FlAsH-FALI Inactivation of a Protein at the Third-Instar Neuromuscular Junction

Korte inhoud:INTRODUCTION Fluorescein-assisted light inactivation (FALI) is a powerful method for studying acute loss of protein function, even if the corresponding mutations lead to early lethality. In this protocol, FALI is mediated by the membrane-permeable FlAsH (4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein) compound that binds with high specificity to the genetically encoded tetracysteine tag and thus allows the inactivation of protein function in vivo with exquisite spatial (<40 Å) and temporal (<30 sec) resolution. It also enables the analysis of kinetically distinct processes such as synaptic vesicle exocytosis and endocytosis. This protocol describes efficient inactivation of a protein using FlAsH-FALI at the neuromuscular junction (NMJ) of third-instar larvae. Note that FlAsH-FALI in other tissues is also theoretically possible with minor adaptations to the protocol described here. We explain controls for positional effects, for unspecific FlAsH binding to endogenous proteins, and for phototoxicity. Following FlAsH-FALI, protein function can be studied using a number of secondary assays, including electrophysiology, immunohistochemistry, and electron microscopy or FM1-43 labeling of synaptic vesicle pools.

Jaar van publicatie:2011

Reviewstatus:Peerreview