Yi Shen

University of Alberta | UAlberta · Department of Chemistry

Fluorescent proteins with pH-sensitive fluorescence are valuable tools for the imaging of exocytosis and endocytosis. The Aequorea green fluorescent protein mutant superecliptic pHluorin (SEP) is particularly well suited to these applications. Here we describe pHuji, a red fluorescent protein with a pH sensitivity that approaches that of SEP, makin...

Background: Genetically encoded calcium ion (Ca2+) indicators (GECIs) are indispensable tools for measuring Ca2+ dynamics and neuronal activities in vitro and in vivo. Red fluorescent protein (RFP)-based GECIs have inherent advantages relative to green fluorescent protein-based GECIs due to the longer wavelength light used for excitation. Longer w...

Potassium ion (K⁺) homeostasis and dynamics play critical roles in biological activities. Here we describe three genetically encoded K⁺ indicators. KIRIN1 (potassium (K) ion ratiometric indicator) and KIRIN1-GR are Förster resonance energy transfer (FRET)-based indicators with a bacterial K⁺ binding protein (Kbp) inserting between the fluorescent p...

Significance Genetically encoded calcium ion (Ca2+) indicators (GECIs) are powerful tools for monitoring intracellular Ca2+ concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of Ca2+ concentration that is associated with the neuronal action potential. H...

Genetically encoded sensors enable quantitative imaging of analytes in live cells. Sensors are commonly constructed by combining ligand-binding domains with one or more sensitized fluorescent protein (FP) domains. Sensors based on a single FP can be susceptible to artifacts caused by changes in sensor levels or distribution in vivo. To develop inte...

Significance: Genetically encoded calcium ion (Ca2+) indicators (GECIs) are powerful tools for monitoring intracellular Ca2+ concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of Ca2+ concentration that is associated with the neuronal action potential....

Genetically encoded pH sensors based on fluorescent proteins are valuable tools for the imaging of cellular events that are associated with pH changes, such as exocytosis and endocytosis. Superecliptic pHluorin (SEP) is a pH-sensitive green fluorescent protein (GFP) variant widely used for such applications. Here, we report the rational design, dev...

Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are indispensable tools for monitoring biochemical changes in cells. Green and red fluorescent protein-based FRET pairs offer advantages over the classically employed cyan and yellow fluorescent protein pairs, such as better spectral separation, lower phototoxicity, an...

Potassium ion (K⁺) plays a critical role as an essential electrolyte in all biological systems. Genetically-encoded fluorescent K⁺ biosensors are promising tools to further improve our understanding of K⁺-dependent processes under normal and pathological conditions. Here, we report the crystal structure of a previously reported genetically-encoded...

Citrate is a central intracellular metabolite with roles in a variety of normal and aberrant biological processes. The methods for quantifying citrate concentration in cells can enable the study of the molecular mechanisms of citrate-related biological processes and diseases. Compared to existing analytical methods such as enzymatic assays and mass...

Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are indispensable tools for monitoring biochemical changes in cells. Green and red fluorescent protein-based FRET pairs offer advantages over the classically employed cyan and yellow fluorescent protein pairs, such as better spectral separation, lower phototoxicity, an...

Genetically-encoded biosensors based on a single fluorescent protein are widely used to visualize analyte levels or enzymatic activities in cells, though usually to monitor relative changes rather than absolute values. We report photochromism-enabled absolute quantification (PEAQ) biosensing, a method that leverages the photochromic properties of b...

Monatomic ions play critical biological roles including maintaining the cellular osmotic pressure, transmitting signals, and catalyzing redox reactions as cofactors in enzymes. The ability to visualize monatomic ion concentration, and dynamic changes in the concentration, is essential to understanding their many biological functions. A growing numb...

Apoptosis of cells and their subsequent removal through efferocytosis occurs in nearly all tissues during development, homeostasis, and disease. However, it has been difficult to track cell death and subsequent corpse removal in vivo. We developed a genetically encoded fluorescent reporter, CharON (Caspase and pH Activated Reporter, Fluorescence ON...

Potassium ions (K ⁺ ) play a critical role as an essential electrolyte in all biological systems. Here we report the crystal structure-guided optimization and directed evolution of an improved genetically encoded fluorescent K ⁺ biosensor, GINKO2. GINKO2 is highly sensitive and specific for K ⁺ and enables in vivo detection of K ⁺ dynamics in multi...

Genetically encoded biosensors based on engineered fluorescent proteins (FPs) are essential tools for monitoring the dynamics of specific ions and molecules in biological systems. Arsenic ion in the +3 oxidation state (As3+) is highly toxic to cells due to its ability to bind to protein thiol groups, leading to inhibition of protein function, disru...

Photocleavable molecules can enable the light-dependent modulation of biomolecular activities with high spatiotemporal precision. We have previously reported a photocleavable protein (PhoCl1) that, uniquely, is a fully genetically encoded photocleavable molecule that can be introduced into cells in the form of its corresponding gene to enable optog...

Intensiometric genetically encoded biosensors, based on allosteric modulation of the fluorescence of a single fluorescent protein, are powerful tools for enabling imaging of neural activities and other cellular biochemical events. The archetypical example of such biosensors is the GCaMP series of Ca²⁺ biosensors, which have been steadily improved o...

The photocleavable protein (PhoCl) is a green-to-red photoconvertible fluorescent protein that, when illuminated with violet light, undergoes main chain cleavage followed by spontaneous dissociation of the resulting fragments. The first generation PhoCl (PhoCl1) exhibited a relative slow rate of dissociation, potentially limiting its utilities for...

Genetically encoded calcium ion (Ca ²⁺ ) indicators (GECIs) are widely-used molecular tools for functional imaging of Ca ²⁺ dynamics and neuronal activities on a single cell level. Here we report the design and development of two new far-red fluorescent GECIs, FR-GECO1a and FR-GECO1c, based on the monomeric far-red fluorescent protein mKelly. We ch...

Genetically-encoded biosensors based on a single fluorescent protein are widely used to visualize analyte levels or enzymatic activities in cells, though usually to monitor relative changes rather than absolute values. We report photochromism-enabled analyte quantification (PEAQ) biosensing, a method that lever-ages photochromic properties of biose...

Optogenetic (photo-responsive) actuators engineered from photoreceptors are widely used in various applications to study cell biology and tissue physiology. In the toolkit of optogenetic actuators, the key building blocks are genetically encodable light-sensitive proteins. Currently, most optogenetic photosensory modules are engineered from natural...

As the technological hurdles are overcome and optogenetic techniques advance to have more control over neurons, therapies based on these approaches will begin to emerge in the clinic. Here, we consider the technical challenges surrounding the transition of this breakthrough technology from an investigative tool to a true therapeutic avenue. The eme...

Motivated by the growing recognition of citrate as a central metabolite in a variety of biological processes associated with healthy and diseased cellular states, we have developed a series of high-performance genetically encoded citrate biosensors suitable for imaging of citrate concentrations in mammalian cells. The design of these biosensors was...

In this paper, we demonstrate that a functional, portable device for the growth of bacteria or amplification of bacteriophage can be created using simple materials. These devices are comprised of packing tape, sheets of paper patterned by hydrophobic printer ink, and a polydimethyl siloxane (PDMS) membrane, which is selectively permeable to oxygen...

Motivated by the growing recognition of citrate as a central metabolite in a variety of biological processes associated with healthy and diseased cellular states, we have developed a series of high-performance genetically encoded citrate biosensors suitable for imaging of citrate concentrations in mammalian cells. The design of these biosensors was...

Genetically encoded fluorescent indicators have transformed the way neuroscientists record neuronal activities and interrogate the nervous system in vivo. In this review, we discuss recent advances and new additions to the toolkit of indicators for calcium ion entry, membrane voltage change, neurotransmitter release, and other neuronal molecular pr...

In the online version of the article [ 1 ], Figure S1 was mistakenly replaced with Figure 1.

For over 20 years, genetically encoded Ca ²⁺ indicators have illuminated dynamic Ca ²⁺ signaling activity in living cells and, more recently, whole organisms. We are just now beginning to understand how they work. Various fluorescence colors of these indicators have been developed, including red. Red ones are promising because longer wavelengths of...

Botulinum neurotoxins (BoNTs) are the most potent toxin known to man and a significant threat as a weapon of bioterrorism. BoNTs contain a metalloprotease domain that blocks neurotransmitter release in nerve terminals, resulting in a descending, flaccid paralysis with a 5–10% mortality rate. Existing treatment options cannot access or neutralize to...

Genetically encoded Ca ²⁺ indicators (GECIs) are widely used to illuminate dynamic Ca ²⁺ signaling activity in living cells and tissues. Various fluorescence colors of GECIs are available, including red. Red GECIs are promising because longer wavelengths of light scatter less in tissue, making it possible to image deeper. They are engineered from a...

Botulinum neurotoxins (BoNTs) are among the most potent toxins known and are also used to treat an increasing number of medical disorders. There are seven well-established serotypes (BoNT/A-G), which all act as zinc-dependent endopeptidases targeting specific members of the SNARE proteins required for synaptic vesicle exocytosis in neurons. A new t...

Potassium ion (K ⁺ ) homeostasis and dynamics play critical roles in regulating various biological activities, and the ability to monitor K ⁺ spatial-temporal dynamics is critical to understanding these biological functions. Here we report the design and characterization of a Förster resonance energy transfer (FRET)-based genetically encoded K ⁺ in...

Genetically-encoded calcium ion (Ca ²⁺ ) indicators (GECIs) are indispensable tools for measuring Ca ²⁺ dynamics and neuronal activities in vitro and in vivo. Red fluorescent protein (RFP)-based GECIs enable multicolor visualization with blue or cyan-excitable fluorophores and combined use with blue or cyan-excitable optogenetic actuators. Here we...

Peptide fragments of X-LC-HN under limited proteolysis analyzed by TMT labeling and quantitative mass spectrometry. His6-tagged recombinant X-LC-HN was labeled with the light TMT. Equal amounts of X-LC-HN samples were exposed to Lys-C and then labeled with the heavy TMT. Both samples were then digested with chymotrypsin, combined, and subjected to...

Supplementary Figures

Botulinum neurotoxins are known to have seven serotypes (BoNT/A–G). Here we report a new BoNT serotype, tentatively named BoNT/X, which has the lowest sequence identity with other BoNTs and is not recognized by antisera against known BoNTs. Similar to BoNT/B/D/F/G, BoNT/X cleaves vesicle-associated membrane proteins (VAMP) 1, 2 and 3, but at a nove...

MCherry, the Discosoma sp. mushroom coral-derived monomeric red fluorescent protein (RFP), is a commonly used genetically encoded fluorophore for live cell fluorescence imaging. We have used a combination of protein design and directed evolution to develop mCherry variants with low cytotoxicity to Escherichia coli and altered excitation and emissio...

Amino acid substitutions in new RFPs described in this work. (PDF)

Numerical data for all figures. (XLSX)

Representative fluorescence images of cells expressing mCherry and mEGFP fusions. (PDF)

FRET; protein engineering

The tetrameric red fluorescent protein from Discosoma sp. coral (DsRed) has previously been engineered to produce dimeric and monomeric fluorescent variants with excitation and emission profiles that span the visible spectrum. The brightest of the effectively monomeric DsRed variants is tdTomato - a tandem fusion of a dimeric DsRed variant. Here we...

Optical Probes in Biology. Jin Zhang, Sohum Mehta, and Carsten Schultz (Eds.). CRC Press, Boca Raton, FL; 2015, 511 pages. ISBN 1466510110 - Volume 21 Issue 6 - Yi Shen

The inherent advantages of red-shifted fluorescent proteins and fluorescent protein-based biosensors for the study of signaling processes in neurons and other tissues have motivated the development of a plethora of new tools. Relative to green fluorescent proteins (GFPs) and other blue-shifted alternatives, red fluorescent proteins (RFPs) provide t...

We have developed a versatile new class of genetically encoded fluorescent biosensor based on reversible exchange of the heterodimeric partners of green and red dimerization-dependent fluorescent proteins. We demonstrate the use of this strategy to construct both intermolecular and intramolecular ratiometric biosensors for qualitative imaging of ca...

We report a photochromic and thermochromic fluorescent protein that exhibits a reversible and striking visible colour switch between yellow and red. The protein has been characterized in terms of its light, temperature and pH-dependence. Based on a mutational analysis we propose that the colour switch mechanism involves chromophore protonation coup...

Ca2+ is a key intermediary in a variety of signaling pathways and undergoes dynamic changes in its cytoplasmic concentration due to release from stores within the endoplasmic reticulum (ER) and influx from the extracellular environment. In addition to regulating cytoplasmic Ca2+ signals, these responses also affect the [Ca2+] within the ER and mito...

Naturally occurring fluorescent proteins (FPs) cloned from marine organisms often suffer from many drawbacks for cell biology applications, including poor folding efficiency at 37 °C, slow chromophore formation and obligatory quaternary structure. Many of these drawbacks can be minimized or eliminated by using protein engineering and directed evolu...

In this paper, we demonstrate that a functional, portable device for the growth of bacteria or amplification of bacteriophage can be created using simple materials. These devices are comprised of packing tape, sheets of paper patterned by hydrophobic printer ink, and a polydimethyl siloxane (PDMS) membrane, which is selectively permeable to oxygen...