In vivo single-molecule tracking of voltage-gated calcium channels with split-fluorescent proteins in crispr-engineered c. elegans

Abstract

How voltage-gated calcium channels (VGCCs) mediate signal transductions in response to changes in membrane potential is primarily studied in in vitro and ex vivo systems via biochemical and electrophysiological methods. With the emergence of single-molecule (SM) fluorescence microscopy techniques, it is now possible to characterize the molecular organization and the biophysical dynamics of ion channels in cells with precisions on the order of a few nanometers. However, performing such SM measurements within excitable tissues in intact animals is challenging. Here we describe protocols for an in vivo and tissue-specific SM imaging technique called complementation-activated light microscopy (CALM). By combining native expression of CRISPR-engineered split-fluorescent protein (split-FP) fusions and controlled fluorescence activation of split-FPs in vivo, CALM enables researchers to study the dynamics of individual calcium channels with a precision better than 30 nm directly within neuromuscular synapses of adult Caenorhabditis elegans (C. elegans) nematodes or at the sarcolemma of their body-wall muscle cells. With the availability of various split-FP spectral variants and of tissue-specific fluorescent markers, CALM can be extended to multicolor and nanoscale dynamic studies of virtually any membrane proteins and channels expressed at physiological levels in live animals.