Visualizing endocytic recycling and trafficking in live neurons by subdiffractional tracking of internalized molecules

Abstract

Our understanding of endocytic pathway dynamics is restricted by the diffraction limit of light microscopy. Although super-resolution techniques can overcome this issue, highly crowded cellular environments, such as nerve terminals, can also dramatically limit the tracking of multiple endocytic vesicles such as synaptic vesicles (SVs), which in turn restricts the analytical dissection of their discrete diffusional and transport states. We recently introduced a pulse-chase technique for subdiffractional tracking of internalized molecules (sdTIM) that allows the visualization of fluorescently tagged molecules trapped in individual signaling endosomes and SVs in presynapses or axons with 30- to 50-nm localization precision. We originally developed this approach for tracking single molecules of botulinum neurotoxin type A, which undergoes activity-dependent internalization and retrograde transport in autophagosomes. This method was then adapted to localize the signaling endosomes containing cholera toxin subunit-B that undergo retrograde transport in axons and to track SVs in the crowded environment of hippocampal presynapses. We describe (i) the construction of a custom-made microfluidic device that enables control over neuronal orientation; (ii) the 3D printing of a perfusion system for sdTIM experiments performed on glass-bottom dishes; (iii) the dissection, culturing and transfection of hippocampal neurons in microfluidic devices; and (iv) guidance on how to perform the pulse-chase experiments and data analysis. In addition, we describe the use of single-molecule-tracking analytical tools to reveal the average and the heterogeneous single-molecule mobility behaviors. We also discuss alternative reagents and equipment that can, in principle, be used for sdTIM experiments and describe how to adapt sdTIM to image nanocluster formation and/or tubulation in early endosomes during sorting events. The procedures described in this protocol take ∼1 week.

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

“…By applying super-resolution imaging approaches to study their dynamic nanoscale organization and vesicle dynamics (Joensuu et al, 2017;Joensuu et al, 2016), a more comprehensive picture of the dynamics of the exocytic molecular machinery will be developed, thereby revealing the principles that govern regulated exocytosis.…”

p110δ PI3-Kinase Inhibition Perturbs APP and TNFα Trafficking, Reduces Plaque Burden, Dampens Neuroinflammation, and Prevents Cognitive Decline in an Alzheimer's Disease Mouse Model

“…In both situations, the glass surfaces were coated with poly-L-lysine (Sigma-Aldrich). Primary neuronal culture was performed as described previously (Joensuu et al, 2017). Briefly, the dissected hippocampi were digested with 0.25% trypsin-EDTA and DNase I for 20 min at 37°C, followed by mechanical trituration.…”

Radial contractility of Actomyosin-II rings facilitates cargo trafficking and maintains axonal structural stability following cargo-induced transient axonal expansion

“…1c). This was followed by a thorough wash in culture medium to remove the excess fluorescent probe and confocal time-lapse imaging and automatic tracing of the fluorescently tagged cargoes as previously described (Joensuu et al, 2017; Wang et al, 2016; Wang et al, 2015). To investigate different stages of axonal trafficking, live-imaging was performed in two distinct axonal regions: (1) the axon shafts adjacent to the soma chamber (Fig.…”

“…To test this hypothesis, we examined the diameter of axons in the presence or absence of cargoes at the ultrastructural level. In order to eliminate confounding factors related to the analysis of dendrites, experiments were only performed on axonal bundles formed within the channels of microfluidic devices (Joensuu et al, 2017; Wang et al, 2016; Wang et al, 2015), as shown in Fig. 2a.…”

“…To objectively and quantitatively analyze the effect of the contractility on the two motion states, we employed a two-state hidden Markov model (HMM) to annotate these CTB trajectories into undirected (D) and directed (DV) states (Fig. 7k), as previously described (Joensuu et al, 2017). The separating efficacy of this model was demonstrated by the fact that the step size of the undirected (0.1168 ± 0.45 µm) and directed transport states (0.4352 ± 1.53 µm) were distinct from each other.…”