Fig 5. Retrograde trafficking of tagged Nrg in Lis1 knock down and Glued^Δ96B mutant background.

(a) Axonal transport of Nrg^eGFP vesicles in GFs with postdevelopmental Lis1 knock down. Kymographs showing trajectory of Nrg^eGFP vesicles in a photobleached area flanked by unbleached areas in the GF axon over a 10 min time period. Videos were obtained at one frame per second. Ascending and descending slopes represent retrograde and anterograde vesicles, respectively. Vertical lines are stationary vesicles. Scale bar is 4 μm. (b) Quantification of Nrg^eGFP vesicles in wild type and in flies expressing UAS-Lis1^RNAiH with GF-Split-Gal4. The lower expression, magnification and one frame per second imaging rate allows to quantify only slow (< 1μm/sec) moving Nrg^eGFP vesicles. The average flux (left graph) are the average numbers of retrograde vesicles that entered the photobleached area per minute, with n indicating the number of GFs analyzed. The maximum net displacement of retrograde Nrg^eGFP vesicles during the imaging period was analyzed and the average travel velocity was expressed in μm/second. Ten and 15 axons were assessed in wild type and Lis1 knock down background, respectively, with n indicating the number of vesicles analyzed. The average number of stationary vesicles per micron axon length was assessed in the unbleached regions. A total axon length of 169 μm and 180 μm was assessed in wild type and Lis1 knock down background, respectively, with n indicating the number axons analyzed. Error bars show standard error of the mean and significant differences are indicated with asterisks (*** = p<0.001, Kruskal-Wallis One Way Analysis Of Variance and Mann-Whitney Rank Sum Test). (c) Expression of Nrg^mCherry without and with UAS-Lis1^RNAiH and UAS-Glued^Δ96B using the R68A06 driver line. Left panel, quantification of fast (> 1μm/sec) retrograde vesicles in eight (n) GF axons of each genotype. Error bars show standard error of the mean and significant differences are indicated with asterisks (*** = p<0.001, Kruskal-Wallis One Way Analysis Of Variance and Mann-Whitney Rank Sum Test). Right panel, kymographs of Nrg^mCherry vesicles without and with Lis1^RNAiH and Glued^Δ96B expression. Videos were obtained at 4 frames per second and a 2 minute time period after phototobleaching is depicted. Some fast retrograde vesicles are indicated with arrows. Scale bar is 10 μm.