Fig. 7

Lam1 Contact Directs Kif5c560-YFP to the Contacting Neurite In Vitro and In Vivo

(A–C) RGCs were dissociated from ath5:GAP-RFP embryos injected with Kif5c560-YFP mRNA at the one-cell stage, plated on poly-L-lysine with scattered 1 μM Lam1-coated beads (A and B, pseudocolored yellow), or on islands of Lam1 stained with Texas red dye (C, magenta substrate). When a single neurite contacted Lam1 (A), this quickly caused the YFP signal to accumulate specifically in that neurite (arrow). When multiple neurites contacted Lam1 (B and C), this caused the YFP signal to accumulate specifically in Lam1-contacting neurites, and to oscillate preferentially between these neurites (arrows). (Di) Blastomeres were transplanted from ath5:GAP-RFP transgenic embryos that were injected with Kif5c560-YFP mRNA into lamα1 morphant embryos or mosaic embryos grown to 24 hpf, and 6 μM Lam1-coated beads were implanted into the right eye. (Dii) The highlighted RGC exhibits typical Kif5c560-YFP oscillations during Stage 2 (cyan phase, marked by red arrowheads). Upon contact with the Lam1 bead (white arrowhead; bead is pseudocolored yellow), the contacting process is stabilized and does not retract, and YFP signal is concentrated at the contact point. This neurite then extends to form the axon (green phase). Note that the individual confocal z-slices were cropped to remove signal not associated with the cell. A reconstruction of the uncropped frames is shown in Movie S16. (E) Kymograph of the cell shown in (D), demonstrating the specific Kif5c560-YFP signal accumulation upon neurite contact with the Lam1-coated bead (contact marked by white arrow; bead pseudocolored yellow). Frames are taken from Movie S13. Contact with Lam1 Directs Specific Kif5c560-YFP Accumulation at the Tip of a Neurite In Vitro (Part 1), Movie S14. Contact with Lam1 Directs Specific Kif5c560-YFP Accumulation at the Tip of a Neurite In Vitro (Part 2), Movie S15. Contact with Lam1 Directs Specific Kif5c560-YFP Accumulation at the Tip of a Neurite In Vitro (Part 3) and [Movie S16. Lam1 Contact Directs Stable and Specific Kif5c560-YFP Accumulation in the Contacting Neurite Followed by Axon Extension In Vivo]. Time is shown in hr:min; scale bars = 10 μM.