Figure 11.

Depletion of DCX and DCLK reduces the depth of microtubule invasion into growth cone filopodia. We performed time-lapse imaging of EGFP-EB3 to evaluate microtubule invasion into growth cone filopodia. Neurons were co-transfected with EGFP-EB3 cDNA and either control siRNA or DCX+DCLK siRNA, cultured for 3 days and then re-plated in preparation for time-lapse imaging of EB3 comets (see Materials and Methods for details). (A and B) Still-frames from time-lapse video sequences of a control and a DCX+DCLK-depleted growth cone. Numerous EB3 comets are apparent in the growth cones, some of which have entered filopodia (white arrowheads). To quantify the depth of comet invasion into filopodia, we used the time-lapse sequences to track and record the most distal portion of the filopodia reached by the EGFP-EB3 comet. The quantitative analyses, based on analyses of 13 control and 13 depleted growth cones, are depicted in the graph. On average, the relative distance that microtubules invaded filopodia was significantly less in depleted neurons compared with control neurons (P < 0.0001, χ²-test). Scale bar = 6 μm.