A bright cyan fluorescence calcium indicator for mitochondrial calcium with minimal interference from physiological pH fluctuations

Mitochondria are crucial for maintaining calcium balance in cells, but their calcium dynamics are challenging to study due to pH changes in the mitochondrial matrix, and the lack of cyan-colored GECIs hinders the monitoring of calcium signals across different organelles and cell types. In this study, researchers used the bright cyan fluorescent protein mTurquoise2 to create a new GECI called TurCaMP. They found that the deprotonation of the chromophore is the key mechanism for TurCaMP’s calcium-dependent fluorescence changes, leading to the development of an inverse GECI with a strong basal fluorescence that is stable in the physiological pH range of 6 to 9. This new tool has the potential to accurately monitor mitochondrial calcium dynamics and open up new avenues for research in mitochondrial biology.Key Findings:

1. Enhanced Basal Fluorescence: The optimized TurCaMPb variant is approximately 3.1 times brighter than Tq-Ca-FLITs and 2.6 times brighter than TurCaMP0.1, making it suitable for visualizing calcium transients in small subcellular compartments.
2. pH Stability: TurCaMPb’s fluorescence is stable across the pH range of 7 to 9, rendering it suitable for monitoring calcium signals amidst pH fluctuations, such as in the mitochondrial matrix.
3. Inverse Calcium Response: TurCaMPb exhibits an inverse response to calcium transients, enabling accurate calcium dynamics monitoring with a high signal-to-noise ratio.
4. Mitochondrial Targeting: The introduction of a mitochondrial matrix targeting sequence results in mt-TurCaMPb, which is tailored for the specific detection of calcium elevations within mitochondria.

Applications: TurCaMPb’s distinct characteristics render it a crucial tool for investigating mitochondrial calcium signaling and its involvement in various cellular processes. It facilitates:

1. Precise Mitochondrial Calcium Dynamics Monitoring: TurCaMPb’s pH insensitivity and inverse calcium response allow for exact measurements of calcium changes within mitochondria, even in pH-variable environments.

This research introduces TurCaMPb as an invaluable resource for probing mitochondrial calcium signaling and its role in cellular processes. Its unique properties make it perfect for accurate and multiplexed calcium dynamics monitoring, paving the way for new research directions in mitochondrial biology and related fields. The work entitled “Exploring lysosomal biology: current approaches and methods” was published on Biophysics Reports (published on January, 2024).