Nwk in live NMJs localizes to puncta that transiently interact with SNX16 endosomes. (A) elav^C155;Nwk-mRFP expression in α-Nwk IBs of Drosophila larval fillet extracts. The single asterisk indicates endogenous Nwk; the double asterisk indicates Nwk-mRFP. Molecular masses are given in kilodaltons. tub, tubulin. (B) Localization of Nwk-mRFP in live third instar NMJs. Arrows indicate Nwk-mRFP puncta. (C) Localization of Nwk-mRFP in fixed third instar NMJs. Nwk collapses to discrete structures upon fixation, and Nwk puncta are not strongly detected by α-Nwk antibodies. (D) Nwk-mRFP puncta colocalize with GFP-Rab11 at NMJs. (E) Localization of Nwk-mRFP and SNX16-GFP in live third instar larval NMJs. Time-lapse recordings of these NMJs are shown in Videos 1, 2, and 3. The white box indicates the area shown in the time-lapse images below. Arrows track a pair of Nwk-mRFP and SNX16-GFP puncta. (F) The dynamin inhibitor dynasore drives association of Nwk and SNX16 particles. The quantification shows the mean fraction of Nwk particles with a SNX16 particle within 1 µm of their center. Error bars are means ± SEM, and the number of samples averaged in each measurement is indicated at the base of each bar. **, P < 0.01. Bars, 10 µm.