RBO facilitates FM1-43-dye endocytosis at the neuromuscular synapse. (A)
Schematic of dye-loading, temperature and imaging protocols used at the larval
NMJ. In protocol 1, FM1-43 was loaded for 2 minutes with 60 mM [K+]
at 25°C and then unloaded for 2 minutes with 60 mM [K+] at
37°C. In protocol 2, following a 10-minute incubation at 37°C, dye was
loaded for 2 minutes at 37°C and then unloaded for 2 minutes at 25°C.
(B) Representative images of wild type (OR), syx3-69 and
rbots single mutants, and the
rbots;syx3-69 double mutant following
protocol 1. (C) Top three panels show representative images comparing FM1-43
loading and unloading following protocol 2. Bottom panel shows
rbots;syx3-69 37°C unloading after
5 minutes of loading at 25°C. Scale bars: 10 μm. (D) Quantification of
loading (L) and unloading (UL) with both protocols. Error bars show mean
± s.e.m. ***Significance of P<0.001.