Monomeric Fluorescent Protein Research Articles

Creating a novel method for chicken primordial germ cell health monitoring using the fluorescent ubiquitination-based cell cycle indicator reporter system

The most current in vitro genetic methods, including gene preservation, gene editing and developmental modelling, require a significant number of healthy cells. In poultry species, primordial germ cells (PGCs) are great candidates for all the above-mentioned purposes, given their easy culturing and well-established freezing method for chicken. However, the constant monitoring of cultures can be financially challenging and consumes large amounts of solutions and accessories. This study aimed to introduce the Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) complex into the chicken PGCs. FUCCI is a powerful transgenic tool based on the periodic protein expression changes during the cell cycle. It includes chromatin licensing and DNA replication factor 1 attached monomeric Kusabira-Orange and Geminin-attached monomeric Azami-Green fluorescent proteins, that cause the cells to express a red signal in the G1 phase and a green signal in S and G2 phases. Modification of the chicken PGCs was done via electroporation and deemed to be successful according to confocal microscopy, DNA sequencing and timelapse video analysis. Stable clone cell lines were established, cryopreserved, and injected into recipient embryos to prove the integrational competency. The cell health monitoring was tested with medium change experiments, that proved the intended reactions of the FUCCI transgene. These results established the future for FUCCI experiments in chicken, including heat treatment and toxin treatment.

Characterization and structural basis for the brightness of mCLIFY: a novel monomeric and circularly permuted bright yellow fluorescent protein.

We present mCLIFY: a monomeric, bright, yellow, and long-lived fluorescent protein (FP) created by circular permutation of YPet, the brightest yellow FP from Aequorea Victoria for use in cellular and in vitro single molecule studies. mCLIFY retains the enhanced photophysical properties of YPET as a monomer at concentrations ≤ 40 μM. In contrast, we determined that YPet has a dimerization dissociation constant (K D 1-2) of 3.4 μM. Dimerization of YPet can cause homo-FRET, which underlies quantitative errors due to dimerization and homo-FRET. We determined the atomic structure of mCLIFY at 1.57 Å resolution and used its similarity with Venus for guided chromophore-targeted substitution studies to provide insights into its enhanced photophysical properties. The mutation V58L within the chromophore pocket improved quantum yield and extinction coefficient, making mCLIFY ~30% brighter than Venus. The extensive characterization of the photophysical and structural properties of YPet and mCLIFY presented here allowed us to reveal the basis of their long lifetimes and enhanced brightness and the basis of YPet's dimerization.

Bright and stable monomeric green fluorescent protein derived from StayGold.

The high brightness and photostability of the green fluorescent protein StayGold make it a particularly attractive probe for long-term live-cell imaging; however, its dimeric nature precludes its application as a fluorescent tag for some proteins. Here, we report the development and crystal structures of a monomeric variant of StayGold, named mBaoJin, which preserves the beneficial properties of its precursor, while serving as a tag for structural proteins and membranes. Systematic benchmarking of mBaoJin against popular green fluorescent proteins and other recently introduced monomeric and pseudomonomeric derivatives of StayGold established mBaoJin as a bright and photostable fluorescent protein, exhibiting rapid maturation and high pH/chemical stability. mBaoJin was also demonstrated for super-resolution, long-term live-cell imaging and expansion microscopy. We further showed the applicability of mBaoJin for neuronal labeling in model organisms, including Caenorhabditis elegans and mice.

A monomeric StayGold fluorescent protein

StayGold is an exceptionally bright and stable fluorescent protein that is highly resistant to photobleaching. Despite favorable fluorescence properties, use of StayGold as a fluorescent tag is limited because it forms a natural dimer. Here we report the 1.6 Å structure of StayGold and generate a derivative, mStayGold, that retains the brightness and photostability of the original protein while being fully monomeric.

Fluorescent protein lifetimes report densities and phases of nuclear condensates during embryonic stem-cell differentiation

Fluorescent proteins (FP) are frequently used for studying proteins inside cells. In advanced fluorescence microscopy, FPs can report on additional intracellular variables. One variable is the local density near FPs, which can be useful in studying densities within cellular bio-condensates. Here, we show that a reduction in fluorescence lifetimes of common monomeric FPs reports increased levels of local densities. We demonstrate the use of this fluorescence-based variable to report the distribution of local densities within heterochromatin protein 1α (HP1α) in mouse embryonic stem cells (ESCs), before and after early differentiation. We find that local densities within HP1α condensates in pluripotent ESCs are heterogeneous and cannot be explained by a single liquid phase. Early differentiation, however, induces a change towards a more homogeneous distribution of local densities, which can be explained as a liquid-like phase. In conclusion, we provide a fluorescence-based method to report increased local densities and apply it to distinguish between homogeneous and heterogeneous local densities within bio-condensates.

A sweet new set of inducible and constitutive promoters in Haloferax volcanii.

Inducible promoters are one of cellular and molecular biology's most important technical tools. The ability to deplete, replete, and overexpress genes on demand is the foundation of most functional studies. Here, we developed and characterized a new xylose-responsive promoter (Pxyl), the second inducible promoter system for the model haloarcheon Haloferax volcanii. Generating RNA-seq datasets from cultures in the presence of four historically used inducers (arabinose, xylose, maltose, and IPTG), we mapped upregulated genomic regions primarily repressed in the absence of the above inducers. We found a highly upregulated promoter that controls the expression of the xacEA (HVO_B0027-28) operon in the pHV3 chromosome. To characterize this promoter region, we cloned msfGFP (monomeric superfold green fluorescent protein) under the control of two upstream regions into a modified pTA962 vector: the first 250 bp (P250) and the whole 750 bp intergenic fragments (P750). The P250 sequence drove the expression of msfGFP constitutively, and its expression did not respond to the presence or absence of xylose. However, the P750 promoter showed not only to be repressed in the absence of xylose but also expressed higher levels of msfGFP than the previously described inducible promoter PtnaA in the presence of the inducer. Finally, we validated the inducible Pxyl promoter by reproducing morphological phenotypes already described in the literature. By overexpressing the tubulin-like FtsZ1 and FtsZ2, we observed similar but slightly more pronounced morphological defects than the tryptophan-inducible promoter PtnaA. FtsZ1 overexpression created larger, deformed cells, whereas cells overexpressing FtsZ2 were smaller but mostly retained their shape. In summary, this work contributes a new xylose-inducible promoter that could be used simultaneously with the well-established PtnaA in functional studies in H. volcanii in the future.

Rational Design of the β‐Bulge Gate in a Green Fluorescent Protein Accelerates the Kinetics of Sulfate Sensing**

AbstractDetection of anions in complex aqueous media is a fundamental challenge with practical utility that can be addressed by supramolecular chemistry. Biomolecular hosts such as proteins can be used and adapted as an alternative to synthetic hosts. Here, we report how the mutagenesis of the β‐bulge residues (D137 and W138) in mNeonGreen, a bright, monomeric fluorescent protein, unlocks and tunes the anion preference at physiological pH for sulfate, resulting in the turn‐off sensor SulfOFF‐1. This unprecedented sensing arises from an enhancement in the kinetics of binding, largely driven by position 138. In line with these data, molecular dynamics (MD) simulations capture how the coordinated entry and gating of sulfate into the β‐barrel is eliminated upon mutagenesis to facilitate binding and fluorescence quenching.

Rational Design of the β-Bulge Gate in a Green Fluorescent Protein Accelerates the Kinetics of Sulfate Sensing.

Detection of anions in complex aqueous media is a fundamental challenge with practical utility that can be addressed by supramolecular chemistry. Biomolecular hosts such as proteins can be used and adapted as an alternative to synthetic hosts. Here, we report how the mutagenesis of the β-bulge residues (D137 and W138) in mNeonGreen, a bright, monomeric fluorescent protein, unlocks and tunes the anion preference at physiological pH for sulfate, resulting in the turn-off sensor SulfOFF-1. This unprecedented sensing arises from an enhancement in the kinetics of binding, largely driven by position 138. In line with these data, molecular dynamics (MD) simulations capture how the coordinated entry and gating of sulfate into the β-barrel is eliminated upon mutagenesis to facilitate binding and fluorescence quenching.

Efficient generation of mNeonGreen Plasmodium falciparum reporter lines enables quantitative fitness analysis.

CRISPR editing has enabled the rapid creation of fluorescent Plasmodium transgenic lines, facilitating a deeper understanding of parasite biology. The impact of genetic perturbations such as gene disruption or the introduction of drug resistance alleles on parasite fitness is typically quantified in competitive growth assays between the query line and a wild type reference. Although fluorescent reporter lines offer a facile and frequently used method to measure relative growth, this approach is limited by the strain background of the existing reporter, which may not match the growth characteristics of the query strains, particularly if these are slower-growing field isolates. Here, we demonstrate an efficient CRISPR-based approach to generate fluorescently labelled parasite lines using mNeonGreen derived from the LanYFP protein in Branchiostoma lanceolatum, which is one of the brightest monomeric green fluorescent proteins identified. Using a positive-selection approach by insertion of an in-frame blasticidin S deaminase marker, we generated a Dd2 reporter line expressing mNeonGreen under the control of the pfpare (P. falciparum Prodrug Activation and Resistance Esterase) locus. We selected the pfpare locus as an integration site because it is highly conserved across P. falciparum strains, expressed throughout the intraerythrocytic cycle, not essential, and offers the potential for negative selection to further enrich for integrants. The mNeonGreen@pare line demonstrates strong fluorescence with a negligible fitness defect. In addition, the construct developed can serve as a tool to fluorescently tag other P. falciparum strains for in vitro experimentation.

A Bright Monomeric Near-Infrared Fluorescent Protein with an Excitation Peak at 633 nm for Labeling Cellular Protein and Reporting Protein-Protein Interaction.

Bright monomeric near-infrared fluorescent proteins (NIR-FPs) are useful as markers for labeling proteins and cells and as sensors for reporting molecular activities in living cells and organisms. However, current monomeric NIR-FPs are dim under excitation with common 633/635/640 nm lasers, limiting their broad use in cellular/subcellular level imaging. Here, we report a bright monomeric NIR-FP with maximum excitation at 633 nm, named mIFP663, engineered from Xanthomonas campestris pv Campestris phytochrome (XccBphP). mIFP663 has high molecular brightness with a large extinction coefficient (86,600 M-1 cm-1) and a decent quantum yield (19.4%), and high cellular brightness that is 3-6 times greater than those of spectrally similar NIR-FPs in HEK293T cells in the presence of exogenous BV. Moreover, we demonstrate that mIFP663 is able to label critical cellular and viral proteins without perturbing subcellular localization and virus replication, respectively. Finally, with mIFP663, we engineer improved bimolecular fluorescence complementation (BiFC) and new bioluminescent resonance energy transfer (BRET) systems to detect protein-protein interactions in living cells.

Transgenic mice encoding modern imaging probes: Properties and applications.

SUMMARYModern biology is increasingly reliant on optical technologies, including visualization and longitudinal monitoring of cellular processes. The major limitation here is the availability of animal models to track the molecules and cells in their natural environment in vivo. Owing to the integrity of the studied tissue and the high stability of transgene expression throughout life, transgenic mice encoding fluorescent proteins and biosensors represent unique tools for in vivo studies in norm and pathology. We review the strategies for targeting probe expression in specific tissues, cell subtypes, or cellular compartments. We describe the application of transgenic mice expressing fluorescent proteins for tracking protein expression patterns, apoptotic events, tissue differentiation and regeneration, neurogenesis, tumorigenesis, and cell fate mapping. We overview the possibilities of functional imaging of secondary messengers, neurotransmitters, and ion fluxes. Finally, we provide the rationale and perspectives for the use of transgenic imaging probes in translational research and drug discovery.

Single Molecule Analysis Reveals Dimer Formation of the Norepinephrine Transporter at the Plasma Membrane of Live Cells

BackgroundMonoamine transporters are the principal regulators of extracellular monoamine levels in the brain. At the plasma membrane, monoamine transporters form oligomers, which may play an important role in transporter trafficking, drug‐induced monoamine efflux, and drug tolerance development. Using single molecule microscopy, we previously reported that the serotonin transporter (SERT) co‐exists as monomers, dimers, and higher‐order oligomers, while the dopamine transporter (DAT) forms monomers and dimers only. Furthermore, whereas SERT oligomers are kinetically trapped at the plasma membrane by the phospholipid phosphatidylinositol (4,5)‐bisphosphate (PIP2), DAT dimerization is PIP2 independent. The subunit stoichiometry of the norepinephrine transporter (NET) is currently unknown. Based on a high degree of sequence homology between DAT and NET, we aimed to test the hypothesis that NET oligomerization resembles the one of DAT.MethodsWe fused monomeric green fluorescent protein (mGFP) to the N‐terminus of human NET and expressed the construct in Chinese hamster ovary cells. We assessed NET oligomerization using single molecule total internal reflection fluorescence (TIRF) microscopy in live cells. In order to determine the role of cell membrane lipids in NET oligomerization, we pharmacologically depleted the cell membrane of cholesterol and PIP2 before imaging.ResultsSingle molecule brightness analysis revealed that NET co‐exists as monomers (~60%) and dimers (~40%) at the plasma membrane. NET oligomerization displayed signs of transporter density dependence and subunit exchange. Cholesterol depletion did not affect NET dimerization, whereas PIP2 depletion resulted in a small but significant decrease in the average oligomeric state (Student's t‐test, P<0.05).ConclusionNET forms monomers and dimers but no higher‐order oligomers, thereby resembling the oligomerization of DAT. However, unlike DAT, NET dimers exchange subunits over time and dimerization is affected by the transporter density and PIP2 concentrations. This study therefore demonstrates that the oligomerization behavior of each monoamine transporters has unique features.

Construction of a fusion protein consisting of α-1,3-glucan-binding domains and tetrameric red fluorescent protein, which is involved in the aggregation of α-1,3-glucan and inhibition of fungal biofilm formation

Agl-KA, an α-1,3-glucan-hydrolyzing enzyme from Bacillus circulans KA-304, has three α-1,3-glucan-binding domains DS1, CB6, and DS2 (DCD). While their individual binding activities toward insoluble α-1,3-glucan and fungal cell-wall are weak, the three domains in combination bind strongly to the α-1,3-glucan and the cell-wall. In this study, we constructed DCD-tetraRFP by fusing DCD with DsRed-Express2, a tetrameric red fluorescent protein. DCD-tetraRFP forms a tetramer in an aqueous solution and contains twelve substrate-binding domains in one complex. We also constructed DCD-monoGFP by fusing DCD with AcGFP1, a monomeric green fluorescent protein. The molecular weight of DCD-tetraRFP and DCD-monoGFP were compared. The results of gel filtration chromatography and dynamic light scattering indicated that DCD-tetraRFP was larger than DCD-monoGFP, suggesting that DCD-tetraRFP had a tetrameric structure. In addition, DCD-tetraRFP bound to insoluble α-1,3-glucan strongly, and the amount of DCD-tetraRFP binding to 0.01% α-1,3-glucan was about twice of DCD-monoGFP. The Kd values of DCD-tetraRFP (measurements per subunit) and DCD-monoGFP were 0.16 and 0.84μM, respectively. Adding DCD-tetraRFP to a suspension of α-1,3-glucan caused glucan aggregation; however, adding DCD-monoGFP did not. These data suggested that DCD-tetraRFP had four DCDs sterically arranged in different directions so that DCD-tetraRFP cross-linked with the substrate, causing aggregation. Lastly, the aggregates of DCD-tetraRFP and α-1,3-glucan captured Aspergillus oryzae conidia and decreased their biofilm formation by 80% in a 24-well dish.

Transition Metals Induce Quenching of Monomeric Near-Infrared Fluorescent Proteins.

Transition metals such as zinc and copper are essential in numerous life processes, and both deficiency and toxic overload of these metals are associated with various diseases. Fluorescent metal sensors are powerful tools for studying the roles of metal ions in the physiology and pathology of biological systems. Green fluorescent protein (GFP) and its derivatives are highly utilized for protein-based sensor design, but application to anaerobic systems is limited because these proteins require oxygen to become fluorescent. Bacteriophytochrome-based monomeric near-infrared fluorescent proteins (miRFPs) covalently bind a bilin cofactor, which can be added exogenously for anaerobic cells. miRFPs can also have emission wavelengths extending to >700 nm, which is valuable for imaging applications. Here, we evaluated the suitability of miRFP670 and miRFP709 as platforms for single fluorescent protein metal ion sensors. We found that divalent metal ions like Zn2+, Co2+, Ni2+, and Cu2+ can quench from ∼6-20% (Zn2+, Co2+, and Ni2+) and up to nearly 90% (Cu2+) of the fluorescence intensity of pure miRFPs and have similar impacts in live Escherichia coli cells expressing miRFPs. The presence of a 6× histidine tag for purification influences metal quenching, but significant Cu2+-induced quenching and a picomolar binding affinity are retained in the absence of the His6 tag in both cuvettes and live bacterial cells. By comparing the Cu2+ and Cu+-induced quenching results for miRFP670 and miRFP709 and through examining absorption spectra and previously reported crystal structures, we propose a surface metal binding site near the biliverdin IXα chromophore.

Mice expressing fluorescent PAR2 reveal that endocytosis mediates colonic inflammation and pain

G protein-coupled receptors (GPCRs) regulate many pathophysiological processes and are major therapeutic targets. The impact of disease on the subcellular distribution and function of GPCRs is poorly understood. We investigated trafficking and signaling of protease-activated receptor 2 (PAR2) in colitis. To localize PAR2 and assess redistribution during disease, we generated knockin mice expressing PAR2 fused to monomeric ultrastable green fluorescent protein (muGFP). PAR2-muGFP signaled and trafficked normally. PAR2 messenger RNA was detected at similar levels in Par2-mugfp and wild-type mice. Immunostaining with a GFP antibody and RNAScope in situ hybridization using F2rl1 (PAR2) and Gfp probes revealed that PAR2-muGFP was expressed in epithelial cells of the small and large intestine and in subsets of enteric and dorsal root ganglia neurons. In healthy mice, PAR2-muGFP was prominently localized to the basolateral membrane of colonocytes. In mice with colitis, PAR2-muGFP was depleted from the plasma membrane of colonocytes and redistributed to early endosomes, consistent with generation of proinflammatory proteases that activate PAR2 PAR2 agonists stimulated endocytosis of PAR2 and recruitment of Gαq, Gαi, and β-arrestin to early endosomes of T84 colon carcinoma cells. PAR2 agonists increased paracellular permeability of colonic epithelial cells, induced colonic inflammation and hyperalgesia in mice, and stimulated proinflammatory cytokine release from segments of human colon. Knockdown of dynamin-2 (Dnm2), the major colonocyte isoform, and Dnm inhibition attenuated PAR2 endocytosis, signaling complex assembly and colonic inflammation and hyperalgesia. Thus, PAR2 endocytosis sustains protease-evoked inflammation and nociception and PAR2 in endosomes is a potential therapeutic target for colitis.

Discovery of a monomeric green fluorescent protein sensor for chloride by structure-guided bioinformatics.

Chloride is an essential anion for all forms of life. Beyond electrolyte balance, an increasing body of evidence points to new roles for chloride in normal physiology and disease. Over the last two decades, this understanding has been advanced by chloride-sensitive fluorescent proteins for imaging applications in living cells. To our surprise, these sensors have primarily been engineered from the green fluorescent protein (GFP) found in the jellyfish Aequorea victoria. However, the GFP family has a rich sequence space that could already encode for new sensors with desired properties, thereby minimizing protein engineering efforts and accelerating biological applications. To efficiently sample this space, we present and validate a stepwise bioinformatics strategy focused first on the chloride binding pocket and second on a monomeric oligomerization state. Using this, we identified GFPxm163 from GFPxm found in the jellyfish Aequorea macrodactyla. In vitro characterization shows that the binding of chloride as well as bromide, iodide, and nitrate rapidly tunes the ground state chromophore equilibrium from the phenolate to the phenol state generating a pH-dependent, turn-off fluorescence response. Furthermore, live-cell fluorescence microscopy reveals that GFPxm163 provides a reversible, yet indirect readout of chloride transport via iodide exchange. With this demonstration, we anticipate that the pairing of bioinformatics with protein engineering methods will provide an efficient methodology to discover and design new chloride-sensitive fluorescent proteins for cellular applications.

Highly Efficient SARS-CoV-2 Infection of Human Cardiomyocytes: Spike Protein-Mediated Cell Fusion and Its Inhibition.

ABSTRACTSevere cardiovascular complications can occur in coronavirus disease of 2019 (COVID-19) patients. Cardiac damage is attributed mostly to the aberrant host response to acute respiratory infection. However, direct infection of cardiac tissue by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also occurs. We examined here the cardiac tropism of SARS-CoV-2 in spontaneously beating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). These cardiomyocytes express the angiotensin-converting enzyme 2 (ACE2) receptor but not the transmembrane protease serine 2 (TMPRSS2) that mediates spike protein cleavage in the lungs. Nevertheless, SARS-CoV-2 infection of hiPSC-CMs was prolific; viral transcripts accounted for about 88% of total mRNA. In the cytoplasm of infected hiPSC-CMs, smooth-walled exocytic vesicles contained numerous 65- to 90-nm particles with canonical ribonucleocapsid structures, and virus-like particles with knob-like spikes covered the cell surface. To better understand how SARS-CoV-2 spreads in hiPSC-CMs, we engineered an expression vector coding for the spike protein with a monomeric emerald-green fluorescent protein fused to its cytoplasmic tail (S-mEm). Proteolytic processing of S-mEm and the parental spike were equivalent. Live cell imaging tracked spread of S-mEm cell-to-cell and documented formation of syncytia. A cell-permeable, peptide-based molecule that blocks the catalytic site of furin and furin-like proteases abolished cell fusion. A spike mutant with the single amino acid change R682S that disrupts the multibasic furin cleavage motif was fusion inactive. Thus, SARS-CoV-2 replicates efficiently in hiPSC-CMs and furin, and/or furin-like-protease activation of its spike protein is required for fusion-based cytopathology. This hiPSC-CM platform enables target-based drug discovery in cardiac COVID-19.IMPORTANCE Cardiac complications frequently observed in COVID-19 patients are tentatively attributed to systemic inflammation and thrombosis, but viral replication has occasionally been confirmed in cardiac tissue autopsy materials. We developed an in vitro model of SARS-CoV-2 spread in myocardium using induced pluripotent stem cell-derived cardiomyocytes. In these highly differentiated cells, viral transcription levels exceeded those previously documented in permissive transformed cell lines. To better understand the mechanisms of SARS-CoV-2 spread, we expressed a fluorescent version of its spike protein that allowed us to characterize a fusion-based cytopathic effect. A mutant of the spike protein with a single amino acid mutation in the furin/furin-like protease cleavage site lost cytopathic function. Of note, the fusion activities of the spike protein of other coronaviruses correlated with the level of cardiovascular complications observed in infections with the respective viruses. These data indicate that SARS-CoV-2 may cause cardiac damage by fusing cardiomyocytes.

Engineering an efficient and bright split Corynactis californica green fluorescent protein

Split green fluorescent protein (GFP) has been used in a panoply of cellular biology applications to study protein translocation, monitor protein solubility and aggregation, detect protein–protein interactions, enhance protein crystallization, and even map neuron contacts. Recent work shows the utility of split fluorescent proteins for large scale labeling of proteins in cells using CRISPR, but sets of efficient split fluorescent proteins that do not cross-react are needed for multiplexing experiments. We present a new monomeric split green fluorescent protein (ccGFP) engineered from a tetrameric GFP found in Corynactis californica, a bright red colonial anthozoan similar to sea anemones and scleractinian stony corals. Split ccGFP from C. californica complements up to threefold faster compared to the original Aequorea victoria split GFP and enable multiplexed labeling with existing A. victoria split YFP and CFP.

Protein and solutes freeze-concentration in water/glycerol mixtures revealed by pulse EPR

Lyophilization can extend protein drugs stability and shelf life, but it also can lead to protein degradation in some cases. The development of safe freeze-drying approaches for sensitive proteins requires a better understanding of lyophilization on the molecular level. The evaluation of the freezing process and its impact on the protein environment in the nm scale is challenging because feasible experimental methods are scarce. In the present work we apply pulse EPR as a tool to study the local concentrations of the solute in the 20 nm range and of the solvent in the 1 nm range for a spin labeled 27 kDa monomeric green fluorescent protein, mEGFP, and the 172 Da TEMPOL spin probe, frozen in different water/glycerol-d5 mixtures. For average glycerol volume fractions, φgly-d5avg, ≥ 0.4 we observed transparent glassy media; the local concentration and the 1 nm solvent shell of TEMPOL and the protein correspond to those of a uniform vitrified glass. At φgly-d5avg ≤ 0.3 we observed partial ice crystallization, which led to ice exclusion of glycerol and TEMPOL with freeze-concentration up to the glycerol maximal-freeze local volume fraction, φgly-d5loc, of 0.64. The protein concentration and its shell behavior was similar except for the lowest φgly-d5avg (0.1), which showed a 4.7-fold freeze-concentration factor compared to sevenfold for TEMPOL, and also a smaller φgly-d5loc. We explain this behavior with an increased probability for proteins to get stuck in the ice phase during fast freezing at higher freeze-concentration and the related large-scale mass transfer.

A switch-on molecular biosensor for detection of caspase-3 and imaging of apoptosis of cells

Apoptosis is a form of programmed cell death that is essential for maintaining internal environmental stability. Disordered apoptosis can cause a variety of diseases; therefore, sensing apoptosis can provide help in study of mechanism of the relevant diseases and drug development. It is known that caspase-3 is a key enzyme involved in apoptosis and the expression of its activity is an indication of apoptosis. Here, we present a genetically encoded switch-on mNeonGreen2-based molecular biosensor. mNeonGreen2 is the brightest monomeric green fluorescent protein. The substrate of caspase-3, DEVD amino acid residues, is inserted in it, while cyclized by insertion of Nostoc punctiforme DnaE intein to abolish the fluorescence (inactive state). Caspase-3-catalyzed cleavage of DEVD linearizes mNeonGreen2 and rebuilds the natural barrel structure to restore the fluorescence (activated state). The characterization exhibited that the Caspase-3 biosensor has shortened response time, higher sensitivity, and prolonged functional shelf life in detection of caspase-3 amongst the existing counterparts. We also used the Caspase-3 biosensor to evaluate the effect of several drugs on the induction of apoptosis of HeLa and MCF-7 tumor cells and inhibition of Zika virus invasion.Supporting InformationThe supporting information is available online at 10.1007/s11427-021-1986-7. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.