Antibody-driven capture of synaptic vesicle proteins on the plasma membrane enables the analysis of their interactions with other synaptic proteins

Katharina N Richter,Christina Patzelt,Silvio O Rizzoli,Nhu T N Phan

doi:10.1038/s41598-019-45729-4

Katharina N Richter, Christina Patzelt + Show 2 more

Open Access

https://doi.org/10.1038/s41598-019-45729-4

Copy DOI

Abstract

Many organelles from the secretory pathway fuse to the plasma membrane, to exocytose different cargoes. Their proteins are then retrieved from the plasma membrane by endocytosis, and the organelles are re-formed. It is generally unclear whether the organelle proteins colocalize when they are on the plasma membrane, or whether they disperse. To address this, we generated here a new approach, which we tested on synaptic vesicles, organelles that are known to exo- and endocytose frequently. We tagged the synaptotagmin molecules of newly exocytosed vesicles using clusters of primary and secondary antibodies targeted against the luminal domains of these molecules. The antibody clusters are too large for endocytosis, and thus sequestered the synaptotagmin molecules on the plasma membrane. Immunostainings for other synaptic molecules then revealed whether they colocalized with the sequestered synaptotagmin molecules. We suggest that such assays may be in the future extended to other cell types and other organelles.

Highlights

The question of whether cellular proteins are able to interact or colocalize with each other has been solved by a multitude of biochemical assays, ranging from co-immunoprecipitation, co-fractionation or other gel- and blot-based binding assays[1,2] to optical methods such as fluorescent resonance energy transfer (FRET3) or bimolecular fluorescence complementation[4]
Organelles can be isolated and purified[5,6], and the interactions between the molecules can be tested by biochemical tools[7,8], but this approach does not reveal the potential colocalization of the proteins during the steps between exocytosis and endocytosis, when the organelle molecules are fused to the plasma membrane
The optical methods mentioned above could in principle be employed, but they generally do not offer the resolution needed to differentiate between molecules on the plasma membrane, or molecules on organelles that are docked to the plasma membrane

Summary

Introduction

The question of whether cellular proteins are able to interact or colocalize with each other has been solved by a multitude of biochemical assays, ranging from co-immunoprecipitation, co-fractionation or other gel- and blot-based binding assays[1,2] to optical methods such as fluorescent resonance energy transfer (FRET3) or bimolecular fluorescence complementation[4]. Super-resolution imaging could be performed to address this[9], but one would still have difficulties differentiating between membrane-bound and organelle-bound molecules To address this issue, we decided to generate an assay in which we immobilize or sequester the proteins of interest on the plasma www.nature.com/scientificreports/. The preferred biological system was the synaptic bouton, where synaptic vesicles regularly fuse to the plasma membrane to release their neurotransmitters, and are endocytosed and refilled with neurotransmitter[11,12] This system relies on accurate exo- and endocytosis, and was especially convenient for the present investigation, since numerous imaging tools are available for the synapse, including a variety of antibodies for synaptic proteins (e.g.13,14). The antibodies can be taken up by vesicles recycling both during stimulated activity and spontaneously[24], and do not seem to affect the vesicles in the short- or medium-term (a few days25), a rabbit polyclonal antibody against the same target has been shown to perturb synaptic activity[26]

Objectives

Methods

Results

Conclusion