<em>In Vivo</em> Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Abstract

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

Need for speed: Super-resolving the dynamic nanoclustering of syntaxin-1 at exocytic fusion sites

“…Syntaxin-1 is organized as nanoclusters in the NMJs of Drosophila larvae (Bademosi et al, 2017;Ullrich et al, 2015). To understand how activity-dependent neurotransmitter release alters the nanocluster organization of syntaxin-1 in the presynaptic membrane, Bademosi et al (2017Bademosi et al ( , 2018aBademosi et al ( , 2018b) developed a new protocol to image syntaxin-1-mEos2 in the NMJs of fixed and live third instar Drosophila larvae (Fig. 2).…”

“…This technique allows for the quantification of the size distribution of protein clusters on the plasma membrane (Klar and Hell, 1999;Sieber et al, 2006;Sieber et al, 2007;van den Bogaart et al, 2011). PALM allows for the characterization of the size and distribution of protein clusters in fixed and live cells and allows single particle tracking (sptPALM) in live cells (Bademosi et al, 2018a;Bademosi et al, 2017;Bademosi et al, 2018b;Manley et al, 2008;Vanhauwaert et al, 2017;Yang et al, 2012). Unlike PALM, stochastic optical reconstruction microscopy (STORM) uses photoswitchable organic fluorophores to immunolabel endogenous proteins (Bar-On et al, 2012;Rust et al, 2006).…”

Tracking Single Molecule Dynamics in the AdultDrosophilaBrain

“…Here, synaptic vesicles in the fly larval neuromuscular junction are more readily docked and primed compared to chromaffin cells, suggesting important differences in the physiological relevance of the two systems for studying neurotransmission. We recently described single molecule imaging in intact motor nerve terminals of filleted Drosophila larvae (Bademosi et al, 2016(Bademosi et al, , 2018a. In that study we tagged the pre-synaptic protein syntaxin1A (Sx1a) with photoconvertible mEos2 and found that genetic stimulation of motoneurons resulted in increased mobility of Sx1A in the motor nerve terminals, suggesting increased mobilization of the presynaptic machinery when neurons are activated.…”

Tracking single molecule dynamics in the anesthetizedDrosophilabrain

“…Recently, we have described single molecule imaging in motor nerve terminals of filleted larvae of the fruit fly Drosophila melanogaster (Bademosi et al, 2018a, 2016). In that study we tagged the pre-synaptic protein syntaxin1A (Sx1a) with photoconvertible mEos2 and found that genetic activation of motoneurons resulted in increased mobility of Sx1A in the motor nerve terminals, suggesting increased mobilization of the presynaptic machinery when neurons are activated.…”