Membrane-permeable Dye Research Articles

Quantifying the viability of lactic acid bacteria using ratiometric fluorescence assays

Quantifying the viability of lactic acid bacteria (LAB) has become an important task in evaluating the quality, function and storage condition of LAB products. Propidium iodide (PI), as a DNA-binding dye that penetrates only dead bacteria, has been widely used for LAB viability assessments. However, high fluorescence background from live LAB was frequently occurred during PI-based staining, which led to the low sensitivity in probing minor amounts of dead LAB in a dead/live mixture. Here, we found that PI could be adsorbed nonspecifically in LAB periplasm, which contributed to the high fluorescence background of live LAB. CaCl2 was used to reduce this nonspecific adsorption and increase the fluorescence ratio of dead to live LAB. Combined with SYTO 9 (a membrane permeable dye) for double-staining, a ratiometric fluorescent method that could probe dead LAB at a ratio as low as 0.2 % was established. Using Lacticaseibacillus casei as a LAB model, an allometric correlation between the ratiometric fluorescence and theoretical ratio of live to dead LAB was acquired, with a correlation coefficient R2 of 0.993. The as-developed method was successfully applied to the viability assessment of LAB stored in different environments. We believe it would have practical applications in LAB food industry for quality management and control.

Rapid and Sensitive Quantification of Bacterial Viability Using Ratiometric Fluorescence Sensing.

Bacterial viability assessment plays an important role in food-borne pathogen detection and antimicrobial drug development. Here, we first used GelRed as a DNA-binding stain for a bacterial viability assessment. It was found that live bacteria were able to exclude GelRed, which however could easily penetrate dead ones and be absorbed nonspecifically on the bacterial periplasm. Cations were used to reduce the nonspecific adsorption and greatly increase the red fluorescence ratio of dead to live bacteria. Combined with SYTO 9 (a membrane-permeable dye) for double-staining, a ratiometric fluorescent method was established. Using Escherichia coli O157:H7 as a bacteria model, the ratiometric fluorescent method can probe dead bacteria as low as 0.1%. A linear correlation between the ratiometric fluorescence and the theoretical ratio of dead bacteria was acquired, with a correlation coefficient R2 of 0.97. Advantages in sensitivity, accuracy, and safety of the GelRed/SYTO9-based ratiometric fluorescent method against traditional methods were demonstrated. The established method was successfully applied to the assessment of germicidal efficacy of different heat treatments. It was found that even 50 °C treatment could lead to the death of minor bacteria. The as-developed method has many potential applications in microbial researches, and we believe it could be expanded to the viability assessment of mammalian cells.

Multiplexed sequential imaging in living cells with orthogonal fluorogenic RNA aptamer/dye pairs.

Detecting multiple targets in living cells is important in cell biology. However, multiplexed fluorescence imaging beyond two-to-three targets remains a technical challenge. Herein, we introduce a multiplexed imaging strategy, 'sequential Fluorogenic RNA Imaging-Enabled Sensor' (seqFRIES), which enables live-cell target detection via sequential rounds of imaging-and-stripping. In seqFRIES, multiple orthogonal fluorogenic RNA aptamers are genetically encoded inside cells, and then the corresponding cell membrane permeable dye molecules are added, imaged, and rapidly removed in consecutive detection cycles. As a proof-of-concept, we have identified in this study four fluorogenic RNA aptamer/dye pairs that can be used for highly orthogonal and multiplexed imaging in living bacterial and mammalian cells. After further optimizing the cellular fluorescence activation and deactivation kinetics of these RNA/dye pairs, the whole four-color semi-quantitative seqFRIES process can be completed in ∼20 min. Meanwhile, seqFRIES-mediated simultaneous detection of critical signalling molecules and mRNA targets was also achieved within individual living cells. We expect our validation of this new seqFRIES concept here will facilitate the further development and potential broad usage of these orthogonal fluorogenic RNA/dye pairs for multiplexed and dynamic live-cell imaging and cell biology studies.

Molecular engineering of fluorescent dyes for long-term specific visualization of the plasma membrane based on alkyl-chain-regulated cell permeability

Long-term visualization of changes in plasma membrane dynamics during important physiological processes can provide intuitive and reliable information in a 4D mode. However, molecular tools that can visualize plasma membranes over extended periods are lacking due to the absence of effective design rules that can specifically track plasma membrane fluorescent dye molecules over time. Using plant plasma membranes as a model, we systematically investigated the effects of different alkyl chain lengths of FMR dye molecules on their performance in imaging plasma membranes. Our findings indicate that alkyl chain length can effectively regulate the permeability of dye molecules across plasma membranes. The study confirms that introducing medium-length alkyl chains improves the ability of dye molecules to target and anchor to plasma membranes, allowing for long-term imaging of plasma membranes. This provides useful design rules for creating dye molecules that enable long-term visualization of plasma membranes. Using the amphiphilic amino-styryl-pyridine fluorescent skeleton, we discovered that the inclusion of short alkyl chains facilitated rapid crossing of the plasma membrane by the dye molecules, resulting in staining of the cell nucleus and indicating improved cell permeability. Conversely, the inclusion of long alkyl chains hindered the crossing of the cell wall by the dye molecules, preventing staining of the cell membrane and demonstrating membrane impermeability to plant cells. The FMR dyes with medium-length alkyl chains rapidly crossed the cell wall, uniformly stained the cell membrane, and anchored to it for a long period without being transmembrane. This allowed for visualization and tracking of the morphological dynamics of the cell plasma membrane during water loss in a 4D mode. This suggests that the introduction of medium-length alkyl chains into amphiphilic fluorescent dyes can transform them from membrane-permeable fluorescent dyes to membrane-staining fluorescent dyes suitable for long-term imaging of the plasma membrane. In addition, we have successfully converted a membrane-impermeable fluorescent dye molecule into a membrane-staining fluorescent dye by introducing medium-length alkyl chains into the molecule. This molecular engineering of dye molecules with alkyl chains to regulate cell permeability provides a simple and effective design rule for long-term visualization of the plasma membrane, and a convenient and feasible means of chemical modification for efficient transmembrane transport of small molecule drugs.

The extracellular vesicle proteomes of Sorghum bicolor and Arabidopsis thaliana are partially conserved.

Plant extracellular vesicles (EVs) are membrane-bound organelles involved mainly in intercellular communications and defense responses against pathogens. Recent studies have demonstrated the presence of proteins, nucleic acids including small RNAs, and lipids along with other metabolites in plant EVs. Here, we describe the isolation and characterization of EVs from sorghum (Sorghum bicolor). Nanoparticle tracking analysis, dynamic light scattering, and cryo-electron tomography showed the presence of a heterogeneous population of EVs isolated from the apoplastic wash of sorghum leaves. Cryo-electron microscopy revealed that EVs had a median size of 110 nm and distinct populations of vesicles with single or multiple lipid bilayers and low or high amounts of contents. The heterogeneity was further supported by data showing that only a subset of EVs that were stained with a membrane dye, Potomac Gold, were also stained with the membrane-permeant esterase-dependent dye, calcein acetoxymethyl ester. Proteomic analysis identified 437 proteins that were enriched in multiple EV isolations, with the majority of these also found in the EV proteome of Arabidopsis (Arabidopsis thaliana). These data suggest a partial conservation of EV contents and function between the monocot, sorghum, and a distantly related eudicot, Arabidopsis.

Rapid and accurate quantification of viable Bacillus cereus in foods with a Propidium monoazide (PMA) - Fluorescence in situ hybridization (FISH) - Flow cytometry (FCM) method

Fluorescence-labeled antibodies and viability indicators are routinely employed in conjunction with flow cytometry (FCM) to rapidly quantify viable target bacteria, ensuring food safety. However, the specific detection of Bacillus cereus, a prominent bacterium causing food poisoning, proves challenging because of its close phylogenetic relationship with other Bacillus species. Moreover, the accuracy of viability indicators in identifying viable B. cereus has been underexplored. This study established a rapid and specific method for quantifying viable B. cereus. The membrane-permeable dye propidium monoazide was identified as an effective means to differentiate between viable and non-viable B. cereus cells. Subsequently, fluorescence in situ hybridization was introduced to specifically identify viable B. cereus cells. Furthermore, FCM was utilized for the rapid and accurate quantification of labeled viable B. cereus cells. Following optimization, the method demonstrated notable specificity and robust anti-interference ability, with B. cereus recovery rates in milk powder and chicken breast after pretreatment ranging from 96.3% to 97.7%. Viable B. cereus cells were accurately quantified within 1.5 h, with a linear range of 102 to 107 cells/g. In conclusion, this developed method enables the rapid and specific quantification of viable B. cereus, offering a novel approach to the detection of specific foodborne pathogens.

Boron Clusters Alter the Membrane Permeability of Dicationic Fluorescent DNA-Staining Dyes.

Membrane-permeable fluorescent dyes that stain DNA are useful reagents for microscopic imaging, as they can be introduced into living cells to label DNA. However, the number of these dyes, such as Hoechst 33342, is limited. Here, we show that the icosahedral dodecaborate B12Br122-, a superchaotropic carrier that delivers different types of molecules into cells, functions as an excellent carrier for membrane-impermeable fluorescent dyes. Propidium iodide (PI) and 4',6-diamidino-2-phenylindole (DAPI), dicationic membrane-impermeable fluorescent dyes that stain DNA, can permeate cell membranes in the presence of boron clusters. Methyl green (MG), a dicationic dye used in the histological and fluorescent staining of DNA, permeated cell membranes in the presence of boron clusters. In contrast, monocationic membrane-permeable fluorescent dyes, such as acridine orange and pyronin Y, exhibited reduced fluorescence in cells in the presence of boron clusters. Boron clusters do not quench dicationic fluorescent dyes in water in vitro but have quenching effects on monocationic fluorescent dyes. We have demonstrated that the addition of B12Br122- to impermeable dicationic fluorescent DNA-staining dyes, such as DAPI, PI, and MG, which have been widely used for numerous years, imparts membrane permeability to introduce these dyes into living cells.

Double-Labeling Method for Visualization and Quantification of Membrane-Associated Proteins in Lactococcus lactis.

Lactococcus lactis displaying recombinant proteins on its surface can be used as a potential drug delivery vector in prophylactic medication and therapeutic treatments for many diseases. These applications enable live-cell mucosal and oral administration, providing painless, needle-free solutions and triggering robust immune response at the site of pathogen entry. Immunization requires quantitative control of antigens and, ideally, a complete understanding of the bacterial processing mechanism applied to the target proteins. In this study, we propose a double-labeling method based on a conjugated dye specific for a recombinantly introduced polyhistidine tag (to visualize surface-exposed proteins) and a membrane-permeable dye specific for a tetra-cysteine tag (to visualize cytoplasmic proteins), combined with a method to block the labeling of surface-exposed tetra-cysteine tags, to clearly obtain location-specific signals of the two dyes. This allows simultaneous detection and quantification of targeted proteins on the cell surface and in the cytoplasm. Using this method, we were able to detect full-length peptide chains for the model proteins HtrA and BmpA in L. lactis, which are associated with the cell membrane by two different attachment modes, and thus confirm that membrane-associated proteins in L. lactis are secreted using the Sec-dependent post-translational pathway. We were able to quantitatively follow cytoplasmic protein production and accumulation and subsequent export and surface attachment, which provides a convenient tool for monitoring these processes for cell surface display applications.

A new biosensor design reveals conformational changes of single molecules in living cells

Studying the conformational changes of single molecules inside living cells is challenging because current biosensor techniques produce insufficient light. We have developed biosensors for GTPase activity based on bright environment-sensing fluorescent dyes that undergo spectral changes suitable for ratiometric imaging in living cells. For simple construction of biosensors within cells, we used membrane-permeable dyes that attach to a SNAP-tag incorporated in the biosensors. The SNAP/dye was positioned in a linker between the GTPase and a peptide that binds specifically to the active conformation of the GTPase.

An Imaging and Computational Algorithm for Efficient Identification and Quantification of Neutrophil Extracellular Traps.

Neutrophil extracellular traps (NETs) are associated with multiple disease pathologies including sepsis, asthma, rheumatoid arthritis, cancer, systemic lupus erythematosus, acute respiratory distress syndrome, and COVID-19. NETs, being a disintegrated death form, suffered inconsistency in their identification, nomenclature, and quantifications that hindered therapeutic approaches using NETs as a target. Multiple strategies including microscopy, ELISA, immunoblotting, flow cytometry, and image-stream-based methods have exhibited drawbacks such as being subjective, non-specific, error-prone, and not being high throughput, and thus demand the development of innovative and efficient approaches for their analyses. Here, we established an imaging and computational algorithm using high content screening (HCS)—cellomics platform that aid in easy, rapid, and specific detection as well as analyses of NETs. This method employed membrane-permeable and impermeable DNA dyes in situ to identify NET-forming cells. Automated algorithm-driven single-cell analysis of change in nuclear morphology, increase in nuclear area, and change in intensities provided precise detection of NET-forming cells and eliminated user bias with other cell death modalities. Further combination with Annexin V staining in situ detected specific death pathway, e.g., apoptosis, and thus, discriminated between NETs, apoptosis, and necrosis. Our approach does not utilize fixation and permeabilization steps that disturb NETs, and thus, allows the time-dependent monitoring of NETs. Together, this specific imaging-based high throughput method for NETs analyses may provide a good platform for the discovery of potential inhibitors of NET formation and/or agents to modulate neutrophil death, e.g., NETosis-apoptosis switch, as an alternative strategy to enhance the resolution of inflammation.

CRISPR/Cas9 Genome Editing vs. Over-Expression for Fluorescent Extracellular Vesicle-Labeling: A Quantitative Analysis.

Over-expression of fluorescently-labeled markers for extracellular vesicles is frequently used to visualize vesicle up-take and transport. EVs that are labeled by over-expression show considerable heterogeneity regarding the number of fluorophores on single particles, which could potentially bias tracking and up-take studies in favor of more strongly-labeled particles. To avoid the potential artefacts that are caused by over-expression, we developed a genome editing approach for the fluorescent labeling of the extracellular vesicle marker CD63 with green fluorescent protein using the CRISPR/Cas9 technology. Using single-molecule sensitive fluorescence microscopy, we quantitatively compared the degree of labeling of secreted small extracellular vesicles from conventional over-expression and the CRISPR/Cas9 approach with true single-particle measurements. With our analysis, we can demonstrate a larger fraction of single-GFP-labeled EVs in the EVs that were isolated from CRISPR/Cas9-modified cells (83%) compared to EVs that were isolated from GFP-CD63 over-expressing cells (36%). Despite only single-GFP-labeling, CRISPR-EVs can be detected and discriminated from auto-fluorescence after their up-take into cells. To demonstrate the flexibility of the CRISPR/Cas9 genome editing method, we fluorescently labeled EVs using the HaloTag® with lipid membrane permeable dye, JaneliaFluor® 646, which allowed us to perform 3D-localization microscopy of single EVs taken up by the cultured cells.

Abstract 9601: Sarcolipin Reduction Prevents Mitochondrial Dysfunction in Dystrophic Myocardium

Rationale & Hypothesis: Sarcolipin (SLN), an inhibitor of sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) is abnormally high and enriched in the mitochondrial associated membrane (MAM) fractions of dystrophic hearts. We, therefore, hypothesized that in the dystrophic cardiomyocytes, SLN upregulation causes abnormal elevation of intracellular Ca 2+ (Ca 2+ i ) including Ca 2+ mishandling in the MAM region, resulting in sustained elevation of mitochondrial Ca 2+ (Ca 2+ m ), which in turn affects mitochondrial structure and function. Approach: We chose dystrophin mutant and utrophin deficient, mdx:utr -/- mice for our studies and generated SLN haploinsufficient mdx:utr -/- (mdx:utr -/- :sln +/- ) mice. Single-cell Ca 2+ transients and Ca 2+ m efflux were measured in isolated cardiomyocytes (CM's) to determine Ca 2+ i handling and Ca 2+ m content, respectively. Mitochondrial membrane potential was assessed with membrane-permeant dyes in isolated CM's followed by confocal imaging. Mitochondrial respiration was measured using Complex activity assays and Seahorse metabolic profiling in ventricular tissue lysates and isolated mitochondria respectively. Mitochondrial structure, number, and area were evaluated using electron micrographs (EM). Results: Heterozygous deletion of the SLN gene normalized SLN expression and improved Ca 2+ i cycling and prevented Ca 2+ m overload in dystrophic CM's. Furthermore, reducing SLN expression prevents loss of membrane potential and improves mitochondrial respiration in the dystrophic cardiac myocytes. TEM analyses of the dystrophic heart revealed significant cristae loss in the mitochondria globally as well as in the MAM-associated mitochondria. On the other hand, the reduction in SLN expression prevents these changes. Conclusions: In conclusion, reduction in SLN expression preserves the SR-mitochondrial interface and restores the mitochondrial function by mitigating the Ca 2+ overload, membrane potential loss, and structural damage. These changes improve cardiac function and prevent the development of cardiomyopathy in mdx:utr -/- mice. Our findings suggest that SLN reduction could be a potential therapeutic strategy for the treatment of Duchenne muscular dystrophy and associated cardiomyopathy.

Abstract P385: Sarcolipin Reduction Prevents Mitochondrial Dysfunction In Dystrophic Myocardium

Rationale & Hypothesis: Sarcolipin (SLN), an inhibitor of sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) is abnormally high and enriched in the mitochondrial associated membrane (MAM) fractions of dystrophic hearts. We, therefore, hypothesized that in the dystrophic cardiomyocytes, SLN upregulation causes abnormal elevation of intracellular Ca 2+ (Ca 2+ i ) including Ca 2+ mishandling in the MAM region, resulting in sustained elevation of mitochondrial Ca 2+ (Ca 2+ m ) , which in turn affects mitochondrial structure and function. Approach: We chose dystrophin mutant and utrophin deficient, mdx:utr -/- mice for our studies and generated SLN haploinsufficient mdx:utr -/- (mdx:utr -/- :sln +/- ) mice. Single-cell Ca 2+ transients and Ca 2+ m efflux were measured in isolated cardiomyocytes to determine Ca 2+ i handling and Ca 2+ m content, respectively. Mitochondrial membrane potential was assessed with membrane-permeant dyes in isolated cardiomyocytes followed by confocal imaging. Mitochondrial respiration was measured using Complex activity assays and Seahorse metabolic profiling in ventricular tissue lysates and isolated mitochondria respectively. Mitochondrial structure, number, and area were evaluated using electron micrographs. Results: Heterozygous deletion of the SLN gene normalized SLN expression and improved Ca 2+ i cycling and prevented Ca 2+ m overload in dystrophic cardiac myocytes. Furthermore, reducing SLN expression prevents loss of membrane potential and improves mitochondrial respiration in the dystrophic cardiac myocytes. Transmission electron microscopic analyses of the dystrophic heart revealed significant cristae loss in the mitochondria globally as well as in the MAM-associated mitochondria. On the other hand, the reduction in SLN expression prevents these changes. Conclusions: In conclusion, reduction in SLN expression preserves the SR-mitochondrial interface and restores the mitochondrial function by mitigating the Ca 2+ overload, membrane potential loss, and structural damage. These changes improve cardiac function and prevent the development of cardiomyopathy in mdx:utr -/- mice. Our findings suggest that SLN reduction could be a potential therapeutic strategy for the treatment of Duchenne muscular dystrophy and associated cardiomyopathy.

Dual-fluorescent bacterial two-hybrid system for quantitative Protein–Protein interaction measurement via flow cytometry

Characterization of protein–protein interactions (PPIs) is essential for understanding cellular signal transduction pathways. However, quantitative measurement of the binding strength remains challenging. Building upon the classical bacterial adenylate cyclase two-hybrid (BACTH) system, we previously demonstrated that the relative reporter protein expression (RRPE), defined as the level of reporter expression normalized to that of the interacting protein, is an intrinsic characteristic associated with the binding strength between the two interacting proteins. In this study, we inserted fluorescent protein tdTomato in the chromosome as the reporter protein by CRISPR/Cas9 technology and employed a 12-amino acid tetracysteine (TC) to tag one of the interacting proteins, which can be further labeled by a membrane-permeable biarsenical dye. The combined use of tdTomato and TC-tag offers rapid and high-throughput analysis of the expression levels of both the reporter protein and one of the interacting proteins at the single-cell level by multicolor flow cytometry, which simplifies the quantitative measurement of PPI. The use of the as-developed RRPE-tdTomato-TC-BACTH approach was demonstrated in three demanding applications. First, binding affinities could be correctly ranked for discriminating interaction strengths with a tenfold difference or of the same order of magnitude. We demonstrate that the method is sensitive enough to discriminate affinities with a small difference of 1.4-fold. Moreover, residues involved in PPI can be easily mapped and ranked. Lastly, protein interaction inhibitors can be rapidly screened.

Dimerization Drives Proper Folding of Human Alanine:Glyoxylate Aminotransferase But Is Dispensable for Peroxisomal Targeting.

Peroxisomal matrix proteins are transported into peroxisomes in a fully-folded state, but whether multimeric proteins are imported as monomers or oligomers is still disputed. Here, we used alanine:glyoxylate aminotransferase (AGT), a homodimeric pyridoxal 5′-phosphate (PLP)-dependent enzyme, whose deficit causes primary hyperoxaluria type I (PH1), as a model protein and compared the intracellular behavior and peroxisomal import of native dimeric and artificial monomeric forms. Monomerization strongly reduces AGT intracellular stability and increases its aggregation/degradation propensity. In addition, monomers are partly retained in the cytosol. To assess possible differences in import kinetics, we engineered AGT to allow binding of a membrane-permeable dye and followed its intracellular trafficking without interfering with its biochemical properties. By fluorescence recovery after photobleaching, we measured the import rate in live cells. Dimeric and monomeric AGT displayed a similar import rate, suggesting that the oligomeric state per se does not influence import kinetics. However, when dimerization is compromised, monomers are prone to misfolding events that can prevent peroxisomal import, a finding crucial to predicting the consequences of PH1-causing mutations that destabilize the dimer. Treatment with pyridoxine of cells expressing monomeric AGT promotes dimerization and folding, thus, demonstrating the chaperone role of PLP. Our data support a model in which dimerization represents a potential key checkpoint in the cytosol at the crossroad between misfolding and correct targeting, a possible general mechanism for other oligomeric peroxisomal proteins.

A New Biosensor Design Reveals Conformational Changes of Single Molecules in Living Cells

GTPases are switch-like enzymes that interact with downstream effector proteins only when they are in their GTP-bound conformation. They regulate a wide variety of cell functions including migration, apoptosis, proliferation, and membrane trafficking. We have developed biosensors for GTPase activity based on environment-sensing fluorescent dyes, purposefully using dyes that undergo spectral changes suitable for ratiometric imaging in living cells. For simple construction of the biosensors within cells, we used membrane-permeable dyes that attach to a SNAP-tag incorporated in the biosensors.

DNA-Origami-Based Fluorescence Brightness Standards for Convenient and Fast Protein Counting in Live Cells.

Fluorescence microscopy has been one of the most discovery-rich methods in biology. In the digital age, the discipline is becoming increasingly quantitative. Virtually all biological laboratories have access to fluorescence microscopes, but abilities to quantify biomolecule copy numbers are limited by the complexity and sophistication associated with current quantification methods. Here, we present DNA-origami-based fluorescence brightness standards for counting 5-300 copies of proteins in bacterial and mammalian cells, tagged with fluorescent proteins or membrane-permeable organic dyes. Compared to conventional quantification techniques, our brightness standards are robust, straightforward to use, and compatible with nearly all fluorescence imaging applications, thereby providing a practical and versatile tool to quantify biomolecules via fluorescence microscopy.

Visualization of Spirochetes by Labeling Membrane Proteins With Fluorescent Biarsenical Dyes.

Numerous methods exist for fluorescently labeling proteins either as direct fusion proteins (GFP, RFP, YFP, etc.—attached to the protein of interest) or utilizing accessory proteins to produce fluorescence (SNAP-tag, CLIP-tag), but the significant increase in size that these accompanying proteins add may hinder or impede proper protein folding, cellular localization, or oligomerization. Fluorescently labeling proteins with biarsenical dyes, like FlAsH, circumvents this issue by using a short 6-amino acid tetracysteine motif that binds the membrane-permeable dye and allows visualization of living cells. Here, we report the successful adaptation of FlAsH dye for live-cell imaging of two genera of spirochetes, Leptospira and Borrelia, by labeling inner or outer membrane proteins tagged with tetracysteine motifs. Visualization of labeled spirochetes was possible by fluorescence microscopy and flow cytometry. A subsequent increase in fluorescent signal intensity, including prolonged detection, was achieved by concatenating two copies of the 6-amino acid motif. Overall, we demonstrate several positive attributes of the biarsenical dye system in that the technique is broadly applicable across spirochete genera, the tetracysteine motif is stably retained and does not interfere with protein function throughout the B. burgdorferi infectious cycle, and the membrane-permeable nature of the dyes permits fluorescent detection of proteins in different cellular locations without the need for fixation or permeabilization. Using this method, new avenues of investigation into spirochete morphology and motility, previously inaccessible with large fluorescent proteins, can now be explored.

An Assay to Assess Gap Junction Communication in Cell Lines.

This protocol was developed to assess communication in tumor cells and to provide a dependable and standardized assay for the in vitro determination of gap junction function. The method is noninvasive; in this method, the cell population under study is divided such that 1 fraction is loaded with a lipophilic cell plasma membrane permeable dye, calcein acetoxymethyl ester, that is hydrolyzed upon cellular uptake by cytoplasmic esterases to yield calcein, a fluorescent and membrane-impermeable molecule. The other fraction is loaded with 1,1'-dioctadecyl-3,3,3',3' tetramethylindodicarbocyanine perchlorate (DiD)/1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate [Dil; DilC18(3)], which is a lipophilic membrane dye that diffuses laterally to stain the entire cell membrane, is impermeable, and attains an orange-red fluorescence upon incorporation into membranes. The 2 fractions are mixed and incubated under coculture conditions. Calcein with MW 890 kD is transferred to the DiD/DiI-stained cells through gap junctions. The assessment of this uptake is made with confocal imaging and quantitated using flow cytometry. Cell lines representing cancer of the breast as well as a nontransformed cell line developed from the buccal mucosa were analyzed for gap junction competency. Confocal imaging with acquisition at specific time points during the in vitro treatment and flow cytometry gave a qualitative and quantitative analysis of the passage of molecules through the gap junctions. Here, the method has been combined to obtain images as well as quantitation and is a simple and effective approach in assessing the functional competency of gap junction in epithelial cells.

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a super-resolution technique that allows for the determination of the microcompartments that are accessible for proteins by generating localization and tracking maps of these proteins. Moreover, by multi-color localization microscopy, the localization and tracking profiles of proteins in different subcompartments are obtained simultaneously. The technique is specific for live cells and is based on the repetitive imaging of single mobile membrane proteins. Proteins of interest are genetically fused with specific, so-called self-labeling tags. These tags are enzymes that react with a substrate in a covalent manner. Conjugated to these substrates are fluorescent dyes. Reaction of the enzyme-tagged proteins with the fluorescence labeled substrates results in labeled proteins. Here, Tetramethylrhodamine (TMR) and Silicon Rhodamine (SiR) are used as fluorescent dyes attached to the substrates of the enzymes. By using substrate concentrations in the pM to nM range, sub-stoichiometric labeling is achieved that results in distinct signals. These signals are localized with ~15-27 nm precision. The technique allows for multi-color imaging of single molecules, whereby the number of colors is limited by the available membrane-permeable dyes and the repertoire of self-labeling enzymes. We show the feasibility of the technique by determining the localization of the quality control enzyme (Pten)-induced kinase 1 (PINK1) in different mitochondrial compartments during its processing in relation to other membrane proteins. The test for true physical interactions between differently labeled single proteins by single molecule FRET or co-tracking is restricted, though, because the low labeling degrees decrease the probability for having two adjacent proteins labeled at the same time. While the technique is strong for imaging proteins in membrane compartments, in most cases it is not appropriate to determine the localization of highly mobile soluble proteins.