Live Mammalian Cells Research Articles

Warfarin and vitamin K epoxide reductase: a molecular accounting for observed inhibition

AbstractVitamin K epoxide reductase (VKOR), an endoplasmic reticulum membrane protein, is the key enzyme for vitamin K–dependent carboxylation, a posttranslational modification that is essential for the biological functions of coagulation factors. VKOR is the target of the most widely prescribed oral anticoagulant, warfarin. However, the topological structure of VKOR and the mechanism of warfarin's inhibition of VKOR remain elusive. Additionally, it is not clear why warfarin-resistant VKOR mutations identified in patients significantly decrease warfarin's binding affinity, but have only a minor effect on vitamin K binding. Here, we used immunofluorescence confocal imaging of VKOR in live mammalian cells and PEGylation of VKOR's endogenous cytoplasmic-accessible cysteines in intact microsomes to probe the membrane topology of human VKOR. Our results show that the disputed loop sequence between the first and second transmembrane (TM) domain of VKOR is located in the cytoplasm, supporting a 3-TM topological structure of human VKOR. Using molecular dynamics (MD) simulations, a T-shaped stacking interaction between warfarin and tyrosine residue 139, within the proposed TY139A warfarin-binding motif, was observed. Furthermore, a reversible dynamic warfarin-binding pocket opening and conformational changes were observed when warfarin binds to VKOR. Several residues (Y25, A26, and Y139) were found essential for warfarin binding to VKOR by MD simulations, and these were confirmed by the functional study of VKOR and its mutants in their native milieu using a cell-based assay. Our findings provide new insights into the dynamics of the binding of warfarin to VKOR, as well as into warfarin's mechanism of anticoagulation.

Single-Protein-Specific Redox Targeting in Live Mammalian Cells and C. elegans.

T-REX (targetable reactive electrophiles and oxidants) enables electrophile targeting in living systems with high spatiotemporal precision and at single-protein-target resolution. T-REX allows functional consequences of individual electrophile signaling events to be directly linked to on-target modifications. T-REX is accomplished by expressing a HaloTagged protein of interest (POI) and introducing a Halo-targetable bioinert photocaged precursor to a reactive electrophilic signal (RES). Light exposure releases the unfettered RES on demand, enabling precision modification of the POI due to proximity. Using alkyne-functionalized 4-hydroxynonenal (HNE) as a representative RES, this protocol delineates optimized strategies to (1) execute T-REX in live human cells and C. elegans, (2) quantitate the POI's RES-sensitivity by either azido-fluorescent-dye conjugation or (3) enrich using biotin-azide/streptavidin pulldown procedure in both model systems, and (4) identify the site of RES-labeling on the POI using proteomics. Built-in T-REX controls that allow users todirectly confirm on-target/on-site specificity of RES-sensing are also described. © 2018 by John Wiley & Sons, Inc.

A multicolor riboswitch-based platform for imaging of RNA in live mammalian cells.

RNAs directly regulate a vast array of cellular processes, emphasizing the need for robust approaches to fluorescently label and track RNAs in living cells. Here, we develop an RNA imaging platform using the cobalamin riboswitch as an RNA tag and a series of probes containing cobalamin as a fluorescence quencher. This highly modular ‘Riboglow’ platform leverages different colored fluorescent dyes, linkers and riboswitch RNA tags to elicit fluorescent turn-on upon binding RNA. We demonstrate the ability of two different Riboglow probes to track mRNA and small non-coding RNA in live mammalian cells. A side-by-side comparison revealed that Riboglow outperformed the dye binding aptamer Broccoli and performed on par with the gold standard RNA imaging system, the MS2-fluorescent protein system, while featuring a much smaller RNA tag. Together, the versatility of the Riboglow platform and ability to track diverse RNAs suggest broad applicability for a variety of imaging approaches.

Nanoquencher‐Based Selective Imaging of Protein Glutathionylation in Live Mammalian Cells

AbstractChanges in the cellular levels of glutathione (GSH) and protein S‐glutathionylation (PSSG) are closely associated with a number of human diseases. Despite recent advances, few thiol‐reactive, small‐molecule GSH sensors could selectively detect GSH over other endogenous thiols, and none was capable of detecting PSSG in live mammalian cells. By using a dye‐loaded mesoporous silica nanoquencher (qMSN) capped with anti‐GSH antibody capable of highly selective binding toward GSH and glutathionylated proteins over other molecules, we have successfully developed a fluorescence GSH/PSSG nanosensor, which showed unprecedented selectivity toward PSSG even in the presence of GSH, had amplifiable and programmable fluorescence Turn‐ON properties, and could be used to image endogenous PSSG in live mammalian cells under stimulated conditions for the first time.

Nanoquencher-Based Selective Imaging of Protein Glutathionylation in Live Mammalian Cells.

Changes in the cellular levels of glutathione (GSH) and protein S-glutathionylation (PSSG) are closely associated with a number of human diseases. Despite recent advances, few thiol-reactive, small-molecule GSH sensors could selectively detect GSH over other endogenous thiols, and none was capable of detecting PSSG in live mammalian cells. By using a dye-loaded mesoporous silica nanoquencher (qMSN) capped with anti-GSH antibody capable of highly selective binding toward GSH and glutathionylated proteins over other molecules, we have successfully developed a fluorescence GSH/PSSG nanosensor, which showed unprecedented selectivity toward PSSG even in the presence of GSH, had amplifiable and programmable fluorescence Turn-ON properties, and could be used to image endogenous PSSG in live mammalian cells under stimulated conditions for the first time.

FRET-enhanced photostability allows improved single-molecule tracking of proteins and protein complexes in live mammalian cells

A major challenge in single-molecule imaging is tracking the dynamics of proteins or complexes for long periods of time in the dense environments found in living cells. Here, we introduce the concept of using FRET to enhance the photophysical properties of photo-modulatable (PM) fluorophores commonly used in such studies. By developing novel single-molecule FRET pairs, consisting of a PM donor fluorophore (either mEos3.2 or PA-JF549) next to a photostable acceptor dye JF646, we demonstrate that FRET competes with normal photobleaching kinetic pathways to increase the photostability of both donor fluorophores. This effect was further enhanced using a triplet-state quencher. Our approach allows us to significantly improve single-molecule tracking of chromatin-binding proteins in live mammalian cells. In addition, it provides a novel way to track the localization and dynamics of protein complexes by labeling one protein with the PM donor and its interaction partner with the acceptor dye.

Polycondensation of a Perylene Bisimide Derivative and L-Malic Acid as Water-Soluble Conjugates for Fluorescent Labeling of Live Mammalian Cells

Based on simple mixing and polymerization of a hydroxyl-containing derivative of perylene bisimide (PBI) and l-malic acid, here, we demonstrate a new type of dye-polymer conjugate, PBI-poly(α,β-malic acid) (PBI–PMA). Benefiting from the excellent water-solubility of weak polyanionic PMA structure and the high fluorescence of PBI, the PBI-PMA conjugates readily dissolve in water, displaying strong pH-dependent fluorescence with the highest intensity at pH 6. Due to the excellent biocompatibility of PMA, those conjugates showed low cytotoxicity on L929 cells. Using L929 and HeLa cells, we also confirmed that the PBI-PMA-labeled cells display intense fluorescence. Overall, the PBI-PMA conjugate demonstrates high potential as a cell labeling agent with its synthesis ease, good solubility in aqueous medium, low cytotoxicity, and high fluorescence.

Engineering Whole Mammalian Cells for Target-Cell-Specific Invasion/Fusion.

Live mammalian cells are equipped with a synthetic cell invasion system that enables their target‐specific insertion into other live mammalian cells. By conjugating RhoA activator to a transmembrane protein that is segregated from cell–cell interface when specific cell contact occurs, polarization of RhoA activity is synthetically induced inside the cells in response to specific cell contact. This polarization is a sufficient condition for invader cells to selectively penetrate cells expressing a target antigen. Further, when an acid‐responsive fusogenic protein is expressed on invader cells, invader/receiver cell fusion occurs after invasion, and the invader's intracellular contents are released into the recipient's cytosol. It is shown that this system can be used for specific cell ablation. This synthetic‐biology‐inspired cell invasion/fusion system might open the door to using whole mammalian cells for cargo delivery purposes or for ablation of a specific cell type.

GMars-T Enabling Multimodal Subdiffraction Structural and Functional Fluorescence Imaging in Live Cells.

Fluorescent probes with multimodal and multilevel imaging capabilities are highly valuable as imaging with such probes not only can obtain new layers of information but also enable cross-validation of results under different experimental conditions. In recent years, the development of genetically encoded reversibly photoswitchable fluorescent proteins (RSFPs) has greatly promoted the application of various kinds of live-cell nanoscopy approaches, including reversible saturable optical fluorescence transitions (RESOLFT) and stochastic optical fluctuation imaging (SOFI). However, these two classes of live-cell nanoscopy approaches require different optical characteristics of specific RSFPs. In this work, we developed GMars-T, a monomeric bright green RSFP which can satisfy both RESOLFT and photochromic SOFI (pcSOFI) imaging in live cells. We further generated biosensor based on bimolecular fluorescence complementation (BiFC) of GMars-T which offers high specificity and sensitivity in detecting and visualizing various protein-protein interactions (PPIs) in different subcellular compartments under physiological conditions (e.g., 37 °C) in live mammalian cells. Thus, the newly developed GMars-T can serve as both structural imaging probe with multimodal super-resolution imaging capability and functional imaging probe for reporting PPIs with high specificity and sensitivity based on its derived biosensor.

A Sensitive Near-Infrared Fluorescent Sensor for Mitochondrial Hydrogen Sulfide.

Hydrogen sulfide (H2S) is an important gasotransmitter. Although a large number of fluorescent probes for cellular H2S have been reported, only a few can detect H2S in mitochondria, a cellular organelle connecting H2S with mitochondrial function and metabolic pathways. We hereby describe a novel near-infrared fluorescent probe, nimazide, by introducing sulfonyl azide to the core structure of a QSY-21 dark quencher. Nimazide responded quickly to H2S, resulting in robust fluorescence turn-off changes. This conversion displayed high specificity and fast kinetics. More impressively, we observed a robust fluorescence decrease in live cells loaded with mitochondrial nimazide in response to extracellular addition of nanomolar H2S, and successfully imaged biologically generated mitochondrial H2S in live mammalian cells. Nimazide is one of the most sensitive fluorescent probes for mitochondrial H2S.

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells.

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A Cell-Targeted Non-Cytotoxic Fluorescent Nanogel Thermometer Created with an Imidazolium-Containing Cationic Radical Initiator.

A cationic fluorescent nanogel thermometer based on thermo-responsive N-isopropylacrylamide and environment-sensitive benzothiadiazole was developed with a new azo compound bearing imidazolium rings as the first cationic radical initiator. This cationic fluorescent nanogel thermometer showed an excellent ability to enter live mammalian cells in a short incubation period (10 min), a high sensitivity to temperature variations in live cells (temperature resolution of 0.02-0.84 °C in the range 20-40 °C), and remarkable non-cytotoxicity, which permitted ordinary cell proliferation and even differentiation of primary cultured cells.

A Cell‐Targeted Non‐Cytotoxic Fluorescent Nanogel Thermometer Created with an Imidazolium‐Containing Cationic Radical Initiator

AbstractA cationic fluorescent nanogel thermometer based on thermo‐responsive N‐isopropylacrylamide and environment‐sensitive benzothiadiazole was developed with a new azo compound bearing imidazolium rings as the first cationic radical initiator. This cationic fluorescent nanogel thermometer showed an excellent ability to enter live mammalian cells in a short incubation period (10 min), a high sensitivity to temperature variations in live cells (temperature resolution of 0.02–0.84 °C in the range 20–40 °C), and remarkable non‐cytotoxicity, which permitted ordinary cell proliferation and even differentiation of primary cultured cells.

Super‐resolution imaging of transcription in live mammalian cells

Gene activation is thought to involve a multistep process whereby transcription factors bind to distal enhancer sites, recruit the Mediator complex which contacts the pre‐initiating RNA Polymerase II (Pol II) complex assembled at the start site of the gene. The interaction of Mediator and Pol II has yet to be observed in the nucleus of living cells and the dynamics of this interaction are not yet elucidated. Here we use quantitative live cell super‐resolution and light sheet imaging to study the organization and dynamics of endogenous Mediator and Pol II directly in living mouse embryonic stem cells. In addition to forming transient clusters with average lifetimes of 11.1 (± 0.9) s, and 12.1 (± 1.4) s respectively, Mediator and Pol II also form previously uncharacterized large and stable clusters in stem cells (~15 stable clusters per cell). The large and stable Mediator and Pol II clusters gradually disappear within hours after induction of stem cell differentiation. Stable Mediator and Pol II clusters co‐localize with each other, as well as with pluripotency factors. Inhibition of Brd4 bromodomains necessary for enhancer association eliminates both Mediator and Pol II stable clusters, and inhibition of transcription elongation selectively eliminates stable Pol II but not stable Mediator clusters. Tracking of Mediator and Pol II stable clusters suggests they are chromatin associated and they coalesce upon contact, a property associated with phase separated droplets. We conclude that Mediator and Pol II associate in diffraction‐sized condensates with a define lifetime dependent on active transcription in living stem cells.Support or Funding InformationThis project is supported by the NIH Director's New Innovator award #DP2CA195769 to I.I.C. and The Pew Charitable Trusts through the PEW Biomedical Scholars grant to I.I.C.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanogenetics for the remote and non‐invasive control of cancer immunotherapy

While cell-based immunotherapy, especially chimeric antigen receptor (CAR)-expressing T cells, is becoming a paradigm-shifting therapeutic approach for cancer treatment, there is a lack of general methods to remotely and non-invasively regulate genetics in live mammalian cells and animals for cancer immunotherapy within confined local tissue space. To address this limitation, we have identified a mechanically sensitive Piezo1 ion channel (mechanosensor) activatable by ultrasound stimulation and integrated it with engineered genetic circuits (genetic transducer) in live HEK cells to convert the ultrasound-activated Piezo1 into transcriptional activities. We have further engineered Jurkat T cell line and primary T cells (periphery blood mononuclear cells, PBMCs) to remotely sense the ultrasound wave and transduce it into transcriptional activation for the CAR expression to recognize and eradicate target tumor cells. This approach is modular and can be extended for remote-controlled activation of different cell types with a high spatiotemporal precision for therapeutic applications. Support or Funding Information This work is supported by grants from NIH HL121365, GM125379 (S.C. and Y. Wang), CA204704 and CA209629 (Y. Wang), NSF CBET1360341, DMS1361421 (Y. Wang and S.L.), and the Beckman Laser Institute Foundation. The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Design of synthetic genetic circuits remotely activatable by ultrasound This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Enzymatic Site‐Specific Labeling of RNA for Affinity Isolation of RNA‐Protein Complexes

The role of RNA in regulating cellular processes has generated tremendous interest in methods to image RNAs and profile their interactions within cells. Building on our recently developed enzymatic technique, RNA‐TAG (Transglycosylation At Guanosine), we demonstrate the site‐specific labeling and affinity purification of RNA transcripts. This technology takes advantage of a bacterial tRNA guanine transglycosylase (TGT) for the incorporation of nucleobase derivatives bearing functional probes, such as biotin, into an encodable RNA stem loop recognition motif. Affinity purification enables high levels of enrichment of target RNA complexes and analysis of bound proteins. Using this methodology, we have demonstrated that an RNA of interest can be selectively biotinylated in live mammalian cells or cell lysate. Through the development of RNA‐TAG, we aim to create a robust, convenient methodology for the elucidation of RNA function via affinity purification of cellular RNA‐protein complexes.Support or Funding InformationThis work is supported by the NIH under grant R01 GM123285. K.N.B. is supported by the Chemical Biology Interfaces Training Grant, NIH Grant T32 GM112584.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sterically Shielded, Stabilized Nitrile Imine for Rapid Bioorthogonal Protein Labeling in Live Cells.

In pursuit of fast bioorthogonal reactions, reactive moieties have been increasingly employed for selective labeling of biomolecules in living systems, posing a challenge in attaining reactivity without sacrificing selectivity. To address this challenge, here we report a bioinspired strategy in which molecular shape controls the selectivity of a transient, highly reactive nitrile imine dipole. By tuning the shape of structural pendants attached to the ortho position of the N-aryl ring of diaryltetrazoles-precursors of nitrile imines, we discovered a sterically shielded nitrile imine that favors the 1,3-dipolar cycloaddition over the competing nucleophilic addition. The photogenerated nitrile imine exhibits an extraordinarily long half-life of 102 s in aqueous medium, owing to its unique molecular shape that hinders the approach of a nucleophile as shown by DFT calculations. The utility of this sterically shielded nitrile imine in rapid (∼1 min) bioorthogonal labeling of glucagon receptor in live mammalian cells was demonstrated.

Receptor-mediated endocytosis generates nanomechanical force reflective of ligand identity and cellular property.

Whether environmental (thermal, chemical, and nutrient) signals generate quantifiable, nanoscale, mechanophysical changes in the cellular plasma membrane has not been well elucidated. Assessment of such mechanophysical properties of plasma membrane may shed lights on fundamental cellular process. Atomic force microscopic (AFM) measurement of the mechanical properties of live cells was hampered by the difficulty in accounting for the effects of the cantilever motion and the associated hydrodynamic force on the mechanical measurement. These challenges have been addressed in our recently developed control-based AFM nanomechanical measurement protocol, which enables a fast, noninvasive, broadband measurement of the real-time changes in plasma membrane elasticity in live cells. Here we show using this newly developed AFM platform that the plasma membrane of live mammalian cells exhibits a constant and quantifiable nanomechanical property, the membrane elasticity. This mechanical property sensitively changes in response to environmental factors, such as the thermal, chemical, and growth factor stimuli. We demonstrate that different chemical inhibitors of endocytosis elicit distinct changes in plasma membrane elastic modulus reflecting their specific molecular actions on the lipid configuration or the endocytic machinery. Interestingly, two different growth factors, EGF and Wnt3a, elicited distinct elastic force profiles revealed by AFM at the plasma membrane during receptor-mediated endocytosis. By applying this platform to genetically modified cells, we uncovered a previously unknown contribution of Cdc42, a key component of the cellular trafficking network, to EGF-stimulated endocytosis at plasma membrane. Together, this nanomechanical AFM study establishes an important foundation that is expandable and adaptable for investigation of cellular membrane evolution in response to various key extracellular signals.

Spectrally Resolved and Functional Super-resolution Microscopy via Ultrahigh-Throughput Single-Molecule Spectroscopy.

As an elegant integration of the spatial and temporal dimensions of single-molecule fluorescence, single-molecule localization microscopy (SMLM) overcomes the diffraction-limited resolution barrier of optical microscopy by localizing single molecules that stochastically switch between fluorescent and dark states over time. While this type of super-resolution microscopy (SRM) technique readily achieves remarkable spatial resolutions of ∼10 nm, it typically provides no spectral information. Meanwhile, current scanning-based single-location approaches for mapping the positions and spectra of single molecules are limited by low throughput and are difficult to apply to densely labeled (bio)samples. In this Account, we summarize the rationale, design, and results of our recent efforts toward the integration of the spectral dimension of single-molecule fluorescence with SMLM to achieve spectrally resolved SMLM (SR-SMLM) and functional SRM ( f-SRM). By developing a wide-field scheme for spectral measurement and implementing single-molecule fluorescence on-off switching typical of SMLM, we first showed that in densely labeled (bio)samples it is possible to record the fluorescence spectra and positions of millions of single molecules synchronously within minutes, giving rise to ultrahigh-throughput single-molecule spectroscopy and SR-SMLM. This allowed us to first show statistically that for many dyes, single molecules of the same species exhibit near identical emission in fixed cells. This narrow distribution of emission wavelengths, which contrasts markedly with previous results at solid surfaces, allowed us to unambiguously identify single molecules of spectrally similar dyes. Crosstalk-free, multiplexed SRM was thus achieved for four dyes that were merely 10 nm apart in emission spectrum, with the three-dimensional SRM images of all four dyes being automatically aligned within one image channel. The ability to incorporate single-molecule fluorescence measurement with SMLM was next utilized to achieve f-SRM. By encoding functional information into the spectral responses of environment-sensing fluorescent probes, f-SRM transcends the structural information provided by typical SRM techniques and reveals the spatiotemporal distribution of physicochemical parameters with single-molecule sensitivity and nanoscale spatial resolution. As one example, by employing the solvatochromic dye Nile Red to sense local chemical polarity, we revealed nanoscale heterogeneity in the membranes of live mammalian cells. This enabled us to unveil substantial polarity differences between the plasma membrane and the membranes of nanoscale intracellular organelles, a result we determined to be due to differences in local cholesterol levels. With the addition of cholesterol or cholera toxin, we further observed the formation of low-polarity, raftlike nanodomains in the plasma membrane. In another study, we generalized SR-SMLM to fluorogenic single-molecule reactions. As a wide-field technique, SR-SMLM readily captures the emission spectra of individual product fluorescent molecules that are stochastically produced from nonfluorescent reactants at random locations over large sample areas, and therefore, it provides the unique possibility to spectrally identify and characterize single product molecules in a high-throughput fashion. Using the ring-opening reaction of a photochromic spiropyran as an example, we demonstrated that the capability to resolve the emission spectra of single product molecules could unveil rich, multipath reaction pathways. In summary, by integrating the spatial, temporal, and spectral dimensions of single-molecule fluorescence, SR-SMLM and f-SRM add rich spectral and functional dimensions to SRM and thus open up new ways of probing biological and chemical systems at the single-molecule and nanoscale levels.