Development and Characterization of a Red Fluorescent Protein-Based Sensor RZnP1 for the Detection of Cytosolic Zn²

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Imaging

Background:

• Fluorescent sensors are crucial for monitoring intracellular zinc ion (Zn²⁺) dynamics.
• Existing single-wavelength sensors primarily focus on green fluorescent proteins, limiting multi-ion imaging capabilities.

Purpose of the Study:

• To engineer a novel, genetically encoded, single red fluorescent protein-based Zn²⁺ sensor.
• To enable simultaneous imaging of Zn²⁺ with other ions and across subcellular compartments.

Main Methods:

• Development of the Red Zinc Probe (RZnP1) utilizing a single red fluorescent protein.
• Characterization of RZnP1's sensitivity, brightness, dynamic range, and specificity.
• Demonstration of simultaneous Zn²⁺ and Ca²⁺ imaging in HeLa cells using RZnP1 and GCaMP5G.
• Concurrent imaging of cytosolic and mitochondrial Zn²⁺ in rat primary neurons using RZnP1 and mito-GZnP2.

Main Results:

• RZnP1 exhibits high sensitivity (K_d = 438 pM), good brightness (QY = 0.15), and a dynamic range (F_max/F_min = 4.0).
• RZnP1 demonstrates specificity for Zn²⁺ over other biologically relevant metal cations.
• Successful simultaneous imaging of Zn²⁺ with Ca²⁺ and tracking of Zn²⁺ in both cytosol and mitochondria was achieved.

Conclusions:

• RZnP1 expands the available tools for Zn²⁺ sensing with a red fluorescent protein.
• The study demonstrates novel techniques for multi-ion and multi-compartment Zn²⁺ imaging.
• This work facilitates advanced studies of cellular Zn²⁺ signaling and homeostasis.