TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Abstract

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in health and disease. Genetically-encoded pH-sensitive probes directly targeted to synaptic vesicles and Total Internal Reflection Fluorescence Microscopy (TIRFM) provide the spatio-temporal resolution necessary to follow vesicle dynamics. The evanescent field generated by total internal reflection can only excite fluorophores placed in a thin layer (<150 nm) above the glass cover on which cells adhere, exactly where the processes of exo-endocytosis take place. The resulting high-contrast images are ideally suited for vesicles tracking and quantitative analysis of fusion events. In this protocol, SH-SY5Y human neuroblastoma cells are proposed as a valuable model for studying neurotransmitter release at the single-vesicle level by TIRFM, because of their flat surface and the presence of dispersed vesicles. The methods for growing SH-SY5Y as adherent cells and for transfecting them with synapto-pHluorin are provided, as well as the technique to perform TIRFM and imaging. Finally, a strategy aiming to select, count, and analyze fusion events at whole-cell and single-vesicle levels is presented. To validate the imaging procedure and data analysis approach, the dynamics of pHluorin-tagged vesicles are analyzed under resting and stimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causes a parallel raise of the net fluorescence signal recorded in whole cell. Single-vesicle analysis reveals modifications of fusion-event behavior (increased peak height and width). These data suggest that potassium depolarization not only induces a massive neurotransmitter release but also modifies the mechanism of vesicle fusion and recycling. With the appropriate fluorescent probe, this technique can be employed in different cellular systems to dissect the mechanisms of constitutive and stimulated secretion.

DOPAL derived alpha-synuclein oligomers impair synaptic vesicles physiological function

“…Fluorescence intensity variation was measured in BE(2)-M17 cells overexpressing aS, and treated with 100 μM DOPAL, to monitor permeabilisation events. Normally, only vesicles fusing with the plasma membrane become fluorescent and then, after endocytosis, they are immediately switched off32. If DOPAL alters vesicles permeability, we expect an increase in the intensity, and in the persistence of the fluorescent signal associated with the vesicular structure.…”

“…To assess whether the DOPAL-induced oligomerization of aS may cause synaptic vesicle damage, we used a pH-sensitive fluorescent protein directly targeted to synaptic vesicles (synaptobrevin2-pHluorin), as a reporter in a total internal reflection fluorescence microscopy (TIRFM) experiment3233 in live cells. Synaptobrevin2-pHluorin fluorescence intensity increases if pH becomes basic, i.e.…”

The LRRK2 G2385R variant is a partial loss-of-function mutation that affects synaptic vesicle trafficking through altered protein interactions

“…We used the sypHy assay with the SH-SY5Y line which expresses LRRK2 together with a panel of SV- associated proteins. We determined that this line shuffles synapto-pHluorin reporter (sypHy) to VAMP2 positive vesicles (Supplementary Figure 1A,B ) and exposes sypHy on the membrane as a consequence of SV fusion events 25 . Consequently, we over-expressed a panel of LRRK2 derived expression constructs together with sypHy reporter in the SH-SY5Y line (Fig.…”

The LRRK2 N-terminal domain influences vesicle trafficking: impact of the E193K variant

“…Impact of LRRK2 N-terminal domain on vesicle trafficking. Since the N-terminal Armadillo domain is involved in LRRK2 supra-molecular organization 19 , we further investigate the functional role of LRRK2 N-terminal domain by means of TIRF microscopy (TIRFM) coupled with a synaptopHluorin assay as previously described 20 . For this purpose, we over-expressed in N2A neuronal line the sypHy reporter together with a panel of RFP-tagged LRRK2 derived constructs: LRRK2 full-length, LRRK2 lacking the first 913 amino acids (hereinafter LRRK2ΔN-term) as well as a complementary a deletion construct LRRK2 aa 1-983, containing the N-terminal Armadillo and Ankyrin domains (hereinafter N-terminal domain) ( Fig.…”

“…(2020) 10:3799 | https://doi.org/10.1038/s41598-020-60834-5 www.nature.com/scientificreports www.nature.com/scientificreports/ Time lapse microscopy by total internal reflection fluorescence microscopy (TIRFM). We imaged transfected cells 48 h after transfection via TIRF microscopy as in 20 . The microscope (Carl Zeiss Inc.) was equipped with an Argon laser at 25•C using a 100× 1.45 numerical aperture (NA) oil immersion objective.…”