Decoding mCherry: Advancements in Fluorescent Proteins
Fluorescent proteins are essential cell and molecular biology reporters. Their discovery and development have revolutionized biomedical research, allowing phenotypical screenings and tracking of cellular processes for offering unprecedented clarity advantages. They work as label tags for proteins and for reporting cellular status by coupling them to specific gene promoters.
Among these proteins, there is a red fluorescent protein called mCherry which is a remarkable example, especially for its unique properties and applications in molecular and cellular biology as an intracellular probe.
This article aims to clarify what is mCherry protein, delving into its characteristics, such as excitation-emission spectra. By exploring the advancements brought by mCherry, we will highlight its significance in biological research and the potential it holds for the future of biomedical research.
Exploring mCherry: Properties and Applications
mCherry is a red fluorescent protein with a monomeric structure of 236 amino acids and a mass of 26.7 kDa. It’s derived from the DsRed constitutively fluorescent protein found in Discosoma sp. coral.
It belongs to a class of engineered fluorescent proteins, the mFruits family, that have significantly expanded the choice of tools available for live-cell imaging. What distinguishes mCherry from other proteins is its optimal balance of photostability, brightness, and excellent pH resistance, making it the most widely used red fluorescent protein in biomedical research.
mCherry fluorescence is a constitutive and inherent characteristic of this protein. It emits light between 550 and 650 nm and absorbs light between 540 and 590 nm. It has an excitation maximum of 587 nm and an emission maximum of 610 nm, and these characteristics fit perfectly within the UV spectra, the less phototoxic spectrum for cells, and penetrates tissues more effectively than other wavelengths. Its photostability and brightness manage precise and long-term imaging of cellular processes, thus facilitating detailed studies on cell behavior, protein localization, and interactions within the complex cell environment based on phenotypic screening. Moreover, mCherry protein's low molecular weight minimizes structural interferences when fused to other proteins, enhancing its utility in gene expression studies.
Applications of mCherry span from basic biological research to applied sciences, including developmental biology, neurobiology, and cancer research in a vast array of models. In developmental biology, mCherry's properties facilitate the real-time visualization of cellular and tissue dynamics in embryos. Neurobiologists use mCherry protein to trace neuronal circuits and understand synaptic interactions, whereas, in cancer research, mCherry fluorescence helps in tracking tumor cells and immune cells or deriving cancer progression mechanisms.
The Significance of mCherry in Biological Research
mCherry development overcame many limitations associated with earlier fluorescent proteins, such as deprived photostability and spectral overlap, which hindered detailed and simultaneous observations of multiple targets. The distinct excitation and emission spectra of mCherry allow for multi-color labelling techniques, one of its most significant characteristics. This allows for the simultaneous visualization of multiple targets within the same cell, tissue or organ, providing a deeper understanding of the multifaceted interactions within biological systems. Furthermore, the compatibility of mCherry protein with various microscopy and imaging techniques further extends its applicability.
In addition, the versatility of mCherry extends to its application across various biological systems, from prokaryotes to eukaryotes, and for both in vivo and in vitro studies. Its rapid maturation rate enables quick results visualization after transfection or transcription activation. This property is especially beneficial in complex experiments involving protein expression monitoring, such as in the Zebrafish New Alternative Model (NAM), where mCherry fluorescence is used to observe specific organs or areas with high precision, thanks to zebrafish embryos' unique transparency.
An example of using mCherry protein in Zebrafish is the Thyroid Disruption Assay developed by Biobide. This assessment aims to screen the thyroid-disrupting potential of chemicals using transgenic embryos with the mCherry fluorescent protein. They express the red fluorescence in the thyroid gland under the thyroglobulin promoter, and intensifications in thyroglobulin levels correspond to an increase in fluorescence (image 1). The fluorescence signal is assessed by image analysis and compared to the control mean. Benchmark Concentration (BMC) and Thyroid-Disrupting Index (TDI) are calculated to determine the potency and hazard profile of the chemicals. The Thyroid Disruption Assay can also be complemented with gene expression analysis, by characterizing the genes of interest involved in the process. The assay has been validated with different reference toxicants and environmental pollutants and published recently by Biobide.
Image 1. a) Control zebrafish-mCherry transgenic embryo expressing red fluorescence in the thyroid gland under the thyroglobulin promoter. b) zebrafish-mCherry transgenic embryo exposed to a Thyroid-Disrupting Chemical.
Zebrafish-mCherry transgenic models enable time and cost-effective assays, allowing phenotypic screenings based on image analysis on a High-Content Screening (HCS) platform, simultaneously assessing different endpoints and reducing the number of experimental animals used. They possess enough sensitivity to detect thyroid-disrupting chemicals at environmentally relevant concentrations.
mCherry protein applications in Zebrafish exemplify its potential and versatility, demonstrating its value as an indispensable New Alternative Model that complies with biomedical research's 3Rs principle (Replacement, Refinement and Reduction).
Sources
Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird GS, Zacharias DA, Tsien RY. A monomeric red fluorescent protein. Proc Natl Acad Sci U S A. 2002 Jun 11;99(12):7877-82.
Chudakov DM, Matz MV, Lukyanov S, Lukyanov KA. Fluorescent proteins and their applications in imaging living cells and tissues. Physiol Rev. 2010 Jul;90(3):1103-63.
Giepmans BN, Adams SR, Ellisman MH, Tsien RY. The fluorescent toolbox for assessing protein location and function. Science. 2006 Apr 14;312(5771):217-24.
Jaka, O. et al. Screening for chemicals with thyroid hormone-disrupting effects using zebrafish embryo. Reprod Toxicol 121, 108463 (2023).
Shaner NC, Lin MZ, McKeown MR, Steinbach PA, Hazelwood KL, Davidson MW, Tsien RY. Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat Methods. 2008 Jun;5(6):545-51.
