A genomic region containing genomic lap and its full upstream and downstream untranslated regions has been tagged at the C-terminal end with the synthetic Flag-Tetracysteine (4C) peptide using recombineering. 4C is comprised of the Tag:FLAG peptide and a FlAsH binding tetracysteine tag (Tag:CALI(TC)).
When larvae expressing lapN.4C.T:Zzzz\FLAG,T:Zzzz\TC in a lap1/lapsd-3 mutant background are pretreated with FlAsH-FALI and photoinactivated to inactive lap, they show synaptic vesicle endocytosis defects (reduced dye internalisation) in the NMJ when stimulated with potassium. Unlike in wild type and unactivated controls, only small punctae of dye are detected within the synaptic boutons. Inactivation of lap has no effect on synaptic vesicle exocytosis or transmitter release. When lap is inactivated just prior to potassium stimulation boutons are seen that display acute synaptic membrane protrusions ("pseudopodia"), and these are not observed in controls. Normal sized synaptic vesicles are rarely observed and two categories of phenotype are seen 1) boutons packed with multiple intermediate-sized cisternae or 2) boutons exhibiting massive synaptic vesicle depletion, but containing very large cisternae and membrane infoldings. Large balloon-like membrane infoldings that remain contiguous with the plasma membrane and may also contain smaller vesicle-like structures, are also seen.
lapC.4C.Tag:FLAG,Tag:CALI(TC) partially rescues lapsd-3/lap1
Expression of lapC.4C.T:Zzzz\FLAG,T:Zzzz\TC partially rescues the reduction in excitatory junctional potential (EJP) amplitude seen in lap1/lapsd-3 mutants. The larval lethality is also rescued.