Spectrin mutations disrupt the axonal transport of synaptic vesicles. (A) Larvae overexpressing wild-type fly β-spectrin (elav-GAL4/+; FSPWT/+) exhibited normal posture when crawling. (B and C) Mutant larvae (elav-GAL4/+; FSPAM/+ and elav-GAL4/+; FSPGM/+) developed the posterior paralysis phenotype common to axonal transport mutants in Drosophila. Videos of larval crawling can be seen in Videos 1–3. (D–F) The distribution of vesicular syt-GFP along larval segmental axons. Expression of the American and German mutant spectrins cause large syt-GFP accumulations (E and F) when compared with the punctuated pattern seen in axons expressing the wild-type protein (D). Large axonal swellings are indicated by arrows. (G) Quantification of the number of syt-GFP accumulations in 50 µm of axonal length. At least 15 segmental nerves were analyzed per genotype. Data represent mean ± SEM (error bars). Significant differences in the number of axonal jams between wild-type and mutants are denoted by asterisks (***, P < 0.001). (H) Fluorescent images of segmental nerves show the accumulation of the axonal transport cargoes syt and CSP within the axonal jams in segmental nerves from mutant but not wild-type fly β-spectrin larvae. (I) Fluorescent images of segmental axons show the accumulation of the motor protein dynein and the synaptic vesicle integral membrane protein syt in mutant but not wild-type fly β-spectrin larvae. (J) Representative kymographs showing the motion of individual syt-GFP particles in larval segmental axons as a function of time. Kymographs corresponding to larvae expressing wild-type fly β-spectrin (elav-GAL4/+; FSPWT/+) show fluorescent vesicles moving in one defined direction (diagonal lines), and few stationary vesicles appear as vertical lines (I and II). Kymographs from mutant spectrin larvae show stationary GFP particles, whereas the majority of those moving undergo numerous reversals in the direction of movement that appear as zigzag lines (III and IV). Bars, 10 µm.