Live Mammalian Cells Research Articles

BioSpotlight / Citations

BioTechniquesVol. 63, No. 2 BioSpotlight / CitationsOpen AccessBioSpotlight / CitationsPatrick C.H. Lo, & Nathan S. BlowPatrick C.H. LoSearch for more papers by this author, & Nathan S. BlowSearch for more papers by this authorPublished Online:16 Mar 2018https://doi.org/10.2144/000114572AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit Taking charge of extracellular vesiclesExtracellular vesicles (EVs) play important roles in long-distance intercellular communication and are potentially useful as biomarkers. EVs have been isolated from body fluids by centrifugation, size exclusion chromatography, filtration, precipitation, and affinity purification; however, these methods vary in the extent of contamination of the isolated EVs with non-EV protein complexes and RNAs. In this issue of BioTechniques, Maja Kosanović and her colleagues at the University of Belgrade present a new method for isolating EVs that reduces such contamination. Given that they are negatively charged due to their surface molecules, the team tested ion-exchange chromatography (IEC) as an approach for purifying EVs. Using amniotic fluid as the source of EVs, they compared the gold-standard method, consisting of ultracentrifugation followed by sucrose density gradient centrifugation, with their novel method of ultracentrifugation followed by IEC. As demonstrated by protein electrophoresis and immuno- and lectin blotting, the use of IEC significantly reduced the contamination seen with the gold-standard method.See “Ion-exchange chromatography purification of extra-cellular vesicles” on page 65.Rapid and no-wash live-cell stainingLive-cell fluorescent dyes are critical tools for cell biology research, allowing the visualization and tracking of cells in various assays. In this issue, Alexey A. Pakhomov and his colleagues at the Russian Academy of Sciences describe a new fluorescent dye for rapid, no-wash staining of live cells. This dye is derived from boron dipyrromethene (BODIPY), a molecule with numerous other derivatives that are used as fluorescent sensors and for conjugation to biomolecules. Some of these derivatives are lipophilic, binding to the plasma and subcellular membranes, and they tend to aggregate in polar media such as the cytosol and extracellular space, which results in aggregation-caused quenching. The team tested these derivatives for use in live-cell staining and demonstrated that one of them immediately stained the plasma and subcellular membranes of live mammalian cells with high contrast, without the need for washing. In fact, washing the stained cells with PBS removed the dye, allowing the cells to be stained with another dye of the same color.See “BODIPY-based dye for no-wash live-cell staining and imaging” on page 77.Two-tailed miRNA quantificationThe analysis of microRNAs (miRNAs) is challenging given the short sequence lengths of these molecules. In an attempt to design a more specific and sensitive assay to quantify miRNA expression patterns, Androvic et al. developed a new method they named Two-tailed RT-qPCR. This technique uses a unique primer design incorporating two specific hemiprobes, which are complementary to the miRNA of interest and separated by a tether that can fold into a hairpin to prevent non-specific inter-actions. The reverse transcription reaction to generate cDNA is primed from the 3´-end hemiprobe, while the 5´-end hemiprobe is displaced. After reverse transcription, the resulting cDNA is quantified using two different target-specific primers. The authors showed that Two-tailed RT-qPCR has the sensitivity to detect as few as 10 target miRNA molecules while providing a dynamic range of 7 logs. This new approach should be of particular interest to those seeking highly accurate miRNA quantification.Androvic, P. et al. 2017. Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification. Nucleic Acids Res. doi:10.1093/nar/gkx588Massive cell separationSingle-cell sequencing has become commonplace in life science research. By studying single cells, scientists are learning about cellular heterogeneity, lineage determination, and hierarchical relationships. In a new article in BMC Genomics, Goldstein et al. take single-cell sequencing high-throughput with their description of a massively parallel single-cell profiling pipeline. The authors took advantage of the ICELL8 system, which enables the separation of cell mixtures into 5184 nanowells and then uses imaging to locate wells possessing only a single cell. Once a nanowell containing a single cell is identified, in-well mRNA isolation and barcoded-cDNA generation is performed, after which the resulting cDNA from multiple wells is pooled for sequence analysis. Using the pipeline, the authors were able to sequence transcriptomes from more than 1000 cultured human and mouse cells, along with nearly 500 mouse pancreatic islet cells.Goldstein, L.D. et al. 2017. Massively parallel nanowell-based single-cell gene expression profiling. BMC Genomics. 18:519.FiguresReferencesRelatedDetails Vol. 63, No. 2 Follow us on social media for the latest updates Metrics History Published online 16 March 2018 Published in print August 2017 Information© 2017 Author(s)PDF download

Fluorescence imaging mitochondrial copper(II) via photocontrollable fluorogenic probe in live cells

Monitoring mitochondrial derived copper(II) in live cells is highly demanded, but accurately detecting is unmet due to the interference with cytoplasmic copper(II). Herein, we have reported the design, synthesis and characterization of photocontrollable fluorogenic probe, MCu-3, which is equipped with a photo-labile group (nitrobenzyl group) and mitochondria targeting unit (triphenylphosphonium salt). This novel probe showed an intense fluorescence enhancement in response to copper(II) without interference from other metal cations in the biological condition (pH 6–9). The detection limit is 1.7×10−7mol/L in HEPES buffer. The confocal fluorescence imaging results demonstrated MCu-3 can visualize mitochondrial copper(II) in live mammalian cells. The clear advantage of our photocontrollable method is successful to avoid the influence of cytoplasmic copper(II) during mitochondria specific detection.

Dynamic Organization of Chromatin Domains Revealed by Super-Resolution Live-Cell Imaging

The eukaryotic genome is organized within cells as chromatin. For proper information output, higher-order chromatin structures can be regulated dynamically. How such structures form and behave in various cellular processes remains unclear. Here, by combining super-resolution imaging (photoactivated localization microscopy [PALM]) and single-nucleosome tracking, we developed a nuclear imaging system to visualize the higher-order structures along with their dynamics in live mammalian cells. We demonstrated that nucleosomes form compact domains with a peak diameter of ∼160nm and move coherently in live cells. The heterochromatin-rich regions showed more domains and less movement. With cell differentiation, the domains became more apparent, with reduced dynamics. Furthermore, various perturbation experiments indicated that they are organized by a combination of factors, including cohesin and nucleosome-nucleosome interactions. Notably, we observed the domains during mitosis, suggesting that they act as building blocks of chromosomes and may serve as information units throughout the cell cycle.

Simultaneous Imaging of Endogenous Survivin mRNA and On-Demand Drug Release in Live Cells by Using a Mesoporous Silica Nanoquencher.

The design of multifunctional drug delivery systems capable of simultaneous target detection, imaging, and therapeutics in live mammalian cells is critical for biomedical research. In this study, by using mesoporous silica nanoparticles (MSNs) chemically modified with a small-molecule dark quencher, followed by sequential drug encapsulation, MSN capping with a dye-labeled antisense oligonucleotide, and bioorthogonal surface modification with cell-penetrating poly(disulfide)s, the authors have successfully developed the first mesoporous silica nanoquencher (qMSN), characterized by high drug-loading and endocytosis-independent cell uptake, which is able to quantitatively image endogenous survivin mRNA and release the loaded drug in a manner that depends on the survivin expression level in tumor cells. The authors further show that this novel drug delivery system may be used to minimize potential cytotoxicity encountered by many existing small-molecule drugs in cancer therapy.

Optogenetics: Switching with red and blue.

Multiple optogenetic technologies are required to control biological activity simultaneously with different colors of light. Optimizing a near-infrared-induced heterodimerization system, which can be combined with blue-light-controlled domains, enables precise spatiotemporal control of target molecules in live mammalian cells.

Application of Nanosecond Laser Photolysis Protein Footprinting to Study EGFR Activation by EGF in Cells

Mass spectrometry-based protein footprinting emerged as a useful technology to understand protein ligand interactions in vitro. We have previously demonstrated the application of footprinting in live E. coli cells. Here, we further optimized an ultrafast laser photolysis hydroxyl radical footprinting method and applied it to study the interaction of EGF and EGFR in live mammalian cells. This method used a nanosecond laser to photochemically generate a burst of hydroxyl radicals in situ in-cell suspension to oxidize the amino acids on the protein surface. Mass spectrometric analysis of the thus modified peptides was interpreted to probe the solvent-accessible surface areas of the protein in its native biological state with and without EGF activation. Our footprinting data agreed with the two relevant EGFR crystal structures, indicating that this in-cell laser photolysis footprinting technique is a valid approach to study the structural properties of integral membrane proteins directly in the native environment.

Correlative imaging across microscopy platforms using the fast and accurate relocation of microscopic experimental regions (FARMER) method.

Imaging specific regions of interest (ROIs) of nanomaterials or biological samples with different imaging modalities (e.g., light and electron microscopy) or at subsequent time points (e.g., before and after off-microscope procedures) requires relocating the ROIs. Unfortunately, relocation is typically difficult and very time consuming to achieve. Previously developed techniques involve the fabrication of arrays of features, the procedures for which are complex, and the added features can interfere with imaging the ROIs. We report the Fast and Accurate Relocation of Microscopic Experimental Regions (FARMER) method, which only requires determining the coordinates of 3 (or more) conspicuous reference points (REFs) and employs an algorithm based on geometric operators to relocate ROIs in subsequent imaging sessions. The 3 REFs can be quickly added to various regions of a sample using simple tools (e.g., permanent markers or conductive pens) and do not interfere with the ROIs. The coordinates of the REFs and the ROIs are obtained in the first imaging session (on a particular microscope platform) using an accurate and precise encoded motorized stage. In subsequent imaging sessions, the FARMER algorithm finds the new coordinates of the ROIs (on the same or different platforms), using the coordinates of the manually located REFs and the previously recorded coordinates. FARMER is convenient, fast (3-15 min/session, at least 10-fold faster than manual searches), accurate (4.4 μm average error on a microscope with a 100x objective), and precise (almost all errors are <8 μm), even with deliberate rotating and tilting of the sample well beyond normal repositioning accuracy. We demonstrate this versatility by imaging and re-imaging a diverse set of samples and imaging methods: live mammalian cells at different time points; fixed bacterial cells on two microscopes with different imaging modalities; and nanostructures on optical and electron microscopes. FARMER can be readily adapted to any imaging system with an encoded motorized stage and can facilitate multi-session and multi-platform imaging experiments in biology, materials science, photonics, and nanoscience.

Global Mapping of Protein-Lipid Interactions by Using Modified Choline-Containing Phospholipids Metabolically Synthesized in Live Cells.

The protein-lipid interaction is an essential metabolic process that mediates cellular signaling and functions. Existing strategies for large-scale mapping studies of the protein-lipid interaction fall short in their incompatibility with metabolic incorporation or inability to remove unwanted interferences from lipidated proteins. By incorporating an alkyne-containing choline head group and a diazirine-modified fatty acid simultaneously into choline-containing phospholipids synthesized from live mammalian cells, protein-phospholipid interactions have been successfully imaged in live cells. Subsequent in situ profiling of the modified Cho phospholipid-crosslinked proteins followed by quantitative proteomics allowed identification of several hundred putative phospholipid-interacting proteins, some of which were further validated.

Global Mapping of Protein–Lipid Interactions by Using Modified Choline‐Containing Phospholipids Metabolically Synthesized in Live Cells

AbstractThe protein–lipid interaction is an essential metabolic process that mediates cellular signaling and functions. Existing strategies for large‐scale mapping studies of the protein–lipid interaction fall short in their incompatibility with metabolic incorporation or inability to remove unwanted interferences from lipidated proteins. By incorporating an alkyne‐containing choline head group and a diazirine‐modified fatty acid simultaneously into choline‐containing phospholipids synthesized from live mammalian cells, protein–phospholipid interactions have been successfully imaged in live cells. Subsequent in situ profiling of the modified Cho phospholipid‐crosslinked proteins followed by quantitative proteomics allowed identification of several hundred putative phospholipid‐interacting proteins, some of which were further validated.

Lanthanide‐based FRET biosensors for time‐gated imaging and detection of protein‐protein interactions in live mammalian cells

Förster resonance energy transfer (FRET) using fluorescent proteins (FPs) as donors and acceptors is commonly used to microscopically image protein‐protein interactions in living cells. However, overlapping FP spectra lower signal‐to‐noise because the FRET‐sensitized acceptor emission signal is convoluted with donor bleedthrough and directly excited acceptor emission, or crosstalk. Quantitative FRET imaging is even more difficult when the local concentrations of donor‐labeled and acceptor labeled proteins are substantially different. We used the well‐studied interaction between FK binding protein 12 (FKBP12) and the rapamycin binding domain of m‐Tor (FRB) as a model system to show that lanthanide‐based biosensors can overcome the limitations of conventional FP‐FRET. The essential features of this approach include the use of luminescent Tb(III) complexes as FRET donors and the elimination of non‐specific fluorescence background that comes from time‐gated detection of long‐lived (~ms) Tb(III) and Tb(III)‐to‐FP emission signals. We prepared stably transformed NIH 3T3 fibroblast lines that express a single polypeptide consisting of the following elements (N to C): 1) FRB; 2) Escherichia coli dihydrofolate reductase (eDHFR); 3) a linker sequence; 4) GFP; and 5) FKBP12. A Tb(III) complex (TMP‐Lumi4) that selectively binds to eDHFR completes the sensor. The linker comprises alternating repeats of four glutamic acid residues followed by four arginine or lysine residues (ER/K) that form a rigid alpha helix in solution. Stochastic breaks in the helix permit close approach and binding of affinity elements positioned at each end. The effective concentration of each binding element depends on linker length and not solution concentration. Consequently, the fraction of two elements in the closed (high‐FRET) state depends on KD and linker length. We tested constructs with ER/K linker lengths of 10, 20 and 30 nm. Following addition of cell permeable TMP‐Lumi4 and rapamycin to culture medium, two‐color, time‐gated images showed significant, ~3‐fold increases in acceptor denominated FRET ratios relative to baseline (without rapamycin). The high sensor dynamic range permitted robust, time‐gated detection (Z'‐factor &gt; 0.8) of the FKBP12/FRB interaction and its inhibition by ascomycin in cells grown in 96‐well plates.Support or Funding InformationNIGMS R01 081030

Mechanistic understanding of the cysteine capping modifications of antibodies enables selective chemical engineering in live mammalian cells

Protein modifications by intricate cellular machineries often redesign the structure and function of existing proteins to impact biological networks. Disulfide bond formation between cysteine (Cys) pairs is one of the most common modifications found in extracellularly-destined proteins, key to maintaining protein structure. Unpaired surface cysteines on secreted mammalian proteins are also frequently found disulfide-bonded with free Cys or glutathione (GSH) in circulation or culture, the mechanism for which remains unknown. Here we report that these so-called Cys-capping modifications take place outside mammalian cells, not in the endoplasmic reticulum (ER) where oxidoreductase-mediated protein disulfide formation occurs. Unpaired surface cysteines of extracellularly-arrived proteins such as antibodies are uncapped upon secretion before undergoing disulfide exchange with cystine or oxidized GSH in culture medium. This observation has led to a feasible way to selectively modify the nucleophilic thiol side-chain of cell-surface or extracellular proteins in live mammalian cells, by applying electrophiles with a chemical handle directly into culture medium. These findings provide potentially an effective approach for improving therapeutic conjugates and probing biological systems.

Anomalous Diffusion and Compartmentalization on the Surface of Mammalian Cells

Tracking individual proteins on the surface of live mammalian cells reveals complex dynamics involving anomalous diffusion and clustering into nanoscale domains. Theoretical models show that anomalous diffusion can be caused by various processes. By performing time series and ensemble analysis of extensive single-molecule tracks, we show that different subdiffusive processes simultaneously coexist in the plasma membrane. Interestingly, ergodicity breaking is found to be maintained by immobilization events that take place when proteins are transiently captured within endocytic pits.

Engineering live cell surfaces with functional polymers via cytocompatible controlled radical polymerization.

The capability to graft synthetic polymers onto the surfaces of live cells offers the potential to manipulate and control their phenotype and underlying cellular processes. Conventional grafting-to strategies for conjugating preformed polymers to cell surfaces are limited by low polymer grafting efficiency. Here we report an alternative grafting-from strategy for directly engineering the surfaces of live yeast and mammalian cells through cell surface-initiated controlled radical polymerization. By developing cytocompatible PET-RAFT (photoinduced electron transfer-reversible addition-fragmentation chain-transfer polymerization), synthetic polymers with narrow polydispersity (Mw/Mn < 1.3) could be obtained at room temperature in 5 minutes. This polymerization strategy enables chain growth to be initiated directly from chain-transfer agents anchored on the surface of live cells using either covalent attachment or non-covalent insertion, while maintaining high cell viability. Compared with conventional grafting-to approaches, these methods significantly improve the efficiency of grafting polymer chains and enable the active manipulation of cellular phenotypes.

Genetically Encodable Bacterial Flavin Transferase for Fluorogenic Protein Modification in Mammalian Cells.

A bacterial flavin transferase (ApbE) was recently employed for flavin mononucleotide (FMN) modification on the Na+-translocating NADH:quinone oxidoreductase C (NqrC) protein in the pathogenic Gram-negative bacterium Vibrio cholerae. We employed this unique post-translational modification in mammalian cells and found that the FMN transfer reaction robustly occurred when NqrC and ApbE were genetically targeted in the cytosol of live mammalian cells. Moreover, NqrC expression in the endoplasmic reticulum (NqrC-ER) induced the retro-translocation of NqrC to the cytosol, leading to the proteasome-mediated ER-associated degradation of NqrC, which is considered to be an innate immunological response toward the bacterial protein. This unexpected cellular process of NqrC-ER could be exploited for the construction of an in cellulo proteasome inhibitor screening system, and our proposed approach yielded substantially improved results compared to a previous method. In addition, a truncated version of RnfG (half-RnfG) was found to be potentially useful as a genetically encoded tag for monitoring protein-protein interactions in a specific compartment, even in the ER, in a live cell according to its fluorogenic post-translational modification via ApbE. This new genetically encoded system in mammalian cells should serve as a valuable tool for anticancer drug screening and other applications in molecular and synthetic biology.

AKT1, LKB1, and YAP1 Revealed as MYC Interactors with NanoLuc-Based Protein-Fragment Complementation Assay.

The c-Myc (MYC) transcription factor is a major cancer driver and a well-validated therapeutic target. However, directly targeting MYC has been challenging. Thus, identifying proteins that interact with and regulate MYC may provide alternative strategies to inhibit its oncogenic activity. In this study, we report the development of a NanoLuc-based protein-fragment complementation assay (NanoPCA) and mapping of the MYC protein interaction hub in live mammalian cells. The NanoPCA system was configured to enable detection of protein-protein interactions (PPI) at the endogenous level, as shown with PRAS40 dimerization, and detection of weak interactions, such as PINCH1-NCK2. Importantly, NanoPCA allows the study of PPI dynamics with reversible interactions. To demonstrate its utility for large-scale PPI detection in mammalian intracellular environment, we have used NanoPCA to examine MYC interaction with 83 cancer-associated proteins in live cancer cell lines. Our new MYC PPI data confirmed known MYC-interacting proteins, such as MAX, GSK3A, and SMARCA4, and revealed a panel of novel MYC interaction partners, such as RAC-α serine/threonine-protein kinase (AKT)1, liver kinase B (LKB)1, and Yes-associated protein (YAP)1. The MYC interactions with AKT1, LKB1, and YAP1 were confirmed by coimmunoprecipitation of endogenous proteins. Importantly, AKT1, LKB1, and YAP1 were able to activate MYC in a transcriptional reporter assay. Thus, these vital growth control proteins may represent promising MYC regulators, suggesting new mechanisms that couple energetic and metabolic pathways and developmental signaling to MYC-regulated cellular programs.

Rotation of single live mammalian cells using dynamic holographic optical tweezers

We report on a method for rotating single mammalian cells about an axis perpendicular to the optical system axis through the imaging plane using dynamic holographic optical tweezers (HOTs). Two optical traps are created on the opposite edges of a mammalian cell and are continuously transitioned through the imaging plane along the circumference of the cell in opposite directions, thus providing the torque to rotate the cell in a controlled fashion. The method enables a complete 360° rotation of live single mammalian cells with spherical or near-to spherical shape in 3D space, and represents a useful tool suitable for the single cell analysis field, including tomographic imaging.

Plasma Membrane is Compartmentalized by a Self-Similar Cortical Actin Meshwork.

A broad range of membrane proteins display anomalous diffusion on the cell surface. Different methods provide evidence for obstructed subdiffusion and diffusion on a fractal space, but the underlying structure inducing anomalous diffusion has never been visualized because of experimental challenges. We addressed this problem by imaging the cortical actin at high resolution while simultaneously tracking individual membrane proteins in live mammalian cells. Our data confirm that actin introduces barriers leading to compartmentalization of the plasma membrane and that membrane proteins are transiently confined within actin fences. Furthermore, superresolution imaging shows that the cortical actin is organized into a self-similar meshwork. These results present a hierarchical nanoscale picture of the plasma membrane.

Trapping, manipulation, and crystallization of live cells using magnetofluidic tweezers.

Live mammalian cells are captured and manipulated in magnetofluidic traps created in a suspension of biocompatible, magnetic nanoparticles by a coaxial magnetic/non-magnetic "micropen". Upon activation by an external electromagnet, the pen creates microscale gradients of magnetic field and nanoparticle concentration that translate into directional and confining forces acting on the cells. Both individual cells and cell collections can be trapped by this method, allowing, for instance, for the formation of regularly shaped cell assemblies. The method does not entail any local heating artifacts and does not require magnetic tagging of the cells.

ProxyPhos sensors for the detection of negatively charged membranes.

Membrane-embedded negatively charged phospholipids (MENCP) can be used as biomarkers for a range of biological processes, including early detection of apoptosis in animal cells, drug-induced phospholipidosis, and selective detection of bacterial over animal cells. Currently, several technologies for the detection of apoptosis and bacterial cells are based on the recognition of MENCPs, including the AnnexinV stain and PSVue™ probes. As probes, these technologies have limitations, the most significant of which is the need for washing the unbound probe away to achieve optimal signal. In contrast, a turn-on chemosensor selective for MENCP would address this shortcoming, and allow for a more rapid protocol for the detection of apoptosis, bacteria and for other relevant applications. In this work, the aim was to explore whether ProxyPhos chemosensors, previously reported by our group for the detection of proximally phosphorylated peptides and proteins, could be re-purposed for the detection of MENCPs. Six lead ProxyPhos sensors were screened against synthetic vesicles containing biologically relevant negatively charged phospholipids including phosphatidic acid (PA), phosphatidylglycerol (PG), cardiolipin (CL) and phosphatidylserine (PS). Through these screens, ProxyPhos sensors exhibiting high selectivity for the detection of MENCPs over zwitterionic lipids were identified. Particular selectivity was observed for PA and CL. Sensitivity of the lead sensors for MENCPs was suitable for the detection of apoptosis: ProxyPhos detected vesicles containing as little as 2.5% PS and detected camptothecin-induced apoptosis in mammalian cells in flow cytometry experiments. The results suggest that ProxyPhos sensors can be used for the detection of MENCPs in synthetic vesicles and live mammalian cells.

High-speed atomic force microscopy imaging of live mammalian cells.

Direct imaging of morphological dynamics of live mammalian cells with nanometer resolution under physiological conditions is highly expected, but yet challenging. High-speed atomic force microscopy (HS-AFM) is a unique technique for capturing biomolecules at work under near physiological conditions. However, application of HS-AFM for imaging of live mammalian cells was hard to be accomplished because of collision between a huge mammalian cell and a cantilever during AFM scanning. Here, we review our recent improvements of HS-AFM for imaging of activities of live mammalian cells without significant damage to the cell. The improvement of an extremely long (~3 μm) AFM tip attached to a cantilever enables us to reduce severe damage to soft mammalian cells. In addition, a combination of HS-AFM with simple fluorescence microscopy allows us to quickly locate the cell in the AFM scanning area. After these improvements, we demonstrate that developed HS-AFM for live mammalian cells is possible to image morphogenesis of filopodia, membrane ruffles, pits open-close formations, and endocytosis in COS-7, HeLa cells as well as hippocampal neurons.