Using extensive knowledge about the structures and mechanisms of genetically encoded fluorescent biosensors, this Perspective provides guidelines to aid and accelerate the development of an increasingly broad range of high-performance imaging tools.
Fluorescent protein biosensors
Genetically encoded (protein-based) fluorescent biosensors have been developed to enable imaging and monitoring of a variety of metabolites and cellular events, as highlighted in this collection of recent papers from Nature Portfolio. Engineering these biosensors is often non-trivial, requiring careful mutagenesis and insertion of active domains, but insights gained from past studies can be used to inform more rational design, and thus more rapid access to useful variants, in the future.
Fluorescent protein engineering:
An infrared fluorescent protein based on a new monomeric scaffold is described in this paper and is tested for protein fusion in cells and in vivo.
In this Perspective the authors discuss strategies for the development of improved fluorescent proteins, with a focus on probes at the red end of the spectrum. They synthesize the literature on chromophore photochemistry and protein structure to identify residues for targeted mutagenesis, and consider improvements in molecular evolution methodologies to enable improved screening for desired probes.
Fluorescent proteins are widely used to image cellular structures. Here, Shemiakina and colleagues develop an enhanced version of a red fluorescent protein that is monomeric and less cytotoxic, thereby improving the quality of images that can be obtained in the red part of the visible spectrum.
Incorporation of the non-canonical amino acid 3-aminotyrosine into the chromophores of green fluorescent protein-based biosensors systematically red-shifts their fluorescent properties while maintaining brightness, dynamic range and responsiveness.
Improved photostability of fluorescent proteins would benefit many applications but is usually an afterthought in selection screens. Setting photostability as the primary selection criterion in screens for improved fluorescent proteins yielded highly photostable variants of existing orange and red fluorescent proteins without compromising other beneficial characteristics.
Roger Tsien left us on August 24. His untimely passing has saddened and shocked the scientific community. Roger literally and figuratively brightened our world, illuminated the dark matter of biology, and forever changed our view of the interface of chemistry and biology.
NIR-GECO1, the first near-infrared genetically encoded calcium ion (Ca2+) indicator, enables improved Ca2+ imaging in conjunction with blue-light-activated optogenetic tools and multiplexed imaging in cell cultures and tissue slices.
By targeting calcium indicators to primary cilia, micrometer-long protrusions from the cellular plasma membrane, the authors measure Ca2+ signaling in these sensory organelles.
Orange CaMBI, a genetically encoded bioluminescent calcium indicator consisting of calcium-sensing domain CaM, luciferase, and fluorescent proteins, reports calcium dynamics in single cells and reveals calcium oscillations in whole mouse organs.
Engineering of the Ca2+-sensing domain in existing yellow Cameleon Ca2+ indicators is used to create indicators with a range of increased Ca2+ affinities capable of detecting subtle changes in intracellular Ca2+ at low resting levels.
Current calcium-sensitive probes based on red fluorescent proteins are unsuitable for two-photon excitation at the near-infrared wavelengths commonly used for green fluorescent probes. Wu et al. use a structure-guided approach to engineer a red fluorescent probe with optimal two-photon excitation at these wavelengths.
In the construction of single fluorescent protein biosensors, selection of the insertion point of a fluorescent protein into a ligand-binding domain is a rate-limiting step. Here, the authors develop an unbiased, high-throughput approach, called domain insertion profiling with DNA sequencing (DIP-seq), to generate a novel trehalose biosensor.
A fluorescence-based sensor of PKA activity has increased brightness, dynamic range and signal-to-noise ratio over related sensors and is useful for visualizing kinase activity in HeLa cells, primary neurons and the cortex of awake mice.
A ratiometric fluorescent sensor that reports the ATP/ADP concentration ratio in living cells was created by fusing the bacterial regulatory protein GlnK1 to a circularly permuted fluorescent protein. The sensor detected inhibition of cellular metabolism caused by transient removal of glucose from the cellular medium or administration of a glycolytic inhibitor.
A fluorescent sensor combining a mutated form of the 2-Cys peroxiredoxin Tsa2 unable to undergo thioredoxin-mediated reduction with a redox-sensitive GFP protein allows real-time detection of baseline hydrogen peroxide levels in yeast cells.
The genetically encoded GABA sensor iGABASnFR allows visualizing GABA signaling in vivo. Its application is demonstrated in mouse slices, in the awake mouse and in behaving zebrafish.
Genetically encoded calcium sensors have brought neuronal recording to the tiny brains of invertebrates, but the methodology has lagged behind classical electrophysiology in vertebrates. Now Douglas Kim and colleagues have used selective mutagenesis to engineer a new ultrasensitive probe, GCaMP6, demonstrating improved spatial and temporal resolution in vivo, from flies to zebrafish. In addition, in mouse visual cortex GCaMP6 can reliably detect single action potentials and single-spine orientation tuning. GCaMP6 sensors can be used to image large groups of neurons as well as tiny synaptic compartments over multiple imaging sessions separated by months, offering a flexible new tool for brain research and calcium signalling studies.
A collection of genetically encoded tools, each with their own capabilities, limitations and performance characteristics, are available for monitoring and manipulating neuronal activity that could allow visualizing the brain at single-cell resolution.
An improved version of the GCaMP genetically encoded calcium indicator, called GCaMP3, has higher calcium affinity and increased baseline fluorescence, dynamic range and stability. GCaMP3 performs better than existing genetically encoded calcium indicators in several assays and organisms, including in vivo imaging of neuronal signaling in worms, flies and mice.
A genetically encoded FRET-based optical sensor generated from a computational design approach can monitor hippocampal glycine levels in brain tissue to determine differences between spines and shafts and changes induced by high- and low-frequency stimulation.
The ‘jGCaMP7’ sensors are four genetically encoded calcium indicators with better sensitivity than state-of-the-art GCaMP6 and specifically improved for applications such as neuropil or wide-field imaging. The sensors are validated in vivo in both flies and mice.
Red and improved green versions of the genetically encoded dopamine sensor GRABDA have been developed. These neurotransmitter sensors are used alone or in combination with, for example, calcium sensors in behaving fruit flies and rodents.
A single-wavelength genetically encoded sensor of extracellular glutamate is reported. The sensor—iGluSnFR—is bright and photostable under both one- and two-photon illumination and is shown to work for in vivo imaging in worms, zebrafish and mice.