# Analytical Post-Embedding Immunogold–Electron Microscopy with Direct Gold-Labelled Monoclonal Primary Antibodies against RIBEYE A- and B-Domain Suggests a Refined Model of Synaptic Ribbon Assembly

## TL;DR

This study uses advanced imaging to explore how synaptic ribbons are built, finding that two parts of a key protein are similarly positioned within the structure.

## Contribution

The study introduces direct gold-labeled monoclonal antibodies to achieve higher-resolution insights into synaptic ribbon architecture.

## Key findings

- RIBEYE A-domain and B-domain show similar localization within synaptic ribbons in mouse photoreceptor synapses.
- There is no obvious gradient between the center and surface of the synaptic ribbon for either domain.
- The findings suggest a refined model where both domains are similarly positioned relative to the synaptic ribbon midline.

## Abstract

Synaptic ribbons are the eponymous specializations of continuously active ribbon synapses. They are primarily composed of the RIBEYE protein that consists of a unique amino-terminal A-domain and carboxy-terminal B-domain that is largely identical to the ubiquitously expressed transcriptional regulator protein CtBP2. Both RIBEYE A-domain and RIBEYE B-domain are essential for the assembly of the synaptic ribbon, as shown by previous analyses of RIBEYE knockout and knockin mice and related investigations. How exactly the synaptic ribbon is assembled from RIBEYE subunits is not yet clear. To achieve further insights into the architecture of the synaptic ribbon, we performed analytical post-embedding immunogold–electron microscopy with direct gold-labelled primary antibodies against RIBEYE A-domain and RIBEYE B-domain for improved ultrastructural resolution. With direct gold-labelled monoclonal antibodies against RIBEYE A-domain and RIBEYE B-domain, we found that both domains show a very similar localization within the synaptic ribbon of mouse photoreceptor synapses, with no obvious differential gradient between the centre and surface of the synaptic ribbon. These data favour a model of the architecture of the synaptic ribbon in which the RIBEYE A-domain and RIBEYE B-domain are located similar distances from the midline of the synaptic ribbon.