Neurons transfected with morpholinos and pCAG mGFP-Actin (Addgene #21948)
were cultured on the fibronectin-coated glass and imaged after 24 hrs using a PlanApoN 100x/1.49 oil immersion objective on an Olympus IX81 system equipped with a Hamamatsu ORCA-R2 CCD camera. Images were captured every 1 s for up to 3 min. The system was equipped with a focus drift correction mechanism and the imaging was conducted at 37 0 C without CO 2 .
Following generation of substacks of the original movie in Fiji, kymographs were generated using the MetaMorph software (Molecular Devices) using a segmented line tool (width: 5 pixels). Velocities were calculated from the kymographs using the FlowTrack code (obtained from Dr. D. Odde, University of Minnesota; (Chan and Odde 2008) in the Matlab 2007b. Retrogradely moving regions from the heat map generated of the kymograph were selected using a rectangular selection. The analysis was restricted to sections of the time-lapse series where the filopodia were attached and not dynamic.
were cultured on the fibronectin-coated glass and imaged after 24 hrs using a PlanApoN 100x/1.49 oil immersion objective on an Olympus IX81 system equipped with a Hamamatsu ORCA-R2 CCD camera. Images were captured every 1 s for up to 3 min. The system was equipped with a focus drift correction mechanism and the imaging was conducted at 37 0 C without CO 2 .
Following generation of substacks of the original movie in Fiji, kymographs were generated using the MetaMorph software (Molecular Devices) using a segmented line tool (width: 5 pixels). Velocities were calculated from the kymographs using the FlowTrack code (obtained from Dr. D. Odde, University of Minnesota; (Chan and Odde 2008) in the Matlab 2007b. Retrogradely moving regions from the heat map generated of the kymograph were selected using a rectangular selection. The analysis was restricted to sections of the time-lapse series where the filopodia were attached and not dynamic.