Single-cell Protein Expression Research Articles

A novel flow cytometric assay for single-cell analysis of microRNA, messenger RNA and protein expression during cell differentiation and maturation.

Abstract We describe a novel assay that enables analysis of microRNA, mRNA, and protein expression in single cells by flow cytometry. This in situ hybridization-based method employs pairs of target-specific probes, branched DNA amplification technology, and fluorochrome-conjugated label probes, resulting in sensitive detection of different types of RNA targets including mRNA, microRNA, long non-coding RNA and viral RNA. To broaden our understanding of the applicability of this assay, we determined its limit of detection for mRNA and microRNA. Using in vitro bone marrow-derived macrophages as a model, we demonstrate how this assay can be used to study the process of cell differentiation and maturation. This novel assay enables the simultaneous detection of up to four RNA targets in the presence of fluorochrome-conjugated antibodies in single cells and is a powerful new tool that will enable new insights into cellular processes.

Direct Correlation between Motile Behavior and Protein Abundance in Single Cells.

Understanding how stochastic molecular fluctuations affect cell behavior requires the quantification of both behavior and protein numbers in the same cells. Here, we combine automated microscopy with in situ hydrogel polymerization to measure single-cell protein expression after tracking swimming behavior. We characterized the distribution of non-genetic phenotypic diversity in Escherichia coli motility, which affects single-cell exploration. By expressing fluorescently tagged chemotaxis proteins (CheR and CheB) at different levels, we quantitatively mapped motile phenotype (tumble bias) to protein numbers using thousands of single-cell measurements. Our results disagreed with established models until we incorporated the role of CheB in receptor deamidation and the slow fluctuations in receptor methylation. Beyond refining models, our central finding is that changes in numbers of CheR and CheB affect the population mean tumble bias and its variance independently. Therefore, it is possible to adjust the degree of phenotypic diversity of a population by adjusting the global level of expression of CheR and CheB while keeping their ratio constant, which, as shown in previous studies, confers functional robustness to the system. Since genetic control of protein expression is heritable, our results suggest that non-genetic diversity in motile behavior is selectable, supporting earlier hypotheses that such diversity confers a selective advantage.

Label-free Quantification of Proteins in Single Embryonic Cells with Neural Fate in the Cleavage-Stage Frog (Xenopus laevis) Embryo using Capillary Electrophoresis Electrospray Ionization High-Resolution Mass Spectrometry (CE-ESI-HRMS)

Quantification of protein expression in single cells promises to advance a systems-level understanding of normal development. Using a bottom-up proteomic workflow and multiplexing quantification by tandem mass tags, we recently demonstrated relative quantification between single embryonic cells (blastomeres) in the frog (Xenopus laevis) embryo. In this study, we minimize derivatization steps to enhance analytical sensitivity and use label-free quantification (LFQ) for single Xenopus cells. The technology builds on a custom-designed capillary electrophoresis microflow-electrospray ionization high-resolution mass spectrometry platform and LFQ by MaxLFQ (MaxQuant). By judiciously tailoring performance to peptide separation, ionization, and data-dependent acquisition, we demonstrate an ∼75-amol (∼11 nm) lower limit of detection and quantification for proteins in complex cell digests. The platform enabled the identification of 438 nonredundant protein groups by measuring 16 ng of protein digest, or <0.2% of the total protein contained in a blastomere in the 16-cell embryo. LFQ intensity was validated as a quantitative proxy for protein abundance. Correlation analysis was performed to compare protein quantities between the embryo and n = 3 different single D11 blastomeres, which are fated to develop into the nervous system. A total of 335 nonredundant protein groups were quantified in union between the single D11 cells spanning a 4 log-order concentration range. LFQ and correlation analysis detected expected proteomic differences between the whole embryo and blastomeres, and also found translational differences between individual D11 cells. LFQ on single cells raises exciting possibilities to study gene expression in other cells and models to help better understand cell processes on a systems biology level.

Abstract 1853: Single-cell multiplexed profiling of protein-level changes induced by EGFR inhibitor gefitinib

Abstract Single-cell molecular analysis has become increasingly important in biomedical research, especially in the field of cancer research, where cell heterogeneity is a characteristic feature. We developed a multiplexed single-cell protein detection assay for the C1™ system that can isolate and process 96 individual cells in parallel. The assay uses oligonucleotide-conjugated antibodies, which produce a detectable DNA amplicon only when they bind to the target protein. The resulting DNA amplicons are quantified by qPCR on the Biomark™ HD system. Data are analyzed using the Singular™ Analysis Toolset. A total of 50 antibody binders were developed for this assay to target proteins related to apoptosis, cell cycle, cell proliferation, tumor suppressors, biomarkers, stem cells, growth factors and proteins associated with specific cancers, such as breast and prostate cancers. The assay can be performed in a 48-plex format, which yields over 4,600 datapoints in two days. Assay sensitivity and specificity were assessed using recombinant proteins and various cell lysates. To demonstrate the usefulness of this assay, we used it to profile the protein level changes in A431 cells treated with epidermal growth factor receptor (EGFR) inhibitor gefitinib. EGFR is a member of ErbB family of type I tyrosine kinases, whose overexpression and mutations are involved in a variety of cancers. While much effort has been made to investigate the consequences of EGFR activation or inhibition, hardly any information is available regarding how EGFR regulation changes protein expression in single cells. Using a multiplexed protein assay, we discovered some interesting co-expression patterns of proteins in single cells. We also demonstrated that TNFRSF10B was upregulated, while MET, GATA3, BRCA1, CCNB1, Ki-67 and CCNA2 were downregulated by gefitinib treatment. Notably, the reduction of the cell cycle-related proteins CCNB1 and CCNA2 and the cell proliferation marker Ki-67 was consistent with the inhibited cell growth by gefitinib. In addition, we measured mRNA expression to investigate the association between protein and mRNA levels in single cells. Single-cell analysis—determining protein changes and analyzing the correlation between different proteins at the single-cell level—yields valuable information that is not achievable using bulk-cell analysis. Citation Format: Ilona Holcomb, Gajalakshmi Dakshinamoorthy, Benjamin Liu, Marc Unger, Ramesh Ramakrishnan, Haibiao Gong. Single-cell multiplexed profiling of protein-level changes induced by EGFR inhibitor gefitinib. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1853.

Phytoplankton IF-FISH: Species-specific labeling of cellular proteins by immunofluorescence (IF) with simultaneous species identification by fluorescence immunohybridization (FISH)

Phytoplankton rarely occur as unialgal populations. Therefore, to study species-specific protein expression, indicative of physiological status in natural populations, methods are needed that will both assay for a protein of interest and identify the species expressing it. Here we describe a protocol for IF-FISH, a dual labeling procedure using immunofluorescence (IF) labeling of a protein of interest followed by fluorescence in situ hybridization (FISH) to identify the species expressing that protein. The protocol was developed to monitor expression of the cell cycle marker proliferating cell nuclear antigen (PCNA) in the red tide dinoflagellate, Karenia brevis, using a large subunit (LSU) rRNA probe to identify K. brevis in a mixed population of morphologically similar Karenia species. We present this protocol as proof of concept that IF-FISH can be successfully applied to phytoplankton cells. This method is widely applicable for the analysis of single-cell protein expression of any protein of interest within phytoplankton communities.

Protein Counting in Single Cancer Cells.

The cell is the basic unit of biology and protein expression drives cellular function. Tracking protein expression in single cells enables the study of cellular pathways and behavior but requires methodologies sensitive enough to detect low numbers of protein molecules with a wide dynamic range to distinguish unique cells and quantify population distributions. This study presents an ultrasensitive and automated approach for quantifying phenotypic responses with single cell resolution using single molecule array (SiMoA) technology. We demonstrate how prostate specific antigen (PSA) expression varies over several orders of magnitude between single prostate cancer cells and how PSA expression shifts with genetic drift. Single cell SiMoA introduces a straightforward process that is capable of detecting both high and low protein expression levels. This technique could be useful for understanding fundamental biology and may eventually enable both earlier disease detection and targeted therapy.

Abstract B20: Single-cell profiling of EGFR-regulated protein changes involved in cell cycle, cell proliferation and apoptosis

Abstract Single-cell molecular analysis has become increasingly important in biomedical research, especially in the field of cancer research, where cell heterogeneity is a characteristic feature. We developed a multiplexed single-cell protein detection assay for the C1™ system that can isolate and process 96 individual cells in parallel. The assay uses oligonucleotide-conjugated antibody binders and monitors the specific oligonucleotide levels instead of protein levels. The resulting DNA amplicon is detected by qPCR on the Biomark™ HD system. Data are analyzed using Singular™ Analysis Toolset. A total of 50 antibody binders were developed for this assay to target proteins related to apoptosis, cell cycle, cell proliferation, tumor suppressors, biomarkers, stem cells, growth factors and proteins associated with specific cancers, such as breast and prostate cancers. The assay can be performed in a 48-plex format which yields over 4,600 data points in two days. Assay sensitivity and specificity were assessed using recombinant proteins and various cell lysates. To demonstrate the usefulness of this assay, a panel of antibody binders was selected to profile the changes of proteins involved in cell cycle, cell proliferation and apoptosis in single cells. We treated cells that overexpress epidermal growth factor receptor (EGFR) with either the EGF ligand or the gefitinib receptor inhibitor and analyzed the changes in protein levels. Cells without EGFR expression were used as the control. EGFR is a member of ErbB family of Type I tyrosine kinases. EGFR overexpression and its mutations are involved in a variety of cancers. While much effort has been made to investigate the consequences of EGFR activation or inhibition, hardly any information is available regarding how EGFR regulation changes protein expression in single cells. Single-cell analysis—determining protein changes and analyzing the correlation between different proteins at the single-cell level—yields valuable information that is not achievable using bulk-cell analysis. Citation Format: Ilona Holcomb, Gajalakshmi Dakshinamoorthy, Benjamin Liu, Marc Unger, Ramesh Ramakrishnan, Haibiao Gong. Single-cell profiling of EGFR-regulated protein changes involved in cell cycle, cell proliferation and apoptosis. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B20.

Simultaneous Multiplexed Measurement of RNA and Proteins in Single Cells.

SummarySignificant advances have been made in methods to analyze genomes and transcriptomes of single cells, but to fully define cell states, proteins must also be accessed as central actors defining a cell’s phenotype. Methods currently used to analyze endogenous protein expression in single cells are limited in specificity, throughput, or multiplex capability. Here, we present an approach to simultaneously and specifically interrogate large sets of protein and RNA targets in lysates from individual cells, enabling investigations of cell functions and responses. We applied our method to investigate the effects of BMP4, an experimental therapeutic agent, on early-passage glioblastoma cell cultures. We uncovered significant heterogeneity in responses to treatment at levels of RNA and protein, with a subset of cells reacting in a distinct manner to BMP4. Moreover, we found overall poor correlation between protein and RNA at the level of single cells, with proteins more accurately defining responses to treatment.

A method for detecting protein expression in single cells using the C1™ Single-Cell Auto Prep System (TECH2P.874)

Abstract Recent improvements in microfluidics and biochemistry have enabled single-cell molecular analysis, providing new insight into the heterogeneity of cell populations. The C1 Single-Cell Auto Prep System is an automated platform that streamlines the isolation and processing of 96 individual, live cells for RNA and DNA analysis. Single-cell protein profiling is a direct complement to genomic analysis as it provides additional insights into key molecular mechanisms and system biology. To enable this, we adapted a highly multiplexed protein detection system (Proseek Multiplex Oncology, Olink Bioscience) for use on the C1 System. We analyzed 192 individual cells from two human promyelocytic leukemia cell lines (HL60 and K562) and compared these result against 16 FACS-sorted cells in tube format. These results indicated that four key proteins (i.e. CASP3, CSTB, CD69 and MPO) are differentially expressed between the two cell lines and vary from cell-to-cell. For example, the human protein MPO, produced by neutrophils for their microbicidal activity, was differentially expression between individual HL60 and K562 cells, suggesting varying levels of neutrophil activation within the cell populations. This work demonstrates highly-multiplexed protein detection at the single-cell level and shows promise to have sufficient sensitivity, reproducibility and scale to effectively interrogate a statistically significant numbers of individual cells.

High-throughput fingerprinting of human pluripotent stem cell fate responses and lineage bias

Populations of cells create local environments that lead to emergent heterogeneity. This is particularly evident in human pluripotent stem cells (hPSCs) where microenvironmental heterogeneity limits cell fate control. We have developed a high-throughput platform to screen hPSCs in configurable micro-environments, enabling the optimization of colony size, cell density, and additional parameters for rapid and robust cell fate responses. Single-cell protein expression profiling revealed that Oct4 and Sox2 co-staining discriminate pluripotent, neuroectoderm, primitive streak, and extraembryonic cell fates, allowing dose responses of 27 developmental factors to simultaneously delineate lineage-specific concentration optima. This platform also enabled quantification of endogenous signaling pathway activation and differentiation bias (fingerprinting). Short-term (48 h) fingerprinting is predictive of definitive endoderm induction efficiency across 12 cell lines and was used a priori to rescue long-term (>18 day) differentiation of a cell line reticent to cardiac induction. These findings facilitate high-throughput hPSC-based screening and quantification of lineage induction bias.

Scaling advantages and constraints in miniaturized capture assays for single cell protein analysis

Measuring protein expression in single cells is the basis of single cell proteomics. The sensitivity and dynamic range of a single cell immunoassay should ideally be such that proteins that are expressed between 1-10(6) copies per cell can be detected and counted. We have investigated the effect of miniaturizing antibody microarrays by reducing capture spot sizes from 100 μm to 15 μm using dip-pen nanolithography. We demonstrate that protocols developed for printing and passivating antibody capture spots using conventional pin-based contact printing can be directly transferred to dip-pen lithography whilst retaining the capture activity per unit area. Using a simple kinetic model, we highlight how the limit of detection and dynamic range of a sandwich immunoassay, respectively, increase and decrease when spot size is reduced. However, we show that reducing spot size is more effective than increasing assay chamber volume when seeking to multiplex such a microfluidic immunoassay. Although we make particular reference to single cell microfluidic immunoassays, the topics discussed here are applicable to capture assays in general.

Emergence of bimodal cell population responses from the interplay between analog single-cell signaling and protein expression noise

BackgroundCell-to-cell variability in protein expression can be large, and its propagation through signaling networks affects biological outcomes. Here, we apply deterministic and probabilistic models and biochemical measurements to study how network topologies and cell-to-cell protein abundance variations interact to shape signaling responses.ResultsWe observe bimodal distributions of extracellular signal-regulated kinase (ERK) responses to epidermal growth factor (EGF) stimulation, which are generally thought to indicate bistable or ultrasensitive signaling behavior in single cells. Surprisingly, we find that a simple MAPK/ERK-cascade model with negative feedback that displays graded, analog ERK responses at a single cell level can explain the experimentally observed bimodality at the cell population level. Model analysis suggests that a conversion of graded input–output responses in single cells to digital responses at the population level is caused by a broad distribution of ERK pathway activation thresholds brought about by cell-to-cell variability in protein expression.ConclusionsOur results show that bimodal signaling response distributions do not necessarily imply digital (ultrasensitive or bistable) single cell signaling, and the interplay between protein expression noise and network topologies can bring about digital population responses from analog single cell dose responses. Thus, cells can retain the benefits of robustness arising from negative feedback, while simultaneously generating population-level on/off responses that are thought to be critical for regulating cell fate decisions.

CRAC Channels Drive Digital Activation and Provide Analog Control and Synergy to Ca2+-Dependent Gene Regulation

Ca(2+)-dependent gene expression is critical for cell growth, proliferation, plasticity, and adaptation [1-3]. Because a common mechanism in vertebrates linking cytoplasmic Ca(2+) signals with activation of protein synthesis involves the nuclear factor of activated T cells (NFAT) family of transcription factors [4, 5], we have quantified protein expression in single cells following physiological Ca(2+) signals by using NFAT-driven expression of a genetically encoded fluorescent protein. We find that gene expression following CRAC channel activation is an all-or-nothing event over a range of stimulus intensities. Increasing agonist concentration recruits more cells but each responding cell does so in an essentially digital manner. Furthermore, Ca(2+)-dependent gene expression shows both short-term memory and strong synergy, where two pulses of agonist, which are ineffectual individually, robustly activate gene expression provided that the time interval between them is short. Such temporal filtering imparts coincidence detection to Ca(2+)-dependent gene activation. The underlying molecular basis mapped to time-dependent, nonlinear accumulation of nuclear NFAT. Local Ca(2+) near CRAC channels has to rise above a threshold level to drive gene expression, providing analog control to the digital activation process and a means to filter out fluctuations in background noise from activating transcription while ensuring robustness and high fidelity in the excitation-transcription coupling mechanism.

Quantitative detection of protein expression in single cells using droplet microfluidics

We demonstrate that single cells can be controllably compartmentalized within aqueous microdroplets; using such an approach we perform high-throughput screening by detecting the expression of a fluorescent protein in individual cells with simultaneous measurement of droplet size and cell occupancy.

Detection of Green Fluorescent Protein in a Single Bacterium by Capillary Electrophoresis with Laser-Induced Fluorescence

Green fluorescence protein (GFP) is a common reporter used to monitor protein expression in single cells. However, autofluorescence from endogenous components can mask the signal from GFP, particularly at low expression levels in prokaryotes. We employ capillary electrophoresis with laser-induced fluorescence for the analysis of the expression of green fluorescent protein in a single bacterium. Capillary electrophoresis separates GFP from native cellular autofluorescent components, reducing the background signal and improving detection limits. Our system provides 100 ymol (60 copies) limits of detection for GFP. To demonstrate the performance of this instrument, we employ a model system of Deinococcus radiodurans that has been engineered to express GFP under the control of the recA promoter. We report resolution and detection of GFP and autofluorescent components in a single D. radiodurans bacterium. This paper presents the first example of expression of GFP in D. radiodurans and the first detection of GFP in a single bacterium by capillary electrophoresis.

Separation Technologies

Separation Technologies

A novel mammalian expression screen exploiting green fluorescent protein-based transcription detection in single cells

The accumulation of DNA sequence information from large-scale genomic and random library sequencing projects is leading to the rapid identification of many putative genes, virtual transcripts and ESTs of unknown function. There is therefore an increasing need for high throughput, sensitive and robust methods for identification and characterisation of genes, and/or their products, based on function. We describe a high throughput functional expression screen based on semi-quantitative analysis of enhanced green fluorescent protein expression in single cells by confocal microscopy. The assay was implemented in a micro-scale format, requiring around 10 4 cells/test. The system was validated by co-transfection of a series of cDNAs encoding pro-inflammatory cytokine intracellular signal mediators with a d2EGFP reporter containing a cytokine responsive promoter. The majority of the test plasmids gave a detectable signal above background at a pool size of 250–500. Replicate tests indicate that the assay is reproducible at this pool size. At this level we demonstrate that large (>10 6 transformants) libraries can be feasibly screened.

Virus-specific transcription and translation in organs of BALB / Mo mice: Comparative study using quantitative hybridization, in Situ hybridization, and immunocytochemistry

Expression of the endogenous Moloney leukemia virus (=M-MuLV) was studied in a variety of tissues of BALB/Mo mice. Previous experiments using quantitative hybridization techniques have shown that M-MuLV expression occurred predominantly in the lymphatic organs spleen and thymus. Here we have employed in situ hybridization and immunocytochemical techniques to detect virus RNA and protein expression in single cells of a large variety of different organs. The two techniques were employed in parallel and compared with quantitative hybridization experiments using total nucleic acids extracted from the different organs. This allowed estimation of the fraction of positive cells in a given organ and to relate it to the total viral-specific RNA present, and thus to assess the value and the limitations of each method for detection of virus expression. The results confirm that most or all cells of the lymphatic system express regularly high concentrations of viral RNA and proteins. We show furthermore that cells of many nonlymphatic organs can be productively infected with virus expressing viral functions. These include cells of the reproductive system, epithelial cells of intestine, brain, and liver, and hormone-producing cells of the pancreas. With the exception of the reproductive system, the fraction of virus-producing cells in all nonlymphatic organs, however, was much lower than in spleen and thymus. Our results indicated that productive infection with M-MuLV is not restricted to cells of specific tissues, although the frequency of infection may vary considerably. They furthermore demonstrate that regardless of cell type viral proteins are always synthesized once viral RNA has been transcribed. Our results suggest that active cell proliferation and hormone production may be among the cellular parameters that influence susceptibility to productive virus infection.

THE PAY OF POOR-LAW MEDICAL OFFICERS IN IRELAND

<h3>Summary</h3> Basic helix-loop-helix genes, particularly proneural genes, are well-described triggers of cell differentiation, yet limited information exists on their dynamics, notably in human development. Here, we focus on Neurogenin 3 (<i>NEUROG3</i>), which is crucial for pancreatic endocrine lineage initiation. Using a double reporter to monitor endogenous <i>NEUROG3</i> transcription and protein expression in single cells in 2D and 3D models of human pancreas development, we show peaks of expression for the RNA and protein at 22 and 11 hours respectively, approximately two-fold slower than in mice, and remarkable heterogeneity in peak expression levels all triggering differentiation. We also reveal that some human endocrine progenitors proliferate once, mainly at the onset of differentiation, rather than forming a subpopulation with sustained proliferation. Using reporter index-sorted single-cell RNA-seq data, we statistically map transcriptome to dynamic behaviors of cells in live imaging and uncover transcriptional states associated with variations in motility as NEUROG3 levels change, a method applicable to other contexts.