Live Mammalian Cells Research Articles

A dimerization-based fluorogenic dye-aptamer module for RNA imaging in live cells.

Live-cell imaging of RNA has remained a challenge because of the lack of naturally fluorescent RNAs. Recently developed RNA aptamers that can light-up small fluorogenic dyes could overcome this limitation, but they still suffer from poor brightness and photostability. Here, we propose a concept of cell-permeable fluorogenic dimer of sulforhodamine B dyes (Gemini-561) and corresponding dimerized aptamer (o-Coral) that can drastically enhance performance of the current RNA imaging method. The unprecedented brightness and photostability together with high affinity of this complex allowed, for the first time, direct fluorescence imaging in live mammalian cells of RNA polymerase-III transcription products as well as messenger RNAs labelled with a single copy of the aptamer, i.e. without tag multimerization. The developed fluorogenic module enables fast and sensitive detection of RNA inside live cells, while the proposed design concept opens the route to new generation of ultrabright RNA probes.

Detection of Nav1.5 Conformational Change in Mammalian Cells Using the Noncanonical Amino Acid ANAP

Nav1.5 inactivation is necessary for healthy conduction of the cardiac action potential. Genetic mutations of Nav1.5 perturb inactivation and cause potentially fatal arrhythmias associated with long QT syndrome type 3. The exact structural dynamics of the inactivation complex is unknown. To sense inactivation gate conformational change in live mammalian cells, we incorporated the solvatochromic fluorescent noncanonical amino acid 3-((6-acetylnaphthalen-2-yl)amino)-2-aminopropanoic acid (ANAP) into single sites in the Nav1.5 inactivation gate. ANAP was incorporated in full-length and C-terminally truncated Nav1.5 channels using mammalian cell synthetase-tRNA technology. ANAP-incorporated channels were expressed in mammalian cells, and they exhibited pathophysiological function. A spectral imaging potassium depolarization assay was designed to detect ANAP emission shifts associated with Nav1.5 conformational change. Site-specific intracellular ANAP incorporation affords live-cell imaging and detection of Nav1.5 inactivation gate conformational change in mammalian cells.

Multi-threshold and multi-input DNA logic design style for profiling the microRNA biomarkers of real cancers.

Early detection of cancer is very critical because it can reduce the treatment risk and cost. MicroRNAs (miRNAs) have been introduced in recent years as an efficient class of biomarkers for cancer early detection. Now, real-time polymerase chain reaction has been used to profile the miRNA expression, which is costly, time consuming and low accuracy. Most recently, DNA logic gates are used to detect the miRNA expression level that is more accurate and faster than previous methods. The DNA-based logic gates face with serious challenges such as the large complexity and low scalability. In this study, the authors proposed a methodology to design multi-threshold and multi-input DNA-based logic gates in response to specific miRNA inputs in live mammalian cells. The proposed design style can simultaneously recognise multiple miRNAs with different rising and falling thresholds. The design style has been evaluated on the lung cancer biomarkers and the experimental results show the efficiency of the proposed method in terms of accuracy, efficiency and speed.

Biothiol-Activatable Bioluminescent Coelenterazine Derivative for Molecular Imaging in Vitro and in Vivo.

There is a high demand for sensitive biothiol probes targeting cysteine, glutathione, and homocysteine. These biothiols are known as playing essential roles to maintain homeostasis and work as indicators of many diseases. This work presents a bioluminescent probe (named AMCM) to detect biothiols in live mammalian cells and in vivo with a limit of detection of 0.11 μM for cysteine in solution and high selectivity for biothiols, making it suitable for real-time biothiol detection in biological systems. Upon application to live cells, AMCM showed low cytotoxicity and sensitively reported bioluminescence in response to changes of biothiol levels. Furthermore, a bioluminescence resonance energy transfer system consisting of AMCM combined with the near-infrared fluorescent protein iRFP713 was applied to in vivo imaging, with emitted tissue-permeable luminescence in living mice. In summary, this work demonstrates that AMCM is of high practical value for the detection of biothiols in living cells and for deep tissue imaging in living animals.

Membrane fusogenic high-density lipoprotein nanoparticles

Membrane fusion under mildly acidic pH occurs naturally during viral infection in cells and has been exploited in the field of nanoparticle-mediated drug delivery to circumvent endosomal entrapment of the cargo. Herein, we aimed to confer virus-like fusogenic activity to HDL in the form of a ca. 10-nm disc comprising a discoidal lipid bilayer and two copies of a lipid-binding protein at the edge. A series of HDL mutants were prepared with a mixture of three lipids and a cell-penetrating peptide (TAT, penetratin, or Arg8) fused to the protein. In a lipid-mixing assay with anionic liposomes at pH 5.5, one HDL mutant showed the fusogenic activity higher than known fusogenic liposomes. In live mammalian cells, this HDL mutant showed high plasma membrane-binding activity in the presence of serum independent of pH. In the absence of serum, a mildly acidic pH dependency for binding to the plasma membrane and the subsequent lipid mixing between them was observed for this mutant. We propose a novel strategy to develop HDL-based drug carriers by taking advantage of the HDL lipid/protein composite structure.

DNA-templated synthesis of biomimetic cell wall for nanoencapsulation and protection of mammalian cells

Mammalian cells are different from plant and microbial cells, having no exterior cell walls for protection. Environmental assaults can easily damage or destroy mammalian cells. Thus, the ability to develop a biomimetic cell wall (BCW) on their plasma membrane as a shield can advance various applications. Here we demonstrate the synthesis of BCW with a framing template and a crosslinked matrix for shielding live mammalian cells. The framing template is a supramolecular DNA structure. The crosslinked matrix is a polyelectrolyte complex made of alginate and polylysine. As the entire procedure of BCW synthesis is strictly operated under physiological conditions, BCW-covered mammalian cells can maintain high bioactivity. More importantly, the data show that BCW can shield live mammalian cells from not only physical assaults but also biological assaults. Thus, this study has successfully demonstrated the synthesis of BCW on live mammalian cells with great potential of shielding them from environmental assaults.

Molecular Imaging of Retinoic Acids in Live Cells Using Single-Chain Bioluminescence Probes.

Retinoic acid (RA) is a key metabolite necessary for embryonic development and differentiation in vertebrates. We demonstrate the utility of genetically encoded, ligand-activatable single-chain bioluminescence probes for detecting RAs from different biological sources. We examined 13 different molecular designs to identify an efficient single-chain probe that can quantify RA with significant sensitivity. The optimal probe consisted of four components: the N- and C-terminal fragments of artificial luciferase variant-16 (ALuc16), the ligand binding domain of retinoic acid receptor α (RARα LBD), and an LXXLL interaction motif. This probe showed a 5.2-fold greater bioluminescence intensity in response to RA when compared to the vehicle control in live mammalian cells. The probe was highly selective to all-trans-RA (at-RA), and highly sensitive in determining at-RA levels in cells derived from tumor xenografts created using MDA-MB-231 cells engineered to stably express the probe. We also detected RA levels in serum and cerebrospinal fluid. Using this probe, the detection limit for at-RA was ∼10-9.5 M, with a linear range of two orders. We present a highly useful technique to quantitatively image endogenous at-RA levels in live mammalian cells expressing novel single-chain bioluminescence probes.

Fluorogenic Photoaffinity Labeling of Proteins in Living Cells.

Genetically encoded fluorescent proteins or small-molecule probes that recognize specific protein binding partners can be used to label proteins to study their localization and function with fluorescence microscopy. However, these approaches are limited in signal-to-background resolution and the ability to temporally control labeling. Herein, we describe a covalent protein labeling technique using a fluorogenic malachite green probe functionalized with a photoreactive cross-linker. This enables a controlled covalent attachment to a genetically encodable fluorogen activating protein (FAP) with low background signal. We demonstrate covalent labeling of a protein in vitro as well as in live mammalian cells. This method is straightforward, displays high labeling specificity, and results in improved signal-to-background ratios in photoaffinity labeling of target proteins. Additionally, this probe provides temporal control over reactivity, enabling future applications in real-time monitoring of cellular events.

Visualizing mRNA in live mammalian cells.

mRNAs play essential role in gene expression and regulation. Direct visualization of specific intracellular mRNAs can provide key information about mRNA distribution and dynamics that accelerating study of their functionality. This highlights the necessity of characterizing mRNA in mammalian cells with good spatial and temporal resolution. Live cell imaging and tracking of mRNA remains a challenge due to their relatively short half-life and lack of versatile imaging probes. This review provides an overview of current methods of live-cell mRNA imaging, including exogenous hybridization probes and genetically encoded strategies. It then focuses on the development and application of aptamer initiated fluorescence complementation (AiFC) method for real-time imaging of mammalian mRNAs and includes a detailed protocol for observing β-actin mRNA in HeLa cells.

Continuous translation of circularized mRNA improves recombinant protein titer

Recent success in demonstrating the translation of circular RNA open reading frames or circular mRNA, may offer a new avenue for improving recombinant protein production from cell and cell-free expression platforms. Initiation and termination are two rate limiting steps of translation. Circular RNA as a class of RNA is defined by covalent joining of terminal ends to give a closed loop structure. By encoding a gene lacking a stop codon on a circular RNA molecule an infinite open reading frame is generated permitting continuously translating circular mRNA (CTC mRNA). CTC mRNAs have shown promise in enhancing the production of multimeric polyproteins in bacterial cell-free expression systems. Problems arise when homogenous, functional post-translationally modified protein is required. To produce post-translationally modified, secreted protein from an CTC mRNA we investigated co-translational cleavage of nascent polypeptide chains by incorporating a 2 A “self-cleavage” peptide motif. Using a model recombinant human glycoprotein Erythropoietin (EPO) we demonstrate for the first time the ability to produce secreted protein from an infinite circular mRNA in live mammalian cells. Both cell-specific and volumetric productivity were improved by using circular mRNAs. This study pioneers the potential of recombinant protein production from CTC mRNA in mammalian cells.

Measuring Small-molecule Inhibition of Protein Interactions in Live Cells Using FLIM-FRET.

This protocol was designed to quantitatively measure small-molecule displacement of proteins in live mammalian cells using fluorescence lifetime imaging microscopy-Förster resonance energy transfer (FLIM-FRET). Tumour cell survival is often dependent on anti-apoptotic proteins, which bind to and inhibit pro-apoptotic proteins, thus preventing apoptosis. Small-molecule inhibitors that selectively target these proteins (termed BH3-mimetics) are therefore a promising avenue for the treatment of several cancers. Previous techniques used to study the efficacy of these drugs often use truncated versions of both pro- and anti-apoptotic proteins, as they are membrane bound and hydrophobic in nature. As a result, the true efficacy of these drugs to displace full-length pro-apoptotic proteins in their native environment within a cell is poorly understood. This protocol describes FLIM-FRET methods to directly measure the displacement (or lack of displacement) of full-length Bcl-2 family proteins in live mammalian cells.

Electroosmotic extraction coupled to mass spectrometry analysis of metabolites in live cells.

Quantitative mass spectrometry analysis of metabolites at a single-cell level is critical to understanding the cell functionality and heterogeneity. To preserve cell viability after extraction, the extracted volume needs to be precisely controlled at a subpicoliter-to-picoliter level. Recently, we developed a volume-controlled, and highly sensitive approach for live cell analysis at a single-cell level by integrating electroosmotic extraction and nano-electrospray ionization mass spectrometry (nanoESI MS) analysis. Herein, we use outer epidermal cells of Allium cepa as a model system to present the details of our workflow, including detailed descriptions of the experimental setup for live cell analysis, preparation of the extraction nanopipette, establishment of calibration curves, and extraction and quantification of glucose in an individual onion cell. The capability of this procedure for quantitative live cell analysis has been demonstrated by accurate quantification of glucose in Allium cepa. In principle, our approach is applicable to identification and quantification of metabolites in live mammalian cells.

Bio-orthogonal chemistry-based method for fluorescent labelling of ribosomal RNA in live mammalian cells.

A bio-orthogonal chemistry-based approach for fluorescent labelling of ribosomal RNA is described. It involves an adenosine analogue modified with trans-cyclooctene and masked 5'-phosphate group using aryl phosphoramidate. The incorporation into rRNA has been confirmed using agarose gel electrophoresis, as well as a highly sensitive UHPLC-MS/MS method. Fluorescent labelling of rRNA has been achieved in live HeLa cells via an inverse electron demand Diels-Alder reaction with a tetrazine conjugated to an Oregon Green fluorophore. This communication describes the stepwise approach that led to the development and characterization of the probe. The results demonstrate a new strategy towards development of future fluorescent probes to investigate the biochemistry of nucleic acids.

Highly bright and stable NIR-BRET with blue-shifted coelenterazine derivatives for deep-tissue imaging of molecular events in vivo.

Background: Bioluminescence imaging (BLI) is one of the most widely used optical platforms in molecular imaging, but it suffers from severe tissue attenuation and autoluminescence in vivo.Methods: Here, we developed a novel BLI platform on the basis of bioluminescence resonance energy transfer (BRET) for achieving a ~300 nm blue-to-near infrared shift of the emission (NIR-BRET) by synthesizing an array of 18 novel coelenterazine (CTZ) derivatives, named “Bottle Blue (BBlue)” and a unique iRFP-linked RLuc8.6-535SG fusion protein as a probe.Results: The best NIR-BRET was achieved by tuning the emission peaks of the CTZ derivatives to a Soret band of the iRFP. In mammalian cells, BBlue2.3, one of the CTZ derivatives, emits light that is ~50-fold brighter than DBlueC when combined with RLuc8.6-535SG, which shows stable BL kinetics. When we used a caged version of BBLue2.3, it showed a BL half decay time of over 60 minutes while maintaining the higher signal sensitivity. This NIR BL is sufficiently brighter to be used for imaging live mammalian cells at single cell level, and also for imaging metastases in deep tissues in live mice without generating considerable autoluminescence. A single-chain probe developed based on this BLI platform allowed us to sensitively image ligand antagonist-specific activation of estrogen receptor in the NIR region.Conclusion: This unique optical platform provides the brightest NIR BLI template that can be used for imaging a diverse group of cellular events in living subjects including protein‒protein interactions and cancer metastasis.

A novel reactive turn-on probe capable of selective profiling and no-wash imaging of Bruton's tyrosine kinase in live cells.

Bruton's tyrosine kinase, an essential mediator of B cell receptor (BCR) signalling, has been validated as an effective therapeutic target in oncology. Development of chemical sensors capable of precise detection of its expression and function is of great importance for cancer diagnosis and therapy. Here, a dual-purpose probe, IB-4, was developed with excellent inhibitory activity (IC50 = 35 nM), and has been demonstrated to be suitable for simultaneous imaging endogenous BTK activity and studying its target engagement in live cells for the first time. The unique turn-on design endows the probe with excellent sensitivity and selectivity toward BTK under both in vitro and in situ settings, and can be further extended to other irreversible inhibitors for protein characterization, quantification and inhibition studies.

Visualization of Genomic Loci in Living Cells with BiFC-TALE.

Tracking the dynamics of genomic loci is essential for understanding a variety of cellular processes. However, earlier methods have all suffered from a low signal-to-background ratio (SBR), mainly caused by the background fluorescence from diffuse full-length fluorescent proteins in the nucleus. We have developed a novel method (BiFC-TALE) for labeling and tracking genomic loci in live mammalian cells, combining bimolecular fluorescence complementation (BiFC) and transcription activator-like effector (TALE) technologies. Since only the sequences-targeted BiFC fragments can be pulled together by TALE modules to recombine intact fluorescent proteins, the background fluorescence in the living nucleus can be largely reduced, which significantly improves SBR. Using telomere and centromere labeling as examples, this unit describes in detail the design and implementation of BiFC-TALE system. © 2018 by John Wiley & Sons, Inc.

Microinjection of Live Mammalian Cells: A Delivery Method that Provides Added Versatility to the Study of Cellular Function.

Microinjection is a technique that allows for the delivery of a diverse array of compounds and biomolecules into live mammalian cells. As a result, the behavior of injected biomolecules and their resulting effects can be observed in the native environments of mammalian cells in real time. This capability allows for more accurate observations and conclusions about biological systems of interest. This chapter discusses the protocol and guidelines to successfully microinject live mammalian cells to maintain their viability. This chapter outlines considerations into the preparation of samples and the microscopy setup as well as a description of a stepwise presentation of the methods for proper execution of a microinjector. The presentation of the technique serves as a starting point to understanding how to apply microinjection to a cellular system compatible with a wide array of live-cell imaging techniques.

Modeling of Soft Sample Deformation in Atomic Force Microscope Imaging: Live Mammalian Cell Example

AbstractA model of the scanning‐caused soft sample deformation during atomic force microscope contact‐mode (CM) imaging is developed. CM imaging has been widely used for topography characterization of live biological samples such as live cells. However, due to the intrinsic softness of these samples, the sample surface deformation caused by the probe–sample interaction leads to significant error in the topography images obtained, and damage to the live biological sample. Although the deformation can be reduced by imaging at a rather slow scan rate (e.g., less than 0.2 Hz), such a low‐speed imaging not only is time consuming, but also inevitably induces large temporal error. In this work, the scanning‐caused surface deformation of soft samples is modeled and quantified, including live biological samples in CM imaging. Effects of both the scanning and the coupling between the vertical and the lateral deformation on the deformation are characterized. The proposed deformation model is validated by implementing it to quantify the topography image difference caused by the scanning‐caused surface deformation of live prostate cancer cells imaged at two different (high and low) speeds, respectively.

Transduction and transfection of difficult-to-transfect cells: Systematic attempts for the transfection of protozoa Leishmania.

Cell-penetrating peptides (CPPs) are used to internalize different cargoes, including DNA, into live mammalian and plant cells. Despite many cells being easily transfected with this approach, other cells are rather "difficult" or "hard to transfect," including protist cells of the genus Leishmania. Based on our previous results in successfully internalizing proteins into Leishmania tarentolae cells, we used single CPPs and three different DNA-binding proteins to form protein-like complexes with plasmids covered with CPPs. We attempted magnetofection, electroporation, and transfection using a number of commercially available detergents. While complex formation with negatively charged DNA required substantially higher amounts of CPPs than those necessary for mostly neutral proteins, the cytotoxicity of the required amounts of CPPs and auxiliaries was thoroughly studied. We found that Leishmania cells were indeed susceptible to high concentrations of some CPPs and auxiliaries, although in a different manner compared with that for mammalian cells. The lack of successful transfections implies the necessity to accept certain general limitations regarding DNA internalization into difficult-to-transfect cells. Only electroporation allowed reproducible internalization of large and rigid plasmid DNA molecules through electrically disturbed extended membrane areas, known as permeable membrane macrodomains.

Visualizing Spatiotemporal Dynamics of Intercellular Mechanotransmission upon Wounding.

During cell-to-cell communications, the interplay between physical and biochemical cues is essential for informational exchange and functional coordination, especially in multicellular organisms. However, it remains a challenge to visualize intercellular signaling dynamics in single live cells. Here, we report a photonic approach, based on laser microscissors and Förster resonance energy transfer (FRET) microscopy, to study intercellular signaling transmission. First, using our high-throughput screening platform, we developed a highly sensitive FRET-based biosensor (SCAGE) for Src kinase, a key regulator of intercellular interactions and signaling cascades. Notably, SCAGE showed a more than 40-fold sensitivity enhancement than the original biosensor in live mammalian cells. Next, upon local severance of physical intercellular connections by femtosecond laser pulses, SCAGE enabled the visualization of a transient Src activation across neighboring cells. Lastly, we found that this observed transient Src activation following the loss of cell-cell contacts depends on the passive structural support of cytoskeleton but not on the active actomyosin contractility. Hence, by precisely introducing local physical perturbations and directly visualizing spatiotemporal transmission of ensuing signaling events, our integrated approach could be broadly applied to mimic and investigate the wounding process at single-cell resolutions. This integrated approach with highly sensitive FRET-based biosensors provides a unique system to advance our in-depth understanding of molecular mechanisms underlying the physical-biochemical basis of intercellular coupling and wounding processes.