Abstract
Purpose::
Dark-adapted rod photoreceptors usually contain a single large ribbon which is clearly visible as a horseshoe-shaped structure in the photoreceptor terminals already at the light microscopical level. During the light phase, ribbons are significantly smaller and, in addition, electron-dense material pinches off the ribbon resulting in club-shaped and spherical profiles surrounded by synaptic vesicles. As these activity-driven structural changes may alter synaptic function and thus, the release properties of the synapse. The purpose of our study is to find out which of the proteins of the cytomatrix at the active zone (CAZ) undergo dynamic changes and which are stable.
Methods::
We developed an in vitro assay applying EGTA (low calcium at the synapse) or a calcium-ionophore (high calcium at the synapse) to enrich club-shaped / spherical ribbon profiles and large, rod-like ribbon profiles, respectively. With light and electron microscopic immunocytochemistry we screened for proteins that belong to one of the two recently identified molecular compartments of the ribbon complex to evaluate their contribution to ribbon dynamics.
Results::
Under low calcium conditions the ribbons at the photoreceptor synapse became smaller due to transfer of ribbon material into spherical profiles. In contrast, after application of the calcium-ionophore the ribbons were elongated. While the CAZ protein Bassoon and a calcium channel alpha1 subunit seem to be stable components of the ribbon complex other CAZ proteins, e.g. Piccolo, RIBEYE and CtBP1 associate with club-shaped and spherical profiles under EGTA conditions and thus, undergo highly dynamic changes in their localization. In addition, the motor protein KIF3A belongs also to the group of proteins that show activity dependent dynamic changes.
Conclusions::
The proteins of the two molecular compartments of the ribbon complex undergo dynamic changes independently, suggesting activity-driven fine-tuning of photoreceptor ribbon structure and function.
Keywords: retina: distal (photoreceptors, horizontal cells, bipolar cells) • synapse • protein structure/function