Rapid subcellular calcium responses and dynamics by calcium sensor G-CatchER

Area of Science:

• Cell Biology
• Biochemistry
• Molecular Biology

Background:

• Subcellular calcium (Ca2+) transients are crucial for physiological functions.
• Accurate measurement of rapid, local Ca2+ dynamics is essential for understanding cellular processes.
• Existing Ca2+ sensors have limitations in speed, sensitivity, or folding properties.

Purpose of the Study:

• To develop a novel, high-performance green Ca2+ sensor for reporting rapid local endoplasmic reticulum (ER) Ca2+ dynamics.
• To characterize the properties of the new sensor, G-CatchER+, including its Ca2+ binding kinetics and fluorescence response.
• To validate G-CatchER+ as a tool for studying Ca2+ signaling in various cell types and in vivo.

Main Methods:

• Protein engineering and design of a novel green Ca2+ sensor, G-CatchER+.
• Biochemical characterization of G-CatchER+ Ca2+ on-rate and fluorescence lifetime changes.
• Cellular imaging of agonist/antagonist-triggered Ca2+ dynamics in primary neurons and other cell types.
• Localization of G-CatchER+ to specific subcellular compartments, such as the RyR channel lumen (G-CatchER+-JP45).
• Transgenic expression of G-CatchER+ in Drosophila muscle for in vivo studies.

Main Results:

• G-CatchER+ demonstrates significantly improved folding properties compared to existing sensors.
• The sensor exhibits a superior Ca2+ on-rate and a Ca2+-induced fluorescence lifetime increase.
• G-CatchER+ successfully reports agonist/antagonist-triggered Ca2+ dynamics orchestrated by IP3Rs, RyRs, and SERCAs.
• Localization to the RyR channel lumen revealed rapid local Ca2+ release, potentially from calsequestrin.
• In vivo studies in Drosophila demonstrated G-CatchER+'s utility in reporting stimulus-evoked SR Ca2+ dynamics.

Conclusions:

• G-CatchER+ is a powerful new tool for precisely measuring rapid, local ER/SR Ca2+ dynamics.
• The sensor's improved properties enable detailed investigation of Ca2+ signaling pathways.
• G-CatchER+ facilitates research into ER dysfunction and aids in the development of targeted therapeutics.