Caspase Sensor Research Articles

Genetically encoded caspase sensor and RFP-LC3 for temporal analysis of apoptosis-autophagy

The balance between pro-death and pro-survival signaling determines the fate of cells under a variety of pathological and physiological conditions. The pro-cell death signaling, apoptosis, and survival singling, autophagy work in an integrated manner for maintaining cell integrity. Their altered balance drives pathological conditions such as cancer, inflammatory disorders, and neurodegenerative diseases. Dissecting complex crosstalk between autophagy and apoptosis requires simultaneous detection of both events at a single cell level with good temporal resolution in real-time. Here, we have used two distinct fluorescent-based probes of caspase activation and autophagy for generating such sensor cells. Cells stably expressing RFP-LC3 as an autophagy marker were further stably expressed with a FRET-based probe for caspase activation with a nuclear localization signal. The functional validation and live-cell imaging of the sensor cells using selected treatments revealed that stress that induces rapid cell death often fails to induce autophagy signaling, and slow cell death induction triggers simultaneous autophagy signaling with caspase activation. The real-time imaging revealed the time-dependent shift of cells towards caspase activation while autophagy is inhibited confirming basal autophagy confers survival against apoptosis under stress conditions. Confocal imaging also revealed that cells under 3D culture condition maintain increased autophagy over monolayer cultures. High-throughput adaptability of the system extends its application for the screening of compounds that cause caspase activation, autophagy, or both demonstrating the potential utility of the sensor probe for diverse biological applications.

Ligation of Gold Nanoparticles with Self-Assembling, Coiled-Coil Peptides.

The surface of gold nanoparticles (AuNPs) can be conjugated with a wide range of highly functional biomolecules. A common pitfall when utilizing AuNPs is their tendency to aggregate, especially when their surface is functionalized with ligands of low molecular weight (no steric repulsion) or ligands of neutral charge (no electrostatic repulsion). For biomedical applications, AuNPs that are colloidally stable are desirable because they have a high surface area and thus reactivity, resist sedimentation, and exhibit uniform optical properties. Here, we engineer the surface of AuNPs so that they remain stable when decorated with coiled-coil (CC) peptides while preserving the native polypeptide properties. We achieve this by using a neutral, mixed ligand layer composed of lipoic acid poly(ethylene glycol) and lipoic acid poly(ethylene glycol) maleimide to attach the CCs. Tuning the surface fraction of each component within the mixed ligand layer also allowed us to control the degree of AuNP labeling with CCs. We demonstrate the dynamic surface properties of these CC-AuNPs by performing a place-exchange reaction and their utility by designing an energy-transfer-based caspase-3 sensor. Overall, this study optimizes the surface chemistry of AuNPs to quantitatively present functional biomolecules while maintaining colloid stability.

A highly sensitive NanoLuc-based protease biosensor for detecting apoptosis and SARS-CoV-2 infection

Proteases play critical roles in various biological processes, including apoptosis and viral infection. Several protease biosensors have been developed; however, obtaining a reliable signal from a very low level of endogenous protease activity remains a challenge. In this study, we developed a highly sensitive protease biosensor, named FlipNanoLuc, based on the Oplophorus gracilirostris NanoLuc luciferase. The flipped β-strand was restored by protease activation and cleavage, resulting in the reconstitution of luciferase and enzymatic activity. By making several modifications, such as introducing NanoBiT technology and CL1-PEST1 degradation tag, the FlipNanoLuc-based protease biosensor system achieved more than 500-fold luminescence increase in the corresponding protease-overexpressing cells. We demonstrated that the FlipNanoLuc-based caspase sensor can be utilized for the detection of staurosporine-induced apoptosis with sixfold increase in luminescence. Furthermore, we also demonstrated that the FlipNanoLuc-based coronavirus 3CL-protease sensor can be used to detect human coronavirus OC43 with tenfold increase in luminescence and severe acute respiratory syndrome-coronavirus-2 infections with 20-fold increase in luminescence by introducing the stem-loop 1 sequence to prevent the virus inducing global translational shutdown.

Caspase sensors based on NanoLuc

Caspases are a family of evolutionary conserved cysteine proteases that play key roles in programmed cell death and inflammation. Among the methods for the detection of caspase activity, biosensors based on luciferases have advantages in genetical encoding and convenience in assay. In this study, we constructed a new set of caspase biosensors based on NanoLuc luciferase. This kind of sensors, named NanoLock, work in dark-to-bright model, with the help of a NanoLuc quencher peptide (HiBiT-R/D) mutated from HiBiT. Optimized NanoLock responded to proteases with high signal to noise ratio (S/N), 1233-fold activation by tobacco etch virus protease in HEK293 cells and > 500-fold induction to caspase 3 in vitro. We constructed NanoLocks for the detection of caspase 1, 3, 6, 7, 8, 9, and 10, and assays in HEK293 cells demonstrated that these sensors performed better than commercial kits in the aspect of S/N and convenience. We further established a cell line stably expressing NanoLock-casp 6 and provided a proof-of-concept for the usage of this cell line in the high throughput screening of caspase 6 modulator.

Hypoxia Re‐Oxygenation Modelling Using Cancer Cells Expressing Cell Cycle and Cell Death Probes to Understand the Dynamics of Resistance Mechanisms

BackgroundMost solid tumors are difficult to treat with conventional cancer drugs. Non‐responsiveness to drugs and the recurrences even after successful treatment are serious problems. Although, extensive studies have been performed to understand the mechanisms of resistance and tumor recurrences, the complex and variable nature of microenvironmental factors that contribute this remains as a challenging topic.Hypoxia is also one of the key factors that play a crucial role in tumor progression. Hypoxia‐Induced Factor 1 α (HIF 1‐α) are involved in the resistance of hypoxic tumors to radiation therapy as well as conventional drugs. However, the growing tumor is often exposed to hypoxia and re‐oxygenation because of the tumor driven hypoxia and occasional angiogenic response that primarily affect the cell cycle and cell death.HypothesisWe hypothesize that the defined models of cell cycle and cell death in the setting of hypoxia‐reoxygenation will help us to understand the hypoxia‐mediated cell signaling and resistance mechanisms.MethodologyWe have employed cancer cells expressing cell cycle probe FUCCI to study the dynamics of cell cycle progression during hypoxia and reoxygenation using real‐time imaging. Similarly, cancer cells were expressed with real‐time probes for cell death, FRET‐based caspase sensor to track their long‐time fate. These innovative strategies allowed us to visualize both cell cycle and cell death events in hypoxic and re‐oxygenation condition and to track the long‐time fate of cancer cells.Results & ConclusionWe have shown that the hypoxic exposures alter the cells’ ability to undergo apoptosis dynamically after its recovery under normoxia. Initially, the cells adapt to hypoxia through mitophagy dependent manner that alters its cell cycle progression both in normoxia and hypoxia. Despite adaptive mitophagy, hypoxic cells remain sensitive to re‐oxygenation and also to cancer drugs owing to the oxidation damage to organelles.However, rare cells survive during these two events progresses towards expansion with a highly variable mitochondrial density that governs differing functional traits of cell cycle, cell death response, ability to invade and migrate. This functional heterogeneity appears to play a key role in temporally regulated tumor resistance and recurrences.

An In Vivo Fluorescence Resonance Energy Transfer-Based Imaging Platform for Targeted Drug Discovery and Cancer Therapy.

In the present study, an efficient in vivo drug screening platform is established based on FRET technique. We transfected cancer cells with FRET-based caspase-3 (C3) sensor and validated the cell lines by detecting the change in FRET signal caused by the in vitro drug-induced cell apoptosis. Furthermore, the C3 expressing cancer cells were then injected into zebrafish embryos and nude mice to establish the corresponding in vivo xenograft models. We found that cancer cell lines expressing C3 were effective in detecting cell death following drug treatment, including the detection of the tipping point of apoptosis. The drug-induced cell apoptosis was also observed in both zebrafish embryos and nude mice xenograft models. Overall, the FRET-based platform, through in vivo imaging, is potentially useful to improve drug screening efficiency.

Therapeutic Effect of IL-4 Receptor-Targeting Pro-Apoptotic Peptide (AP1-ELP-KLAK) in Glioblastoma Tumor Model.

BackgroundThermal-responsive self-assembled elastin-like polypeptide (ELP)-based nanoparticles are an emerging platform for controlled delivery of therapeutic peptides, proteins and small molecular drugs. The antitumor effect of bioengineered chimeric polypeptide AP1-ELP-KLAK containing an interleukin-4 receptor (IL-4R) targeting peptide and pro-apoptotic peptide (KLAKLAK) was evaluated in glioblastoma (GBM) in vitro and in vivo.Methods and ResultsHerein, the therapeutic effect of AP1-ELP-KLAK was tested in advanced, and less curable glioblastoma cells with higher expression of IL-4R. Glioblastoma cell lines stably expressing different reporter systems i.e., caspase-3 sensor (surrogate marker for cellular apoptosis) or effluc/enhanced firefly luciferase (cellular viability) were established to measure cell death non-invasively. Bioluminescence imaging (BLI) of D54/effluc and U97MG/effluc treated with AP1-ELP-KLAK exhibited higher cell death up to 2~3-fold than the control. Treatment with AP1-ELP-KLAK resulted in time-dependent increase of caspase-3 sensor BLI activity in D54/C cells and D54/C tumor-bearing mice. Intravenous injection of AP1-ELP-KLAK dramatically reduced tumor growth by inducing cellular apoptosis in D54/effluc tumor-bearing mice. Further, the immuno-histological examination of the excised tumor tissue confirmed the presence of apoptotic cells as well as caspase-3 activation.ConclusionCollectively, AP1-ELP-KLAK effectively induced cellular apoptosis of glioblastoma cells and non-invasive imaging provides a window for real-time monitoring of anti-tumor effect with the provision of improving therapeutic efficacy in a glioblastoma mice model.

Intravital molecular imaging reveals the restrained capacity of CTLs in the killing of tumor cells in the liver.

Cytotoxic T lymphocytes (CTLs) and their gene-engineered cells display great application prospects in tumor immunotherapy. The timing of CTL-induced molecular events in tumor cells is unclear, and we also unknow whether the killing efficiency of CTLs is restrained in the liver, an immunotolerant organ with a high tumor incidence.Methods: We used intravital imaging to dynamically monitor the fluorescence resonance energy transfer (FRET) signals of caspase-3 and calcium sensor in tumor cells after transferring CTLs into tumor-bearing mice.Results: Our data show that several CTLs attacked on one tumor cell, and on average each CTL killed 1.24 ± 0.11 tumor cells per day in the liver, which was much less efficient than that in the spleen (3.18 ± 0.26 tumor cells/CTL/day). The killing efficiency of CTLs is restrained in the liver and can be reversed by blocking immunosuppressive cytokine. Tumor cells exposed to CTLs appeared to have prolonged calcium influx, which occurred dozens of minutes before caspase-3 activity.Conclusion: The quantitative characterization of these molecular and cellular events provides accurate information to evaluate the efficiency of cellular immunotherapy against tumors and understand the impact of an organ's immune status.

Mitochondria targeted redox GFP reveals time and dose dependent onset and progression of mitochondrial oxidation with diverging cell death decisions during photodynamic therapy.

Photodynamic therapy (PDT) is a successful cancer treatment modality. In vitro, in vivo, and clinical studies with different photosensitizers reveal diverging cell fates, including apoptosis, necrosis, autophagy, and non-specific forms of cell death. The mode of action and efficacy of PDT is mediated through free radical generation and is highly dependent on diverse variables such as nature, dose, metabolism of photosensitizer, irradiation energy, and irradiation cycle. Discovery of newer photosensitizers and optimization of PDT approaches to achieve a clinically relevant form of cell death called apoptosis requires better in vitro real-time methods. Oxidative damage and mitochondrial permeabilization are critical signaling events involved in photodamage and apoptosis. Hence, mitochondrial damage detection is an appropriate target signaling for mechanistic evaluation of PDT. We report mitochondria-targeted redox GFP expressing cells as a sensitive system to test and validate important variables of PDT using the photosensitizer 5-Aminolevulinic acid (5-ALA) as a model. An independent FRET-based caspase sensor cell was also used to study the impact of the photosensitizer dosage and irradiation duration on the mode of cell death. The study reveals that the cancer cells expressing mt-roGFP are extremely sensitive to monitor mitochondrial oxidation induced by PDT. The extent of mitochondrial redox changes induced by PDT can be determined using these sensor cells by real-time image-based approaches. These approaches provide sufficient temporal resolution that is required to fine-tune and optimize the PDT conditions. The degree of oxidation of the probe is highly dependent on the dosage of photosensitizer and duration of light irradiation, which determines the nature of cell death. A real-time caspase sensor probe further confirmed that the caspase-dependent and caspase-independent nature of cell death is in high correlation with the extent of mitochondrial oxidation. A condition that triggers rapid and extreme mito-oxidation seems to favor necrosis, while delayed and slowly progressing redox changes contribute to caspase-dependent apoptosis. The study confirms that temporal analysis of mitochondrial oxidation is a reliable biomarker for fine-tuning PDT conditions to achieve the desired outcome. This can be achieved using stable cancer cell lines expressing mitochondria-targeted roGFP by ratiometric imaging.

Comparative Study on the Chemical Structure and In Vitro Antiproliferative Activity of Anthocyanins in Purple Root Tubers and Leaves of Sweet Potato ( Ipomoea batatas).

The structure and in vitro antiproliferative activity of anthocyanins in the root tubers of a sweet potato variety cv. Bhu Krishna and the purple leaves of a promising accession S-1467 were studied with the objectives of understanding the structure-activity relationship and comparing the leaf and tuber anthocyanins. The chemical structure of anthocyanins was determined by high-resolution electrospray ionization mass spectrometry analysis. A fluorescence-resonance-energy-transfer-based caspase sensor probe had been used to study the antiproliferative property, and analysis of the cell cycle was performed after staining with propidium iodide and subsequent fluorescence-activated cell sorting. Structurally, the anthocyanins in root tubers were identical to those in leaves, but there was a difference in the proportion of various aglycones present in both. This has led to distinguishable differences in the antiproliferative activity of leaf and tuber anthocyanins to various cancer cells. All nine anthocyanins were found in acylated forms in both tubers and leaves. However, peonidin derivatives were major anthocyanins in tubers (33.98 ± 1.41 mg) as well as leaves (27.68 ± 1.07 mg). The cyanidin derivatives were comparatively higher in leaves (20.55 ± 0.91 mg) than tubers (9.44 ± 0.94 mg). The tuber and leaf anthocyanins exhibited potential antiproliferative properties to MCF-7, HCT-116, and HeLa cancer cells, and the structure of anthocyanins had a critical role in it. The leaf anthocyanins exhibited significantly higher activity against colon and cervical cancer cells, whereas tuber anthocyanins had a slightly greater effect against breast cancer cells.

Rapid identification of antimicrometastases drugs using integrated model systems with two dimensional monolayer, three dimensional spheroids, and zebrafish xenotransplantation tumors.

Treating systemic metastases at the micrometastatic stage is a potential strategy to inhibit cancer metastasis. This study aims to establish an apoptosis sensor-based platform for rapid, effective, and noninvasive identification of drugs that can inhibit the proliferation of micrometastatic cancer cells. We stably transfected the plasmid DNA encoding the fluorescence resonance energy transfer-based caspase-3 sensor into highly metastatic melanoma B16F10 cells. The resulting B16F10-C3 cells were applied for screening of antiproliferative and proapoptotic drugs in two-dimensional (2D) monolayer, three-dimensional (3D) spheroids, and zebrafish xenotransplantation tumors. All studies were conducted in 96-well plates in a high throughput manner. Fourteen compounds including six chemotherapeutic drugs and eight kinase inhibitors were tested. Thirteen compounds failed the tests due to: Drug resistance, low efficacy, poor pharmacokinetic profile, and/or high side effects to zebrafish. The only compound that passed all tests was pan-phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, which inhibited the proliferation of B16F10-C3 cells in both 2D and 3D cultures. More important, it significantly reduced the xenograft tumor size in zebrafish by decreasing the viability of metastatic cancer cells. Our study suggests that the PI3K/AKT pathway is a potential therapeutic target for the reactivation of tumor dormancy and proliferation of micrometastases. Moreover, this integrated approach is effective for rapid identification of systemic antimetastases drugs.

Guests involved CB[8] capped silver nanoparticles as a means of electrochemical signal enhancement for sensitive detection of Caspase-3

Cysteine aspartase protease (Caspase-3) plays an important role in various diseases. In this work, silver nanoparticles (AgNPs) with electronic properties have been utilized as signal amplification elements for fabricating electrochemical Caspase-3 biosensor. In the presence of Caspase-3, the cleavage of substrate results in the exposure of a free phenylalanine at the end of the peptide, which can bind CB[8] specifically through host-guest reaction. To enhance the electrochemical signal, AgNPs further reacts with CB[8]. Through layer-by-layer assembly of AgNPs, nanostructure was formed via electrostatic cross-linking of AgNPs and CB[8], which can not only prevent the AgNPs’ agglomeration, but also enhance the electrochemical signals. Furthermore, different guests (methylviologen and anthracene) were studied for its electrical transfer efficiency between electrode and AgNPs redox center. Among them, it was found methylviologen/CB[8] complex can increase the electrical conductivity and thus further enhance the Caspase-3 sensor’s sensitivity. Under the optimized detection condition, the detection limit for Caspase-3 is 24.62 pg mL−1. Attribute to its simple and sensitive properties, the fabricated Caspase-3 electrochemical sensor has great potential in clinical applications.

Long-term fluorescence lifetime imaging of a genetically encoded sensor for caspase-3 activity in mouse tumor xenografts

Caspase-3 is known for its role in apoptosis and programmed cell death regulation. We detected caspase-3 activation in vivo in tumor xenografts via shift of mean fluorescence lifetimes of a caspase-3 sensor. We used the genetically encoded sensor TR23K based on the red fluorescent protein TagRFP and chromoprotein KFP linked by 23 amino acid residues (TagRFP-23-KFP) containing a specific caspase cleavage DEVD motif to monitor the activity of caspase-3 in tumor xenografts by means of fluorescence lifetime imaging-Forster resonance energy transfer. Apoptosis was induced by injection of paclitaxel for A549 lung adenocarcinoma and etoposide and cisplatin for HEp-2 pharynx adenocarcinoma. We observed a shift in lifetime distribution from 1.6 to 1.9ns to 2.1 to 2.4ns, which indicated the activation of caspase-3. Even within the same tumor, the lifetime varied presumably due to the tumor heterogeneity and the different depth of tumor invasion. Thus, processing time-resolved fluorescence images allows detection of both the cleaved and noncleaved states of the TR23K sensor in real-time mode during the course of several weeks noninvasively. This approach can be used in drug screening, facilitating the development of new anticancer agents as well as improvement of chemotherapy efficiency and its adaptation for personal treatment.

Identifying and monitoring neurons that undergo metamorphosis-regulated cell death (metamorphoptosis) by a neuron-specific caspase sensor (Casor) in Drosophila melanogaster.

Activation of caspases is an essential step toward initiating apoptotic cell death. During metamorphosis of Drosophila melanogaster, many larval neurons are programmed for elimination to establish an adult central nervous system (CNS) as well as peripheral nervous system (PNS). However, their neuronal functions have remained mostly unknown due to the lack of proper tools to identify them. To obtain detailed information about the neurochemical phenotypes of the doomed larval neurons and their timing of death, we generated a new GFP-based caspase sensor (Casor) that is designed to change its subcellular position from the cell membrane to the nucleus following proteolytic cleavage by active caspases. Ectopic expression of Casor in vCrz and bursicon, two different peptidergic neuronal groups that had been well-characterized for their metamorphic programmed cell death, showed clear nuclear translocation of Casor in a caspase-dependent manner before their death. We found similar events in some cholinergic neurons from both CNS and PNS. Moreover, Casor also reported significant caspase activities in the ventral and dorsal common excitatory larval motoneurons shortly after puparium formation. These motoneurons were previously unknown for their apoptotic fate. Unlike the events seen in the neurons, expression of Casor in non-neuronal cell types, such as glial cells and S2 cells, resulted in the formation of cytoplasmic aggregates, preventing its use as a caspase sensor in these cell types. Nonetheless, our results support Casor as a valuable molecular tool not only for identifying novel groups of neurons that become caspase-active during metamorphosis but also for monitoring developmental timing and cytological changes within the dying neurons.

Bright Bioluminescent BRET Sensor Proteins for Measuring Intracellular Caspase Activity

FRET-based caspase activity probes have become important tools to monitor apoptotic cell signaling. However, their dependence on external illumination is incompatible with light sensitive cells and hampers applications that suffer from autofluorescence and light scattering. Here we report the development of three caspase sensor proteins based on Bioluminescence Resonance Energy Transfer (BRET) that retain the advantages of genetically encoded, ratiometric optical probes but do not require external illumination. These sensors consist of the bright and stable luciferase NanoLuc and the fluorescent protein mNeonGreen, fused together via a linker containing a recognition site for caspase-3, -8, or -9. In vitro characterization showed that each caspase sensor displayed a robust 10-fold decrease in BRET ratio upon linker cleavage, with modest caspase specificity. Importantly, whereas scattering and background fluorescence precluded FRET-based detection of intracellular caspase activity in plate-reader assays, such measurements could be easily performed using our caspase BRET sensors in a high throughput format. The brightness of the BRET sensors also enabled long-term single-cell imaging, allowing BRET-based recording of cell heterogeneity in caspase activity in a heterogenic cell population.

Bioengineered three-dimensional co-culture of cancer cells and endothelial cells: A model system for dual analysis of tumor growth and angiogenesis.

Angiogenesis marks the transformation of a benign local tumor into a life-threatening disease. Many in vitro assays are available on two-dimensional (2D) platforms, however, limited research has been conducted to investigate the behavior of tumors and endothelial cells (ECs) grown on three-dimensional (3D) platforms. This study provides a 3D co-culture spheroid of tumor cells with ECs to study the interplay between ECs and tumor cells. In a 3D co-culture with HepG2 hepatocellular carcinoma (HCC) cells, ECs differentiate to form tubule networks when in co-culture. Addition of angiogenic factors or angiogenesis inhibitors to the model system enhanced or inhibited endothelial differentiation in the 3D model, enabling investigations of the cellular signaling pathways utilized in HCC development. The 3D model demonstrated similar protein expression levels as a HCC xenograft, as well as exhibited upregulation of essential signaling proteins such as Akt/mTor in the 3D model, which is not reflected in the 2D model. The effects of several anti-angiogenic agents, such as sorafenib, sunitinib, and axitinib were analyzed in the 3D co-culture model by utilizing fluorescent proteins and a fluorescence resonance energy transfer (FRET)-based caspase-3 sensor in the ECs, which can detect apoptosis in real time. The apoptotic capability of a drug to inhibit angiogenesis in the 3D model can be easily distinguished via the FRET sensor, and dual screening of anti-angiogenesis and anti-tumor drugs can be achieved in a single step via the 3D co-culture model. In summary, a 3D co-culture model is constructed, where a HCC tumor microenvironment with a hypoxic core and true gradient penetration of drugs is achieved for drug screening purposes and in vitro studies utilizing a small HCC tumor. Biotechnol. Bioeng. 2017;114: 1865-1877. © 2017 Wiley Periodicals, Inc.

High expression of MnSOD promotes survival of circulating breast cancer cells and increases their resistance to doxorubicin.

Understanding the survival mechanism of metastatic cancer cells in circulation will provide new perspectives on metastasis prevention and also shed new light on metastasis-derived drug resistance. In this study, we made it feasible to detect apoptosis of circulating tumor cells (CTCs) in real-time by integrating a fluorescence resonance energy transfer (FRET)-based caspase sensor into one in vitro microfluidic circulatory system, and two in vivo models: zebrafish circulation and mouse lung metastatic model. Our study demonstrated that fluid shear stresses triggered apoptosis of breast cancer cells in circulation by elevating the mitochondrial production of the primary free radical, superoxide anion. Cancer cells with high levels of manganese superoxide dismutase (MnSOD) exhibited stronger resistance to shear force-induced apoptosis and formed more lung metastases in mice. These metastasized cells further displayed higher resistance to chemotherapeutic agent doxorubicin, which also generates superoxide in mitochondria. Specific siRNA-mediated MnSOD knockdown reversed all three phenotypes. Our findings therefore suggest that MnSOD plays an important integrative role in supporting cancer cell survival in circulation, metastasis, and doxorubicin resistance. MnSOD can serve as a new biomarker for identifying metastatic CTCs and a novel therapeutic target for inhibiting metastasis and destroying doxorubicin-resistant breast cancer cells.

BioSpotlight / Citations

BioTechniquesVol. 60, No. 2 BioSpotlight / CitationsOpen AccessBioSpotlight / CitationsNathan S. Blow & Nijsje DormanNathan S. BlowSearch for more papers by this author & Nijsje DormanSearch for more papers by this authorPublished Online:16 Mar 2018https://doi.org/10.2144/000114375AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit Caspase sensors move into the redFluorescent biosensors have proven to be useful tools for understanding cellular physiology. For example, several such biosensors have been developed to study biochemical signaling during programmed cell death. In this issue of BioTechniques, Konstantin Lukyanov and his colleagues from the Institute of Bioorganic Chemistry in Moscow, Russia sought to further enhance the apoptosis biosensor toolkit with their development of a new caspase-3 biosensor. Although other caspase-3 biosensors are available, Lukyanov's team was interested in designing very far red-shifted sensors that would enable multi-parameter microscopy and whole-body imaging. The authors fused one of two far-red fluorescent proteins, mKate2 or eqFP650, to the infrared fluorescent protein iRFP through a linker containing a DEVD caspase-3 cleavage site. Upon caspase-3 activation, cleavage at the DEVD site releases the fluorescent proteins, thus changing intracellular fluorescence levels. The authors demonstrated the ability of their biosensors to detect caspase-3 activation and also demonstrated simultaneous imaging of an EGFP biosensor.See “Genetically encoded far-red fluorescent sensors for caspase-3 activity”Trimming optimizes NGS dataNew developments in next-generation sequencing (NGS) methodologies are emerging as researchers with diverse interests embrace the technology. For environmental studies, the high-throughput and sequencing depth offered by NGS systems means researchers can explore the greater diversity of the microbial world. But there are challenges in adapting traditional sequencing approaches to NGS instrumentation. In this issue, Anita Mäki and her colleagues from the University of Jyväskylä in Finland present a new method for sequencing long PCR amplicons using NGS. Traditionally, several steps are required to prepare such samples for sequencing, including barcode indexing, adapter ligation, and size selection. Mäki and colleagues devised a clever two-step PCR-based protocol for barcoding and trimming of long PCR products that also optimizes data output. Their strategy uses a 5′ universal sequencing adapter along with a universal barcoded M13-tailed primer. The 3′ adapter is then ligated to sheared amplicon fragments, a step that also enhances the number of 5′-end reads. The authors validated their technique for use in environmental studies by sequencing a variety of archaeal 16S rRNA genes.See “A practical method of barcoding and size-trimming PCR templates for amplicon sequencing”Reversible fluorescent labeling of proteinsThe palette of options for fluorescently labeling proteins has never been more expansive, but notably absent is a strategy for instantaneous, fully reversible labeling. Plamont et al. fill this gap with Y-FAST (yellow fluorescence-activating and absorption-shifting tag) and the fluorogen HMBR (4-hydroxy-3-methylbenzylidene-rhodanine). Y-FAST is an engineered version of the 14-kDa photoactive yellow protein that, by binding HMBR, specifically activates its fluorescence. Y-FAST is about half the size of GFP-like proteins, active upon folding, resolutely monomeric, and works in various cell types and organelles. However, its key advantage is that its fluorescence can be rapidly turned on and off. Fluorescent labeling is evident within 10 seconds of adding HMBR to culture medium, and the signal disappears just as quickly when cells are washed. The authors provide a movie showing 10 on-off cycles of the fluorescent label, and (on a slower scale) demonstrate on/off switching in zebrafish embryos. This property suggests applications of Y-FAST/HMBR for multiplex imaging by sequential labeling.M.A. Plamont et al. Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo. Proc Natl Acad Sci U S A. [Epub ahead of print, December 28, 2015; doi: 10.1073/pnas.1513094113.]Optogenetics for forward genetic screensThe blue light-activated mini singlet oxygen generator (miniSOG) has been used to generate reactive oxygen species (ROS) for cell ablation and protein inactivation. Because ROS can mutate DNA, Noma and Jin examined whether miniSOG could offer an alternative to chemical mutagens for forward genetic screens in C. elegans. To direct miniSOG to DNA, the authors fused the photosensitizer to a histone protein. Without photoinduction, worms transgenic for His-mSOG had longevity and reproduction equivalent to the wild type. Exposure to 30 minutes of blue light from an LED source, however, produced mutant phenotypes. In genetic suppressor screens, the mutation rate was ∼4-fold lower than that of EMS, a popular chemical mutagen. However, His-mSOG mutagenesis generates a different spectrum of mutation types, for example G:C to T:A transversions, which are rarely obtained with EMS mutagenesis. This miniSOG-based technique should be extendable to other DNA-binding protein fusions, and to Drosophila and zebrafish.K. Noma & Y. Jin. 2015. Optogenetic mutagenesis in Caenorhabditis elegans. Nat Commun. 6:8868.FiguresReferencesRelatedDetails Vol. 60, No. 2 Follow us on social media for the latest updates Metrics History Published online 16 March 2018 Published in print February 2016 Information© 2016 Author(s)PDF download

Application of a fluorescence resonance energy transfer (FRET)-based biosensor for detection of drug-induced apoptosis in a 3D breast tumor model.

Two-dimensional (2D) cultures are commonly used for testing drug effects largely because of their easy maintenance. But they do not represent the spatial interactions of the cells within a tumor. Three-dimensional (3D) cultures can overcome those limitations thus mimicking the architecture of solid tumor. However, it is not easy to evaluate drug effects in 3D culture for a long time. This necessitates the development of a real-time and longitudinal analysis of 3D platforms. In this study, we transfected the plasmid DNA encoding the fluorescence resonance energy transfer (FRET)-based biosensor into human breast cancer cells and generated two cell lines of MCF7-C3 and MDA-MB-231-C3 (231-C3) cells. We used them to determine the activation of caspase-3, whereby healthy cells appear green and apoptotic cells appear blue by FRET imaging. As the caspase sensors can be constantly produced within the cells and quickly respond to caspase activation, we hypothesized that these sensor cells will allow longitudinal detection of apoptosis. MCF7-C3 and 231-C3 spheroids were generated and subjected to histological examination, gene expression studies, drug treatment, and FRET analyses. Our results demonstrated that MCF7-C3 cells formed tight 3D spheroids, and mimicked in vivo tumor architecture. The mRNA level of tumorigenic markers such as MMP-9, SOX2, and OCT4A were much higher in cells cultured in 3D than in 2D. Finally, upon treatment with paclitaxel, the FRET effect was reduced at the rim of MCF7-C3 spheroids in a dose and time-dependent manner demonstrating these sensor cells can be used to determine drug-induced apoptosis in a 3D set up. This study supports the possibility of developing a biosensor-based in vitro 3D breast tumor model for determination of anti-cancer drug penetration over a long course of time in a non-invasive manner.

A novel bicistronic sensor vector for detecting caspase-3 activation

Apoptosis is involved in pathological cell death of a wide range of human diseases. One of the most important biochemical markers of apoptosis is activation of caspase-3. Ability to detect caspase-3 activation early in the pathological process is important for determining the timing for interfering with apoptosis initiation and prevention of cell damage. Techniques allowing detection of caspase-3 activity at a single cell level show increased sensitivity, compared to biochemical assays; therefore, we developed a novel bicistronic caspase-3 sensor vector enabling detection of caspase-3 activity in individual cells. We employed green fluorescent protein (GFP) as a reporter for caspase-3 activation in our constructs and assessed the functionality of the generated constructs in transiently transfected Neuro2A and HEK293 cells under basal conditions and following application of okadaic acid (OA) or staurosporine (STS) to induce apoptosis. To ensure responsiveness of the new sensor vector to active caspase-3, we co-transfected the sensor with plasmid(s) overexpressing active caspase-3 and quantified GFP fluorescence using a plate reader. We observed an increase in GFP expression in cells transfected with the new bicistronic caspase-3 sensor in response to both OA and STS. We also showed a significant increase in GFP fluorescence intensity in cells co-expressing the sensor with the plasmid(s) encoding active caspase-3. We generated a novel bicistronic caspase-3 sensor vector which relies on a transcription factor/response element system. The obtained sensor combines high sensitivity of the single cell level detection with the possibility of automated quantification.