Figure 2. Acute inactivation of LAP blocks SV recycling but does not affect SV exocytosis.

A) FM 1-43 dye uptake at third instar larval NMJs is normal in both wild-type or Clap4C-rescued lap mutant flies following 1-min nerve stimulation in the presence of the dye and without 488-nm light exposure. However, acute inactivation of LAP by 488-nm light illumination leads to the appearance of dye-positive punctate (note the normal uptake in wild-type NMJs following the light exposure). Presumably, these are large cisternae that form when CME is blocked. lap = lap¹/lap^sd3. B) For analysis of SV exocytosis we used the lipophilic dye FM 4-64 because it is not bleached by the 488-nm light used to inactivate LAP. FM 4-64 shows typical SV endocytosis in the presence of high K⁺. After the dye is loaded we inactivated LAP and then give a second round of stimulation in the absence of dye, FM 4-64 is exocytosed from the boutons and washed away. Inactivation of LAP has no effect on the ability of the FM dye to be exocytosed. A and B) Protocol for analysis of SV endocytosis and exocytosis using the lipophilic dye FM 1-43 or FM 4-64, respectively. For details, see Materials and Methods. Scale bars = 10 µm.