ECD. The micropipette tip was located at the top approx. 180 µm away from the growth cone and the direction of EphB1-ECD dispersal is indicated by the arrow. (C) Example of cessation of growth cone motile activity after application of EphB2-ECD. Black arrow indicates direction of protein dispersal from the micropipette tip located 70 µm away. Within 5 minutes of application, the growth cone ceased filopodial and lamellipodial movements (black arrowheads) and remained in this ‘frozen’ state without retraction. Cellular material (white arrowhead) accumulated within the growth cone body, indicating continuing axonal transport. Scale bars, 10 µm.