Optical Recording of Cellular Zinc Dynamics with Zinc-Finger-Based Biosensors

Abstract

In addition to serving as an essential structural component, zinc is also involved in intracellular and intercellular signaling pathways to impact a number of cellular functions. Genetically encoded zinc sensors that are specifically targeted to various subcellular compartments (ER, mitochondria, nucleus, plasma membrane, and vesicles) have been proven to provide accurate and sensitive visualization and quantification of zinc. Here we describe the methods to utilize both ratiometric and intensiometric genetically encoded zinc sensors designed based on zinc fingers for imaging and quantification of cellular free, labile zinc concentrations, [Zn]. This chapter explains in detail how to quantify [Zn] in live cells as well as how to monitor zinc influx in INS-1 cells stimulated with high glucose.

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

“…We first adopted the linker regions that were used in the GZnP-family sensors we previously created, ,,, as well as the linkers used in the development of the GCaMP, K-GECO, R-GECO1, and RCaMPh sensors, ,, generating variants RZnP0 to RZnP0.7 (Figure b). Bacterial cell lysate screening has become routine to optimize genetically encoded sensors, but this assay is not sensitive enough to differentiate the small improvements of the prototype sensors due to their dim fluorescence and weak sensor responses.…”

“…This pair of Zap1 Zn 2+ fingers can undergo conformational changes in response to Zn 2+ , which has been evidenced by previous successful applications in constructing FRET Zn 2+ sensors 18,24 and GFP-based Zn 2+ sensors. 6,15,25 Three different RFP variants showed promise for making a functional new sensor because they have been used to develop and optimize Ca 2+ sensors, including mApple used in the RGECO family sensors, 26 mRuby in RCaMP family sensors, and eqFP578 in K-GECO1. 27 We attached the two Zap1 Zn 2+ fingers to the Nand C-termini of each circularly permuted RFP variant separately (Figure 1a).…”