Live Cell Labeling Research Articles

Live Cell Labeling of Native Intracellular Bacterial Receptors Using Aniline-Catalyzed Oxime Ligation

Live cell fluorescent labeling of proteins has become a seminal tool in biology and has led to hallmark discoveries in diverse research areas such as protein trafficking, cell-to-cell interactions, and intracellular network dynamics. One of the main challenges, however, remains the ability to label intracellular proteins using fluorescent ligands with high specificity, all the while retaining viability of the targeted cells. Here, we present the first example of live cell labeling and imaging of an intracellular bacterial receptor involved in cell-to-cell communication (i.e., quorum sensing), using a novel two-step approach involving covalent attachment of a reactive mimic of the primary endogenous Pseudomonas aeruginosa quorum-sensing signal to its receptor, LasR, followed by aniline-catalyzed oxime formation between the modified receptor and a fluorescent BODIPY derivative. Our results indicate that LasR is not distributed evenly throughout the cytoplasmic membrane but is instead concentrated at the poles of the P. aeruginosa cell.

Design and development of quantum dots and other nanoparticles based cellular imaging probe

One goal of nanotechnology is to prepare cellular nanoprobes for various biological applications where conventional molecular probes fall short of long-term stability and simultaneous detection of multiple signals. Successful development of cellular nanoprobes requires the availability of a library of functional nanoparticles, knowledge of their interactions with cells and mechanism of cellular entry and to modulate these interactions by appropriate design of surface functionality. Although a great deal of research has been done in past 15 years, only limited success has been achieved in live cell labeling with high specificity, sub-cellular targeting and single molecule trafficking. This article focuses on the author's effort in making cellular imaging nanoprobes from different nanoparticles and discusses the most critical issues in the context of current knowledge, such as different variables that often influence labeling, non-specific binding/uptake of nanoprobes and specific live cell labeling. Finally, the important role of coating chemistry to overcome these problems has been highlighted and some successful labeling results have been summarized.

Peptide Length and Leaving-Group Sterics Influence Potency of Peptide Phosphonate Protease Inhibitors

The ability to follow enzyme activity in a cellular context represents a challenging technological frontier that impacts fields ranging from disease pathogenesis to epigenetics. Activity-based probes (ABPs) label the active form of an enzyme via covalent modification of catalytic residues. Here we present an analysis of parameters influencing potency of peptide phosphonate ABPs for trypsin-fold S1A proteases, an abundant and important class of enzymes with similar substrate specificities. We find that peptide length and stability influence potency more than sequence composition and present structural evidence that steric interactions at the prime-side of the substrate-binding cleft affect potency in a protease-dependent manner. We introduce guidelines for the design of peptide phosphonate ABPs and demonstrate their utility in a live-cell labeling application that specifically targets active S1A proteases at the cell surface of cancer cells.

Regulation of STIM1 and SOCE by the Ubiquitin-Proteasome System (UPS)

The ubiquitin proteasome system (UPS) mediates the majority of protein degradation in eukaryotic cells. The UPS has recently emerged as a key degradation pathway involved in synapse development and function. In order to better understand the function of the UPS at synapses we utilized a genetic and proteomic approach to isolate and identify novel candidate UPS substrates from biochemically purified synaptic membrane preparations. Using these methods, we have identified Stromal interacting molecule 1 (STIM1). STIM1 is as an endoplasmic reticulum (ER) calcium sensor that has been shown to regulate store-operated Ca2+ entry (SOCE). We have characterized STIM1 in neurons, finding STIM1 is expressed throughout development with stable, high expression in mature neurons. As in non-excitable cells, STIM1 is distributed in a membranous and punctate fashion in hippocampal neurons. In addition, a population of STIM1 was found to exist at synapses. Furthermore, using surface biotinylation and live-cell labeling methods, we detect a subpopulation of STIM1 on the surface of hippocampal neurons. The role of STIM1 as a regulator of SOCE has typically been examined in non-excitable cell types. Therefore, we examined the role of the UPS in STIM1 and SOCE function in HEK293 cells. While we find that STIM1 is ubiquitinated, its stability is not altered by proteasome inhibitors in cells under basal conditions or conditions that activate SOCE. However, we find that surface STIM1 levels and thapsigargin (TG)-induced SOCE are significantly increased in cells treated with proteasome inhibitors. Additionally, we find that the overexpression of POSH (Plenty of SH3′s), an E3 ubiquitin ligase recently shown to be involved in the regulation of Ca2+ homeostasis, leads to decreased STIM1 surface levels. Together, these results provide evidence for previously undescribed roles of the UPS in the regulation of STIM1 and SOCE function.

Quenched Substrates for Live-Cell Labeling of SNAP-Tagged Fusion Proteins with Improved Fluorescent Background

Recent developments in fluorescence microscopy raise the demands for bright and photostable fluorescent tags for specific and background free labeling in living cells. Aside from fluorescent proteins and other tagging methods, labeling of SNAP-tagged proteins has become available thereby increasing the pool of potentially applicable fluorescent dyes for specific labeling of proteins. Here, we report on novel conjugates of benzylguanine (BG) which are quenched in their fluorescence and become highly fluorescent upon labeling of the SNAP-tag, the commercial variant of the human O(6)-alkylguanosyltransferase (hAGT). We identified four conjugates showing a strong increase, i.e., >10-fold, in fluorescence intensity upon labeling of SNAP-tag in vitro. Moreover, we screened a subset of nine BG-dye conjugates in living Escherichia coli and found them all suited for labeling of the SNAP-tag. Here, quenched BG-dye conjugates yield a higher specificity due to reduced contribution from excess conjugate to the fluorescence signal. We further extended the application of these conjugates by labeling a SNAP-tag fusion of the Tar chemoreceptor in live E. coli cells and the eukaryotic transcription factor STAT5b in NIH 3T3 mouse fibroblast cells. Aside from the labeling efficiency and specificity in living cells, we discuss possible mechanisms that might be responsible for the changes in fluorescence emission upon labeling of the SNAP-tag, as well as problems we encountered with nonspecific labeling with certain conjugates in eukaryotic cells.

An experimental model for studying the biomechanics of embryonic tendon: Evidence that the development of mechanical properties depends on the actinomyosin machinery

Tendons attach muscles to bone and thereby transmit tensile forces during joint movement. However, a detailed understanding of the mechanisms that establish the mechanical properties of tendon has remained elusive because of the practical difficulties of studying tissue mechanics in vivo. Here we have performed a study of tendon-like constructs made by culturing embryonic tendon cells in fixed-length fibrin gels. The constructs display mechanical properties (toe–linear–fail stress–strain curve, stiffness, ultimate tensile strength, and failure strain) as well as collagen fibril volume fraction and extracellular matrix (ECM)/cell ratio that are statistically similar to those of embryonic chick metatarsal tendons. The development of mechanical properties during time in culture was abolished when the constructs were treated separately with Triton X-100 (to solubilise membranes), cytochalasin (to disassemble the actin cytoskeleton) and blebbistatin (a small molecule inhibitor of non-muscle myosin II). Importantly, these treatments had no effect on the mechanical properties of the constructs that existed prior to treatment. Live-cell imaging and 14C-proline metabolic labeling showed that blebbistatin inhibited the contraction of the constructs without affecting cell viability, procollagen synthesis, or conversion of procollagen to collagen. In conclusion, the mechanical properties per se of the tendon constructs are attributable to the ECM generated by the cells but the improvement of mechanical properties during time in culture was dependent on non-muscle myosin II-derived forces.

Development of SNAP-tag-mediated live cell labeling as an alternative to GFP inPorphyromonas gingivalis

Porphyromonas gingivalis is an anaerobic periodontal pathogen that resides in the complex multispecies microbial biofilm known as dental plaque. Effective reporter tools are increasingly needed to facilitate physiological and pathogenetic studies of dental biofilm. Fluorescent proteins are ideal reporters for conveniently monitoring biofilm growth, but are restricted by several environmental factors, such as a requirement of oxygen to emit fluorescence. We developed a fluorescent reporter plasmid, known as the SNAP-tag, for labeling P. gingivalis cells, which encode an engineered version of the human DNA repair enzyme O(6)-alkylguanine-DNA alkyltransferase. Fluorescent substrates containing O(6)-benzylguanine covalently and specifically bind to the enzyme via stable thioether bonds. For the present study, we constructed a replicative plasmid carrying SNAP26b under the control of the P. gingivalis endogenous trxB promoter. The P. gingivalis-expressing SNAP26 protein was successfully labeled with specific fluorophores under anaerobic conditions. Porphyromonas gingivalis biofilm formation was investigated using flow cells and confocal laser scanning microscopy. A specific distribution of a strong fluorescence signal was demonstrated in P. gingivalis-SNAP26 monospecies and bispecies biofilms with Streptococcus gordonii-GFPmut3(*). These findings show that the SNAP-tag can be applied to studies of anaerobic bacteria in biofilm models and is a useful and advantageous alternative to existing labeling strategies.

Effect of six different peri‐implantitis disinfection methods on in vivo human oral biofilm

The aim of this human in vivo pilot study was to evaluate the efficacy of six antimicrobial agents on the surface decontamination of an oral biofilm attached to titanium implants. For in vivo biofilm formation, we fixed titanium specimens to individual removable acrylic upper jaw splints (14 specimens in every splint), which were worn by four volunteers overnight for 12 h. The specimens were then treated with different antimicrobial agents for 1 min (Sodium hypochlorite, Hydrogen peroxide 3%, Chlorhexidingluconate 0.2%, Plax, Listerine, citric acid 40%). Afterwards, we quantified the total bacterial load and the viability of adhering bacteria by live or dead cell labelling in combination with fluorescence microscopy. The total bacterial load on the titanium surfaces was significantly higher after incubation in the control solution phosphate-buffered saline (PBS) than after disinfection in sodium hypochlorite, hydrogen peroxide, chlorhexidine, Plax, Listerine, and citric acid. Furthermore, a significantly lower ratio between dead and total adhering bacteria (bactericidal effect) was found after incubation in control PBS, Plax mouth rinse, and citric acid than after incubation in sodium hypochlorite, hydrogen peroxide, chlorhexidine, and Listerine. All tested antiseptics seem to be able to reduce the total amount of microorganisms accumulating on titanium surfaces. Furthermore, sodium hypochlorite, hydrogen peroxide, chlorhexidine, and Listerine showed a significant bactericidal effect against adhering bacteria.

Conjugated Polymer Nanoparticles for Fluorescent Labeling of Live Cells and Delivery of Biological Molecules into Plant Cells

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

NTAㆍNi2+-Functionalized Quantum Dots for VAMP2 Labeling in Live Cells

An efficient method for labeling individual proteins in live cells is required for investigations into biological mechanisms and cellular processes. Here we describe the preparation of small quantum dots (QDs) that target membrane surface proteins bearing a hexahistidine-tag (His 6 -tag) via specific binding to an nitrilotriacetic acid complex of nickel(II) (NTA·Ni 2+ ) on the QD surfaces. We showed that the NTA·Ni 2+ -QDs bound to His-tag functionalized beads as a cellular mimic with high specificity and that QDs successfully targeted His 6 -tagged vesicle-associated membrane proteins (VMAP) on cell surfaces. This strategy provides an efficient approach to monitoring synaptic protein dynamics in spatially restricted and confined biological environments.

The effects of copper additives on the quantity and cell viability of adherent Staphylococcus epidermidis in silicone implants

This in vitro study evaluated the antibacterial effect of copper additives in silicone implants. Specimens of a standard silicone material used in breast augmentation and modified copper-loaded silicone specimens were prepared and incubated in a Staphylococcus epidermidis suspension (2 h, 37°C). After the quantification of adhering staphylococci using a biofluorescence assay (Resazurin), the viability of the adhering bacterial cells was quantified by live or dead cell labeling in combination with fluorescence microscopy. In the Resazurin fluorometric quantification, a higher amount of adhering S. epidermidis cells was detected on pure silicone (4612 [2319/7540] relative fluorescence units [rfu]) than on silicone with copper additives (2701 [2158/4153] rfu). Additionally, a significantly higher amount of adhering bacterial cells (5.07% [2.03%/8.93%]) was found for pure silicone than for silicone with copper additives (1.72% [1.26%/2.32%]); (p < 0.001). Calculations from live or dead staining showed that the percentage of dead S. epidermidis cells adhered on pure silicone (52.1%) was significantly lower than on silicone with copper additives (79.7%); (p < 0.001). In vitro, silicone material with copper additives showed antibacterial effects against S. epidermidis. Copper-loaded silicone may prevent bacterial colonization, resulting in lower infection rates of silicone implants.

Biarsenical Labeling of Tetracysteine-Tagged Proteins for Tracking Existing and Newly Synthesized Pools of Proteins

INTRODUCTIONGenetically tagging proteins of interest with fluorescent labels such as green fluorescent protein is a valuable technique for studying protein localization and interactions in live cells. However, the relatively large size of fluorescent fusion proteins can limit the utility of this approach, and sometimes the tag can render the protein of interest nonfunctional. Small genetic tagging with tetracysteine (TC) and subsequent labeling with membrane-permeable biarsenical dyes can overcome these limitations. This protocol describes the method to detect TC-tagged proteins by biarsenical labeling in live cells. A small amino acid tag containing four cysteines (CCPGCC) is introduced to a protein of interest. Cells expressing proteins with this tag are then treated with membrane-permeable dyes (FlAsH-EDT2 or ReAsH-EDT2), and the two arsenic groups of the dyes each bind to the two thiols in the TC motif. Once bound, FlAsH and ReAsH are converted to a fluorescent state (green and red fluorescence at excitation maxima of 508 and 593 nm, respectively) and can be detected by fluorescence microscopy. The method is particularly suitable for investigating protein dynamics that are difficult to study using a fluorescent protein tag. It also is a powerful method for visualizing trafficking of newly synthesized proteins in real time, because existing and newly synthesized pools of proteins can be labeled differentially with the red and green dyes. A method for labeling virally produced proteins is also presented.

Direct pH Measurements by using Subcellular Targeting of 5(and 6-) Carboxyseminaphthorhodafluor in Mammalian Cells

As a means of reliably measuring intracellular pH, we have precisely targeted 5(and 6-) carboxyseminaphthorhodafluor to cellular subcompartments. This was accomplished by combining the well-established pH-sensitive dye with a protein-based reporter system. When expressed in cells, the reporter protein is designed to covalently bind ligands composed of a functional group and a reactive linker. In order to make a pH-sensitive ligand, we chemically coupled the pH sensor to a reactive linker. Several ligands of differing linker lengths were made and tested for their pH responsiveness in vitro. The most responsive of these ligands was then evaluated for its efficacy in live cell labeling and its use as an intracellular pH sensor for ratiometric confocal microscopy. Here we show that we could target this pH sensor within mammalian cells exclusively to either the nucleus or cytoplasm. Exhibiting the versatility of this reporter technology, we were also able to specifically limit pH sensor labeling to within the trafficking pathway of integrins and directly measure pH of this environment. Results correspond well with previously published reports. Both the simplicity and flexibility of the technology used in this study make possible the development of diverse targeted microenvironmental sensors or other moieties of interest.

Labeling and imaging of GLUT4 in live L6 cells with quantum dots

GLUT4 is sequestered in intracellular storage compartments in a basal state and is rapidly translocated to the cell surface in response to insulin stimulation. Regulation of GLUT4 distribution is key for maintaining whole-body glucose homeostasis. To investigate the complicated intracellular movement of GLUT4 vesicles and their interactions with organelles in detail, new probes suitable for long-term tracking of cellular events are required. In this study, we introduce for the first time quantum dots (QDs) as a superior probe into the research of the mechanisms of GLUT4 translocation. QDs are light-emitting semiconductor nanoparticles with unique optical and spectroscopic properties, such as broad absorption, narrow and tunable emission, resistance to photobleaching, strong luminescence, and long luminescent lifetimes. Owing to their remarkable photophysical properties and relatively small size, QDs are emerging as an alternative to conventional dyes for fluorescence-based applications. We have developed a procedure for labeling and imaging GLUT4 in live cells with streptavidin-conjugated quantum dot (QD-SA) and demonstrated that QDs contained in cytoplasm have no obvious negative influence on L6 cells. This study provides a sensitive, nontoxic, long-term imaging platform for observing the dynamics and regulated characteristics of GLUT4 transport.

Live Cell Labeling of Glial Progenitor Cells Using Targeted Quantum Dots

This study describes the development of targeted quantum dots (T-QDs) as biomarkers for the labeling of glial progenitor cells (GPCs) that over express platelet derived growth factor (PDGF) and its receptor PDGFR (GPC(PDGF)). PDGFR plays a critical role in glioma development and growth, and is also known to affect multiple biological processes such as cell migration and embryonic development. T-QDs were developed using streptavidin-conjugated quantum dots (S-QDs) with biotinylated antibodies and utilized to label the intracellular and extracellular domains of live, cultured GPC(PDGF) cells via lipofection with cationic liposomes. Confocal studies illustrate successful intracellular and extracellular targeted labeling within live cells that does not appear to impact upstream PDGFR dynamics during real-time signaling events. Further, T-QDs were nontoxic to GPC(PDGF) cells, and did not alter cell viability or proliferation over the course of 6 days. These results raise new applications for T-QDs as ultra sensitive agents for imaging and tracking of protein populations within live cells, which that will enable future mechanistic study of oncogenic signaling events in real-time.

A Proteomic-scale, Mass Spec-based Method to Probe Forced Unfolding within Cells

Cysteine is a relatively hydrophobic amino acid and is often buried within protein folds or assemblies. We use Cys labeling of live cells under different conditions, including mechanical stress and temperature, to identify by Mass Spec those proteins and sites that are perturbed. For intracellular proteins, Cys are not tied up in disulfides, and so their reactivity to many fluorescent dyes in solution is readily determined as a function of temperature under both native and high urea conditions. When a protein is folded at low temperature, the reaction rate tends to be faster in urea, and the ratio of rates yields a thermal unfolding curve that looks very similar to results from solution methods such as Circular Dichroism. Unlike these other methods, however, the Cys labeling approach can also be applied to intact cells by using membrane-permeable, cell-viable dyes at low dose. Labeling studies of several cell types, including stem cells, lung cancer cells, and cardiomyocytes, treated under two different conditions, such as stressed vs unstressed or 37C vs fever-like 42C, are followed by electrophoretic separation and detailed LC/MS-MS study. What results is an identification of labeled proteins as well as differentially labeled sites in many proteins, including several structural proteins with major roles in disease and development: Myosin, Filamin, Spectrin, and Vimentin. The Cys shotgun method thus provides unique, proteomic scale measurements of protein structural changes in both solution and intact cells. (Science 317, 663-666, 2007)

Multiplexed labeling of viable cells for high‐throughput analysis of glycine receptor function using flow cytometry

Flow cytometry is an important drug discovery tool because it permits high-content multiparameter analysis of individual cells. A new method dramatically enhanced screening throughput by multiplexing many discrete fixed cell populations; however, this method is not suited to assays requiring functional cellular responses. HEK293 cells were transfected with unique mutant glycine receptors. Mutant receptor expression was confirmed by coexpression of yellow fluorescent protein (YFP). Commercially available cell-permeant dyes were used to label each glycine receptor expressing mutant with a unique optical code. All encoded cell lines were combined in a single tube and analyzed on a flow cytometer simultaneously before and after the addition of glycine receptor agonist. We decoded multiplexed cells that expressed functionally distinct glycine receptor chloride channels and analyzed responses to glycine in terms of chloride-sensitive YFP expression. Here, data provided by flow cytometry can be used to discriminate between functional and nonfunctional mutations in the glycine receptor, a process accelerated by the use of multiplexing. Further, this data correlates to data generated using a microscopy-based technique. The present study demonstrates multiplexed labeling of live cells, to enable cell populations to be subject to further cell culture and experimentation, and compares the results with those obtained using live cell microscopy.

FISH analysis and cytogenetic characterization of male meiotic prophase I in Acricotopus lucidus (Diptera, Chironomidae)

In the chironomid Acricotopus lucidus, two cells with quite different chromosome complements arise from the last unequal spermatogonial mitosis, as a consequence of monopolar migration of the so-called germ line limited chromosomes (Ks). The cell receiving all the Ks, in addition to two sets of the regularly segregating somatic chromosomes (Ss), develops into the primary spermatocyte, while the cell getting only Ss differentiates into an aberrant spermatocyte. Only the primary spermatocyte enters meiosis. These nuclear events in the primary spermatocytes of A. lucidus during prophase I were analyzed by carmine-orcein staining, silver impregnation, live-cell RNA fluorescence labeling, and fluorescence in situ hybridization, using painting probes of the three Ss and the K centromeres. Early prophase I nuclei display large condensed chromatin blocks showing intense carmine staining, dark silver nitrate impregnation and bright DAPI fluorescence. The first clear signs of meiotic prophase progression are loops arising at early pachytene, which originate from the gradually decondensing chromatin blocks. The blocks presumably represent facultative heterochromatin. Chromosome painting demonstrates that the pachytene loops are composed of the two closely paired homologues. Conspicuous telomere attachments of differently painted non-homologous chromosomes were detected. The centromeres of the Ks group together, indicating a classical bouquet arrangement of the paired homologues in pachytene. The clustered centromeres may function as pairing centers to initiate synapsis of the homologues. Nucleolus expression data support the idea that the aberrant spermatocyte nourishes the primary spermatocyte via a connecting cytoplasmic canal.

Tetrazine-Based Cycloadditions: Application to Pretargeted Live Cell Imaging

Bioorthogonal tetrazine cycloadditions have been applied to live cell labeling. Tetrazines react irreversibly with the strained dienophile norbornene forming dihydropyrazine products and dinitrogen. The reaction is high yielding, selective, and fast in aqueous media. Her2/neu receptors on live human breast cancer cells were targeted with a monoclonal antibody modified with a norbornene. Tetrazines conjugated to a near-infrared fluorochrome selectively and rapidly label the pretargeted antibody in the presence of serum. These findings indicate that this chemistry is suitable for in vitro labeling experiments, and suggests that it may prove a useful strategy for in vivo pretargeted imaging under numerous modalities.

Spermatogonial stem cell sensitivity to capsaicin: An in vitro study

BackgroundConflicting reports have been published on the sensitivity of spermatogenesis to capsaicin (CAP), the pungent ingredient of hot chili peppers. Here, the effect of CAP on germ cell survival was investigated by using two testis germ cell lines as a model. As CAP is a potent agonist of the transient receptor potential vanilloid receptor 1 (TRPV1) and no information was available of its expression in germ cells, we also studied the presence of TRPV1 in the cultured cells and in germ cells in situ.MethodsThe rat spermatogonial stem cell lines Gc-5spg and Gc-6spg were used to study the effects of different concentrations of CAP during 24 and 48 h. The response to CAP was first monitored by phase-contrast microscopy. As germ cells appear to undergo apoptosis in the presence of CAP, the activation of caspase 3 was studied using an anti activated caspase 3 antibody or by quantifying the amount of cells with DNA fragmentation using flow cytometry. Immunolocalization was done with an anti-TRPV1 antibody either with the use of confocal microscopy to follow live cell labeling (germ cells) or on Bouin fixed paraffin embedded testicular tissues. The expression of TRPV1 by the cell lines and germ cells was confirmed by Western blots.ResultsInitial morphological observations indicated that CAP at concentrations ranging from 150 uM to 250 uM and after 24 and 48 h of exposure, had deleterious apoptotic-like effects on both cell lines: A large population of the CAP treated cell cultures showed signs of DNA fragmentation and caspase 3 activation. Quantification of the effect demonstrated a significant effect of CAP with doses of 150 uM in the Gc-5spg cell line and 200 uM in the Gc-6spg cell line, after 24 h of exposure. The effect was dose and time dependent in both cell lines. TRPV1, the receptor for CAP, was found to be expressed by the spermatogonial stem cells in vitro and also by premeiotic germ cells in situ.ConclusionCAP adversely affects spermatogonial survival in vitro by inducing apoptosis to those cells and TRPV-1, a CAP receptor, may be involved in this effect as this receptor is expressed by mitotic germ cells.