Figure 7. Axons incubated with NGF- or semaphorin 3A-coated beads display a local increase in F_m/F_c in the region of axon immediately adjacent to the bead.

Cells were rinsed and incubated in very low NGF medium and beads for one hour prior to experiments. Fluorescence images show sites where an NGF-coated bead (A) and a sema3A-coated bead (B) are in contact with otherwise undistinguished regions of axons. Scale bar is 10 μm. (C) The distance between the mitochondria and the bead was measured and used to sort them into three groups; those in the 10 μm of axon centered on the bead (white bars), those between 5 and 50 μm away (gray bars) and those more than 50 μm from the bead (black bars). The mean F_m/F_c of axonal mitochondria was increased within 50 μm of the site of contact with an NGF bead, and this effect was even greater for mitochondria in the 10 μm region centered on the contact site. The increase in F_m/F_c near the NGF bead was partially blocked by either PI3 kinase inhibitor LY294002 or MAPK inhibitor U0126 for 20 minutes. From left to right n = 18, 22, 20, and 16 neurons. (D) The mitochondria in axons treated with sema3A-coated bead were sorted according to distance from the site of axon-bead contact as just described. The mean F_m/F_c of axonal mitochondria was also increased within 50 μm of the site of contact with a sema3A bead, and this effect was even greater for mitochondria adjacent to the contact site. The increase in F_m/F_c near the sema3A bead was partially blocked by PI3 kinase inhibitor LY294002 or MAPK inhibitor U0126. Heat denaturation of both kinds of beads eliminated their effect on F_m/F_c. From left to right n= 18, 17, 21 and 16 neurons.*p< 0.05, ** p< 0.01. Error bars show S.E.M.