Intact Single Cells Research Articles

Experimentally guided models reveal replication principles that shape the mutation distribution of RNA viruses.

Life history theory posits that the sequence and timing of events in an organism's lifespan are fine-tuned by evolution to maximize the production of viable offspring. In a virus, a life history strategy is largely manifested in its replication mode. Here, we develop a stochastic mathematical model to infer the replication mode shaping the structure and mutation distribution of a poliovirus population in an intact single infected cell. We measure production of RNA and poliovirus particles through the infection cycle, and use these data to infer the parameters of our model. We find that on average the viral progeny produced from each cell are approximately five generations removed from the infecting virus. Multiple generations within a single cell infection provide opportunities for significant accumulation of mutations per viral genome and for intracellular selection.

Three-dimensional mid-infrared tomographic imaging of endogenous and exogenous molecules in a single intact cell with subcellular resolution.

Microscopy in the mid-infrared spectral range provides detailed chemical information on a sample at moderate spatial resolution and is being used increasingly in the characterization of biological entities as challenging as single cells. However, a conventional cellular 2D imaging measurement is limited in its ability to associate specific compositional information to subcellular structures because of the interference from the complex topography of the sample. Herein we provide a method and protocols that overcome this challenge in which tilt-series infrared tomography is used with a standard benchtop infrared microscope. This approach gives access to the quantitative 3D distribution of molecular components based on the intrinsic contrast provided by the sample. We demonstrate the method by quantifying the distribution of an exogenous metal carbonyl complex throughout the cell and by reporting changes in its coordination sphere in different locations in the cell.

Lysinibacillus varians sp. nov., an endospore-forming bacterium with a filament-to-rod cell cycle.

Six Gram-stain-positive, motile, filamentous and/or rod-shaped, spherical spore-forming bacteria (strains GY32(T), L31, F01, F03, F06 and F07) showing polybrominated diphenyl ether transformation were investigated to determine their taxonomic status. After spore germination, these organisms could grow more than one hundred microns long as intact single cells and then divide into rod cells and form endospores in 33 h. The cell-wall peptidoglycan of these strains was type A4α, the predominant menaquinone was MK-7 and the major fatty acids were iso-C(16:0), iso-C(15:0) and C(16:1)ω7C. Diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine were detected in the polar lipid profile. Analysis of the 16S rRNA gene sequences indicated that these strains should be placed in the genus Lysinibacillus and they were most closely related to Lysinibacillus sphaericus DSM 28(T) (99% 16S rRNA gene sequence similarity). The gyrB sequence similarity and DNA-DNA relatedness between strain GY32(T) and L. sphaericus JCM 2502(T) were 81% and 52%, respectively. The G+C content of the genomic DNA of strain GY32(T) was 43.2 mol%. In addition, strain GY32(T) showed differences in nitrate reduction, starch and gelatin hydrolysis, carbon resource utilization and cell morphology. The phylogenetic distance from its closest relative measured by DNA-DNA relatedness and DNA G+C content, and its phenotypic properties demonstrated that strain GY32(T) represents a novel species of the genus Lysinibacillus, for which the name Lysinibacillus varians sp. nov. is proposed. The type strain is GY32(T) ( = NBRC 109424(T) = CGMCC 1.12212(T) = CCTCC M 2011307(T)).

In situ drug-receptor binding kinetics in single cells: a quantitative label-free study of anti-tumor drug resistance.

Many drugs are effective in the early stage of treatment, but patients develop drug resistance after a certain period of treatment, causing failure of the therapy. An important example is Herceptin, a popular monoclonal antibody drug for breast cancer by specifically targeting human epidermal growth factor receptor 2 (Her2). Here we demonstrate a quantitative binding kinetics analysis of drug-target interactions to investigate the molecular scale origin of drug resistance. Using a surface plasmon resonance imaging, we measured the in situ Herceptin-Her2 binding kinetics in single intact cancer cells for the first time, and observed significantly weakened Herceptin-Her2 interactions in Herceptin-resistant cells, compared to those in Herceptin-sensitive cells. We further showed that the steric hindrance of Mucin-4, a membrane protein, was responsible for the altered drug-receptor binding. This effect of a third molecule on drug-receptor interactions cannot be studied using traditional purified protein methods, demonstrating the importance of the present intact cell-based binding kinetics analysis.

Microfluidic molecular assay platform for the detection of miRNAs, mRNAs, proteins, and posttranslational modifications at single-cell resolution.

Cell signaling is a dynamic and complex process. A typical signaling pathway may begin with activation of cell surface receptors, leading to activation of a kinase cascade that culminates in induction of messenger RNA (mRNA) and noncoding microRNA (miRNA) production in the nucleus, followed by modulation of mRNA expression by miRNAs in the cytosol, and end with production of proteins in response to the signaling pathway. Signaling pathways involve proteins, miRNA, and mRNAs, along with various forms of transient posttranslational modifications, and detecting each type of signaling molecule requires categorically different sample preparation methods such as Western blotting for proteins, PCR for nucleic acids, and flow cytometry for posttranslational modifications. Since we know that cells in populations behave heterogeneously,(1) especially in the cases of stem cells, cancer, and hematopoiesis, there is need for a new technology that provides capability to detect and quantify multiple categories of signaling molecules in intact single cells to provide a comprehensive view of the cell's physiological state. In this Technology Brief, we describe our automated microfluidic platform with a portfolio of customized molecular assays that can detect nucleic acids, proteins, and posttranslational modifications in single intact cells with >95% reduction in reagent requirement in under 8 h.

Cytodiagnosis of papillary carcinoma of the breast: Report of a case with histological correlation

Papillary lesions of the breast pose diagnostic challenges on aspiration cytology due to overlapping features of benign and malignant entities. Accurate cytologic diagnosis of papillary breast carcinoma cannot usually be made pre-operatively. We present the case of an adult female who underwent fine-needle aspiration (FNA) of a left breast lump. FNA smears were highly cellular showing cohesive clusters, complex papillary fragments and few singly dispersed intact cells. The tumor cells had hyperchromatic nuclei, prominent nucleoli and mild nuclear pleomorphism. A cytologic impression of papillary lesion, possibly malignant (in view of high cellularity, complex papillae and single intact cells) was rendered. The lesion proved to be a papillary carcinoma with microscopic foci of stromal invasion on histologic examination. Papillary carcinoma, an uncommon subtype of breast carcinoma, should be considered while evaluating a papillary lesion with complex branching papillae containing delicate fibrovascular cores and singly lying intact atypical cells.

MiRNA detection at single-cell resolution using microfluidic LNA flow-FISH.

Flow cytometry in combination with fluorescent in situ hybridization (flow-FISH) is a powerful technique that can be utilized to rapidly detect nucleic acids at single-cell resolution without the need for homogenization or nucleic acid extraction. Here, we describe a microfluidic-based method which enables the detection of microRNAs or miRNAs in single intact cells by flow-FISH using locked nucleic acid (LNA)-containing probes. Our method can be applied to all RNA species including mRNA and small noncoding RNA and is suitable for multiplexing with protein immunostaining in the same cell. For demonstration of our method, this chapter details the detection of miR155 and CD69 protein in PMA and ionomycin-stimulated Jurkat cells. We also include instructions on how to set up a microfluidic chip sample preparation station to prepare cells for imaging and analysis on a commercial flow cytometer or a custom-built micro-flow cytometer.

Non-invasive In-cell Determination of Free Cytosolic [NAD+]/[NADH] Ratios Using Hyperpolarized Glucose Show Large Variations in Metabolic Phenotypes

Accumulating evidence suggest that the pyridine nucleotide NAD has far wider biological functions than its classical role in energy metabolism. NAD is used by hundreds of enzymes that catalyze substrate oxidation and, as such, it plays a key role in various biological processes such as aging, cell death, and oxidative stress. It has been suggested that changes in the ratio of free cytosolic [NAD(+)]/[NADH] reflects metabolic alterations leading to, or correlating with, pathological states. We have designed an isotopically labeled metabolic bioprobe of free cytosolic [NAD(+)]/[NADH] by combining a magnetic enhancement technique (hyperpolarization) with cellular glycolytic activity. The bioprobe reports free cytosolic [NAD(+)]/[NADH] ratios based on dynamically measured in-cell [pyruvate]/[lactate] ratios. We demonstrate its utility in breast and prostate cancer cells. The free cytosolic [NAD(+)]/[NADH] ratio determined in prostate cancer cells was 4 times higher than in breast cancer cells. This higher ratio reflects a distinct metabolic phenotype of prostate cancer cells consistent with previously reported alterations in the energy metabolism of these cells. As a reporter on free cytosolic [NAD(+)]/[NADH] ratio, the bioprobe will enable better understanding of the origin of diverse pathological states of the cell as well as monitor cellular consequences of diseases and/or treatments.

Receptor-ligand interactions: binding affinities studied by single-molecule and super-resolution microscopy on intact cells.

Protein–ligand interactions play an important role in many biological processes. Notably, membrane receptors are the starting point for a huge variety of cellular signal transduction pathways. Quantifying the binding affinity of a ligand for its transmembrane receptor is of great importance as it provides information on the potency of the ligand. We developed a new experimental procedure to determine binding affinities of ligands for their membrane receptors directly on intact single cells using super-resolution imaging. Dissociation constants were determined by titrating fluorophore-labelled ligand against cells expressing the target protein and applying single-molecule imaging.

Dynamics of Gαi1 interaction with type 5 adenylate cyclase reveal the molecular basis for high sensitivity of Gi-mediated inhibition of cAMP production

Many physiological and pathophysiological processes are regulated by cAMP. Different therapies directly or indirectly influence the cellular concentration of this second messenger. A wide variety of receptors either activates or inhibits adenylate cyclases in order to induce proper physiological responses. A key event in this signalling system is the direct and dynamic interaction of Gαi1 subunits with adenylate cyclases. We established a FRET-based assay between G-protein subunits and AC5 (type 5 adenylate cyclase) and monitored receptor-stimulated interactions between Gαi1 and AC5 in single intact cells with high temporal resolution. We observed that FRET between Gαi1 and AC5 developed at much lower concentration of agonist compared with the overall Gi-protein activity resulting in a left-shift of the concentration-response curve by approximately one order of magnitude. Furthermore, Gi1-protein-mediated attenuation of AC5-dependent increases in cAMP occurred at comparable low concentrations of agonist. On analysing the dynamics we found the dissociation of the Gαi1 subunits and AC5 to occur significantly slower than the G-protein deactivation and to be insensitive to RGS4 (regulator of G-protein signalling type 4) expression. This led us to the conclusion that AC5, by binding active Gαi1, interferes with G-protein deactivation and reassembly and thereby might sensitize its own regulation.

Abstract 4128: A novel approach to the measurement of tyrosine phosphorylation dynamics in intact single cells using capillary electrophoresis.

Abstract Receptor tyrosine kinases (RTKs) are key components of physiological and pathological cell signaling, and are normally subject to tight regulation by protein tyrosine phosphatases (PTPs). Irregularities on either side of this equilibrium can lead to inflammation, metabolic disease, and cancer. While many established and emerging techniques have been used to characterize these signaling pathways, few approaches are well adapted to the analysis of single primary cells, and fewer still measure enzyme activity directly. We report a new method for measuring the balance between RTK and PTP activity using fluorescent peptide substrates in conjunction with capillary electrophoresis. Briefly, live cells are visualized on an inverted microscope, microinjected with a fluorescent peptide substrate which is acted upon by RTKs and/or PTPs. After the desired time interval, a single cell is lysed using a microsecond laser pulse and cell contents are aspirated into a fused-silica capillary where electrophoresis is performed and reporter species are separated and quantified based on phosphorylation status. We have applied this technique to the characterization of PTP inhibition in single A431 cells by several components of diesel exhaust particles, an important component of air pollution linked to cancer. We have also adapted this system to measure the effects of small-molecule epidermal growth factor receptor (EGFR) inhibitors on RTK activity in tumor cell lines as well as tumor xenograft models, also at the single cell level. This approach provides a unique set of advantages over alternative techniques and is a valuable tool for the study of cell signaling in cancer. The integrated microscopy permits selection of cells for analysis based on morphology, viability, or vital staining, an important consideration for analysis of heterogeneous samples such as tumor specimens, especially when total sample size is small. High-sensitivity detection allows chemical cytometry data from individual cells to be gathered and the presence of phenotypic subpopulations is not lost by observing only the average behavior of a large population. Additionally, peptide substrate reporters can be tuned for enzyme specificity, and the analytical separation allows multiple reporter pairs to be separated and detected in a single experiment, permitting simultaneous analysis of multiple enzymes. The introduction of reporter peptides by microinjection preserves cellular architecture, while being equally applicable to immortalized and primary cells since no genetic manipulation is necessary. This array of advantages makes our approach uniquely well suited to the study of RTK and PTP signaling in both cultured tumor cell lines and patient specimens and a valuable new tool for cancer research. Citation Format: Ryan M. Phillips, David S. Lawrence, Christopher E. Sims, Nancy L. Allbritton. A novel approach to the measurement of tyrosine phosphorylation dynamics in intact single cells using capillary electrophoresis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4128. doi:10.1158/1538-7445.AM2013-4128

Temporal and spatial characterization of nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a quality control mechanism responsible for "surveying" mRNAs during translation and degrading those that harbor a premature termination codon (PTC). Currently the intracellular spatial location of NMD and the kinetics of its decay step in mammalian cells are under debate. To address these issues, we used single-RNA fluorescent in situ hybridization (FISH) and measured the NMD of PTC-containing β-globin mRNA in intact single cells after the induction of β-globin gene transcription. This approach preserves temporal and spatial information of the NMD process, both of which would be lost in an ensemble study. We determined that decay of the majority of PTC-containing β-globin mRNA occurs soon after its export into the cytoplasm, with a half-life of <1 min; the remainder is degraded with a half-life of >12 h, similar to the half-life of normal PTC-free β-globin mRNA, indicating that it had evaded NMD. Importantly, NMD does not occur within the nucleoplasm, thus countering the long-debated idea of nuclear degradation of PTC-containing transcripts. We provide a spatial and temporal model for the biphasic decay of NMD targets.

Inducible growth mode switches influence Valonia rhizoid differentiation

Cell differentiation and cell type commitment are an integral part of plant growth and development. Investigations on how environmental conditions affect the formation of shoots, roots, and rhizoids can help illustrate how plants determine cell fate and overall morphology. In this study, we evaluated the role of substratum and light on rhizoid differentiation in the coenocytic green alga, Valonia aegagropila. Elongating rhizoids displayed varying growth modes and cell shape upon exposure to different substrata and light conditions. It was found that soft substrata and dark incubation promoted rhizoid elongation via tip growth while subsequent exposure to light prevented tip growth and instead induced swelling in the apical region of rhizoids. Swelling was accompanied by the accumulation of protoplasm in the rhizoid tip through expansion of the cell wall and uninhibited cytoplasmic streaming. Subsequent diffuse growth led to the transformation from slender, rod-shaped rhizoids into spherical thallus-like structures that required photosynthesis. Further manipulation of light regimes caused vacillating cell growth redirections. An elongating V. aegagropila rhizoid cell thus appears capable of growth mode switching that is regulated by immediate environmental conditions thereby influencing ultimate cell shape and function. This is the first description of inducible, multiple growth mode shifts in a single intact plant cell that directly impact its differentiation.

Analysis of EGFR Signaling in Single Cells using Capillary Electrophoresis

The importance of protein kinases in cell signaling and human disease is well established. While much is understood about how these enzymes regulate the cellular environment, the direct measurement of kinase activity in single intact cells remains challenging. In the present work, a novel assay for measuring Epidermal Growth Factor Receptor (EGFR) activity in single cells has been developed. EGFR is a receptor tyrosine kinase known to play a role in numerous cancers and is implicated in the inflammatory response to air pollution. The analysis of EGFR activity in clinical samples would provide valuable information regarding mechanisms of disease and potential interventions, however these samples tend to be small, heterogeneous mixtures of primary cells that are difficult to analyze using existing technologies that require large cell populations (e.g. Western blotting, flow cytometry) or expression of a genetically encoded sensor (e.g. FRET). We have developed an assay for measuring the phosphorylation dynamics within single intact cells using fluorescent substrate peptides in conjunction with capillary electrophoresis (CE). A peptide reporter is loaded into the cell of interest, phosphorylated by the intact cellular machinery, and then the cell is lysed and its contents analyzed by CE using high sensitivity laser-induced fluorescence detection. The ability to select individual cells coupled with sensitivity to 10−21 mol allows effective analysis of the clinical samples we are interested in studying. This technology has been validated using purified EGFR kinase domain and cell lysates, where the reporter is resistant to degradation for at least 1 hour. Additionally, phosphorylation (16.9% in 90 seconds) has been observed in an intact A431 cell treated with the phosphatase inhibitor sodium pervanadate. Single cell studies are ongoing and will explore pharmacologic modulation of EGFR as well as protein tyrosine phosphatases.

Ultrastructural Probes of Clusters of Open Regulatory Elements (Core) Within Chromatin

Introduction: Mammalian cells during interphase display clustering of DNase I-sensitive sites within the 10 nm micro-fibrils of active euchromatin (Song L, et al, Genome Research, August 19, 2011). Over 870,000 distinct CORE sites were identified as poised or active in gene transcription across an analysis of seven human cell lines, (ibid). Methods: We have developed a high-resolution electron microscopic technique for detecting DNase I-sensitive sites within intact single human cells. All such sites are confined to the euchromatin portion of the cell nucleus, and can be analyzed for location, number, shape, and size within each single cell analyzed within native biopsied tissue. Such ultrastructural probes can detect sites greater than 10 nm in diameter, and offer a global 3D view in 10 nm thick serial sections of the probed tissue after instant fixation during biopsy. Results: DNase I-sensitive probes were visualized within tissue biopsies of normal human bone marrow or Hodgkin lymphoma lymph nodes before therapy. Single cell analysis revealed all probe sites are confined to the euchromatin portion of the cell nucleus, and are visualized as ellipsoid clusters ranging in size from 25 - 100 nm (small), and 100 - 350 nm (large), to over 350 nm in overall diameter (giant). Within probed clusters, fine granulation is apparent of approximately 10 nm size, and the clusters are found near nuclear pores. Normal bone marrow cells display a reduction in size and number of probed clusters during cell differentiation and maturation, while Hodgkin lymphoma apposed lymphocytes display an increase in size and number of probed giant clusters during in-vivo immune activation. Conclusions: Cluster of open regulatory elements (CORE) ultrastructural probes based on the DNase I-sensitivity of active transcription sites, can be useful in single-cell analysis within biopsied intact tissues.

DIRECT TISSUE AND CELL ANALYSIS OF TULIP BULBS BY COMBINING PRESSURE PROBE AND UV-MALDI-MS

ISHS XXVIII International Horticultural Congress on Science and Horticulture for People (IHC2010): International Symposium on Micro and Macro Technologies for Plant Propagation and Breeding in Horticulture DIRECT TISSUE AND CELL ANALYSIS OF TULIP BULBS BY COMBINING PRESSURE PROBE AND UV-MALDI-MS

Intracellular Magneto-Spatial Organization of Magnetic Organelles Inside Intact Bacterial Cells

Magnetotactic bacteria naturally produce magnetosomes, i.e., biological membrane bound nanomagnets, at ambient conditions. It is important to understand simultaneously the possible size variations and the magnetic behavior of nano-magnets inside intact bacterial cells for both applicational purposes as well as to enhance the basic understanding of biomineralization leading to intracellular nano-magnet synthesis. In this work, we utilize High-resolution Transmission Electron Microscopy and Near-field Scanning Optical Microscopy based measurements on intact non-fixed single cells to rigorously and quantitatively understand the intra-cellular magneto-spatial distribution of nano-magnets synthesized by Magnetospirillum gryphiswaldense. We demonstrate that it is possible to measure the relative magnetic moments along the intracellular magnetosomal chains for intact and non-fixed bacterial cells. Using our in vivo measurements on several single cells, we report that magnetic behavior of intracellular nano-magnets synthesized by magnetotactic bacteria depend on their relative location in the magnetosomal chains. Our work opens promising avenues in the direction of measuring the magnetic behavior of nano-magnets inside living systems by utilizing an operationally straight-forward approach.

Partially purified components of Nardostachys chinensis suppress melanin synthesis through ERK and Akt signaling pathway with cAMP down-regulation in B16F10 cells

Ethnopharmacological relevance Nardostachys chinensis has been used in folk medicine to treat melasma and lentigines in Korea. We investigated the inhibitory activities of Nardostachys chinensis in melanogenesis and its related signaling pathway. Materials and methodsBioassay-guided fractionation of Nardostachys chinensis using solvent partitioning and purification with octadecylsilane open-column chromatography resulted in partial purification. The active 20% methanol chromatographic fraction from the ethyl acetate layer (PPNC) was used to investigate melanogenesis by melanin synthesis, tyrosinase activity assay, cAMP assay, Western blot and flow cytometric analyses in B16F10 mouse melanoma cells. ResultsPPNC markedly inhibits melanin synthesis and tyrosinase activity in a concentration-dependent manner. We also found that PPNC decreases microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein (TRP)-1, and dopachrome tautomerase (Dct) protein expressions and MITF and tyrosinase mRNA levels. Moreover, PPNC reduces intracellular cAMP levels and activates mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/Akt expression in B16F10 cells. The specific MEK/ERK inhibitor PD98059 and PI3K/Akt inhibitor LY294002, block the PPNC-induced hypopigmentation effect, and abrogate the PPNC-suppressed expression of melanogenic proteins such as MITF, tyrosinase, TRP-1, and Dct. Using flow cytometry, we elucidated whether PPNC directly induces ERK phosphorylation at the level of an intact single cell. PPNC shows marked expression of phosphorylated ERK in live B16F10 cells and abrogates PPNC-induced phosphorylated ERK by PD98059 treatment. ConclusionsPPNC stimulates MEK/ERK phosphorylation and PI3K/Akt signaling with suppressing cAMP levels and subsequently stimulating MITF and TRPs down-regulation, resulting in melanin synthesis suppression.

STATISTICAL OPTIMIZATION OF SINGLE-CELL PRODUCTION FROM Taxus cuspidata PLANT CELL AGGREGATES

Flow-cytometric characterization of plant cell culture growth and metabolism at the single-cell level is a method superior to traditional culture average measurements for collecting population information. Investigation of culture heterogeneity and production variability by obtaining information about different culture subpopulations is crucial for optimizing bio-processes for enhanced productivity. Obtaining high yields of intact and viable single cells from aggregated plant cell cultures is an enabling criterion for their analysis and isolation using high-throughput flow cytometric methods. The critical parameters affecting the enzymatic isolation of single cells from aggregated Taxus cuspidata plant cell suspensions were optimized using response-surface methodology and factorial central composite design. Using a design of experiments approach, the output response single-cell yield (SCY, percentage of cell clusters containing only a single cell) was optimized. Optimal conditions were defined for the independent parameters cellulase concentration, pectolyase Y-23 concentration, and centrifugation speed to be 0.045% (w/v), 0.7% (w/v), and 1200 × g, respectively. At these optimal conditions, the model predicted a maximum SCY of 48%. The experimental data exhibited a 72% increase over previously attained values and additionally validated the model predictions. More than 99% of the isolated cells were viable and suitable for rapid analysis through flow cytometry, thus enabling the collection of population information from cells that accurately represent aggregated suspensions. These isolated cells can be further studied to gain insight into both growth and secondary metabolite production, which can be used for bio-process optimization.

Distinct pharmacological properties of morphine metabolites at G i-protein and β-arrestin signaling pathways activated by the human μ-opioid receptor

Morphine and several other opioids are important drugs for the treatment of acute and chronic pain. Opioid-induced analgesia is predominantly mediated by the μ-opioid receptor (MOR). When administered to humans, complex metabolic pathways lead to generation of many metabolites, nine of which may be considered major metabolites. While the properties of the two main compounds, morphine-6-glucuronide and morphine-3-glucuronide, are well described, the activity of other morphine metabolites is largely unknown. Here we performed an extensive pharmacological characterization by comparing efficacies and potencies of morphine and its nine major metabolites for the two main signaling pathways engaged by the human MOR, which occur via G i-protein activation and β-arrestins, respectively. We used radioligand binding studies and FRET-based methods to monitor MOR-mediated G i-protein activation and β-arrestin recruitment in single intact 293T cells. This approach identified two major groups of morphine metabolites, which we classified into “strong” and “weak” receptor ligands. Strong partial agonists morphine, morphine-6-glucuronide, normorphine, morphine-6-sulfate, 6-acetylmorphine and 3-acetylmorphine showed efficacies in the nanomolar range, while the weak metabolites morphine-N-oxide, morphine-3-sulfate, morphine-3-glucuronide and pseudomorphine activated MOR pathways only in the micromolar range. Interestingly, three metabolites, normorphine, 6-acetylmorphine and morphine-6-glucuronide, had lower potencies for Gi-protein activation but higher potencies and efficacies for β-arrestin recruitment than morphine itself, suggesting that they are biased towards β-arrestin pathways.