Contact-FP: A Dimerization-Dependent Fluorescent Protein Toolkit for Visualizing Membrane Contact Site Dynamics.
Gregory E Miner1, Sidney Y Smith1, Wendy K Showalter1
1Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Contact (Thousand Oaks (Ventura County, Calif.))
|February 8, 2024
Summary
Researchers developed Contact-FP, a novel tool using dimerization-dependent fluorescent proteins, to visualize membrane contact site dynamics between organelles in live cells. This technology allows for precise observation of organelle interactions and cellular responses.
Area of Science:
- Cell Biology
- Molecular Biology
- Biophysics
Background:
- Membrane contact sites (MCSs) are crucial for cellular communication and are dynamic, yet challenging to study.
- Existing tools for visualizing MCS dynamics in live cells are limited.
- Dimerization-dependent fluorescent proteins (ddFPs) offer a promising approach for MCS visualization.
Purpose of the Study:
- To engineer and validate a novel suite of ddFPs, named Contact-FP, for visualizing MCS dynamics.
- To demonstrate the utility of Contact-FP probes in studying organelle interactions, specifically between lipid droplets and mitochondria.
- To assess the ability of Contact-FP to visualize complex MCS configurations and perturbations.
Main Methods:
- Engineering of Contact-FP probes targeting various organelles (LDs, ER, mitochondria, peroxisomes, lysosomes, plasma membrane, caveolae, cytoplasm).
- Localization studies to confirm probe targeting to specific organelles.
- Live-cell imaging to visualize MCS morphology and dynamics using Contact-FP pairs.
- Perturbation studies involving overexpression of organelle tethers (e.g., PLIN5).
Main Results:
- Contact-FP probes were successfully engineered and localized to their target organelles.
- Contact-FP pairs specifically localized to the interface between apposed organelles.
- The probes enabled visualization of dynamic changes in MCSs, including responses to PLIN5 overexpression.
- Simultaneous visualization of multiple MCSs involving a single organelle was achieved.
Conclusions:
- Contact-FP provides a versatile and powerful tool for studying the dynamics and morphology of membrane contact sites in live cells.
- This technology overcomes previous limitations in visualizing dynamic organelle interactions.
- Contact-FP probes can be optimized for studying MCSs between a wide range of organelle pairs.
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