Reconstitution of Ribbon-Type Active Zones in a Heterologous Expression System and Insights into Presynaptic Ca2+ Channel Clustering

Core Concepts

The authors reconstituted a minimal ribbon-type active zone model system in HEK293 cells by co-expressing membrane-targeted Bassoon, RIBEYE, RBP2, and CaV1.3 Ca2+ channels. These synthetic ribbon-type active zones recapitulate basic structural and functional aspects of native ribbon synapses and provide a platform to study the molecular interactions of active zone proteins and Ca2+ channels.

Abstract

The authors employed a synthetic biology approach to reconstitute ribbon-type active zones (AZs) in HEK293 cells. They identified a minimal set of proteins - membrane-targeted Bassoon, RIBEYE, RBP2, and CaV1.3 Ca2+ channels - that can assemble into structures resembling native ribbon-type AZs of inner hair cells (IHCs).
Key highlights:

Co-expression of membrane-targeted Bassoon and RIBEYE led to the formation of synthetic ribbon-like structures (SyRibbons) at the plasma membrane, recapitulating the arrangement of these proteins at native IHC ribbon synapses.
Additional expression of CaV1.3 Ca2+ channels and RBP2 resulted in the clustering of CaV1.3 channels underneath the SyRibbons, similar to the organization at native IHC ribbon synapses.
The size and morphology of the SyRibbons and associated CaV1.3 clusters showed similarities but also differences compared to native IHC ribbon synapses, likely due to the lack of tight regulation in the heterologous expression system.
Functionally, co-expression of RBP2 and SyRibbons led to larger whole-cell CaV1.3 currents, and Ca2+ imaging revealed preferential Ca2+ influx at sites underneath the SyRibbons.
The authors conclude that the synthetic ribbon-type AZs provide a valuable platform for studying the molecular interactions of active zone proteins and Ca2+ channels, complementing and potentially reducing experiments on native ribbon synapses.

Stats

"Co-expression of RBP2 and palm-Bassoon seemingly increases the whole-cell Ca2+ current density in HEK293 cells expressing CaV1.3, [Ca]e = 10mM."
"CaV1.3 clusters appear larger on an average in cells expressing CaV1.3, RBP2 and SyRibbons versus in cells expressing only CaV1.3."
"On average, Calbryte590 ΔFmax/F0 was higher for ROIs with SyRibbons than ROIs without them in a given cell."

Quotes

"We identify Ca2+ channels, RBP, membrane-anchored Bassoon, and RIBEYE as minimal components for reconstituting a basic ribbon-type AZ."
"SyRibbons might complement, refine, and reduce experiments on native ribbon synapses asserted from animals."
"We expect the approach to complement, refine, and reduce experiments on native ribbon synapses."

Key Insights Distilled From

Establishing synthetic ribbon-type active zones in a heterologous expression system

by Kapoor,R., S... at www.biorxiv.org 03-19-2024

https://www.biorxiv.org/content/10.1101/2024.03.18.585517v2

Deeper Inquiries

How could the synthetic ribbon-type active zones be further improved to better mimic the structural and functional properties of native ribbon synapses?

In order to enhance the synthetic ribbon-type active zones to better mimic native ribbon synapses, several improvements can be considered:

Incorporation of Additional Proteins: Apart from the proteins already included in the synthetic system, adding other key proteins found in native ribbon synapses, such as CAST and ELKS, could help in better replicating the complex molecular environment of the presynaptic active zone.

Regulation of Protein Expression: Fine-tuning the expression levels of the different proteins within the synthetic system could help in achieving a more accurate representation of the protein stoichiometry and localization observed in native ribbon synapses.

Introduction of Synaptic Vesicles: Including components related to synaptic vesicles and their release machinery would be crucial for studying the complete process of neurotransmitter release at the active zone, which is a fundamental function of ribbon synapses.

Dynamic Imaging Techniques: Implementing live-cell imaging techniques, such as time-lapse imaging or super-resolution microscopy, could provide insights into the dynamic behavior of the synthetic ribbon-type active zones and their interactions with other cellular components.

Functional Assays: Conducting functional assays beyond Ca2+ channel clustering, such as neurotransmitter release assays or electrophysiological recordings, would offer a more comprehensive understanding of the synaptic activity at the synthetic ribbon-type active zones.

What are the potential limitations and challenges of the heterologous expression system used in this study, and how could they be addressed?

The heterologous expression system used in the study has several limitations and challenges:

Non-Native Cellular Environment: HEK293 cells differ significantly from the specialized sensory cells where native ribbon synapses are found, potentially affecting the behavior and interactions of the expressed proteins.

Regulation of Protein Expression: The variability in protein expression levels and stoichiometry in the heterologous system may not accurately reflect the precise organization of proteins at native ribbon synapses.

Functional Differences: The functional properties of the synthetic ribbon-type active zones may not fully replicate the complex neurotransmission mechanisms observed in native ribbon synapses.

Lack of Synaptic Vesicles: The absence of synaptic vesicles in the synthetic system limits the study of complete synaptic transmission processes.

To address these limitations, the following strategies could be implemented:

Cell Line Optimization: Consider using more specialized cell lines that closely resemble the characteristics of sensory cells hosting native ribbon synapses.

Inducible Expression Systems: Employ inducible expression systems to control the timing and levels of protein expression, allowing for more precise experimental conditions.

Co-Culture Systems: Utilize co-culture systems with neuronal cells to create a more physiologically relevant environment for studying synaptic function.

Incorporation of Additional Proteins: Include a broader range of proteins involved in synaptic transmission to capture the complexity of native ribbon synapses.

What other applications or research questions could the synthetic ribbon-type active zones be used to investigate, beyond the study of Ca2+ channel clustering and function?

The synthetic ribbon-type active zones offer a versatile platform for exploring various aspects of synaptic biology and could be applied to investigate the following research questions:

Synaptic Vesicle Dynamics: Study the dynamics of synaptic vesicle trafficking, docking, and fusion at the active zone to understand the mechanisms of neurotransmitter release.

Protein-Protein Interactions: Investigate the interactions between different proteins at the active zone and their roles in regulating synaptic function and plasticity.

Synaptic Plasticity: Explore how changes in protein composition or activity at the active zone impact synaptic plasticity and long-term potentiation/depression.

Drug Screening: Use the synthetic system for screening potential drug candidates targeting proteins involved in synaptic transmission, offering insights into novel therapeutic strategies for neurological disorders.

Comparative Studies: Compare the properties and functions of synthetic ribbon-type active zones with those of conventional synapses to identify unique features and mechanisms specific to ribbon synapses.

By expanding the scope of research questions and applications, the synthetic ribbon-type active zones can serve as a valuable tool for advancing our understanding of synaptic biology and neurological function.