Signaling network responses can be highly heterogeneous across cells in a tissue because of many sources of genetic and non-genetic variance. The emergence of multiplexed single-cell technologies has ...made it possible to evaluate this heterogeneity. In this review, we categorize currently established single-cell signaling network profiling approaches by their methodology, coverage, and application, and we discuss the advantages and limitations of these technologies. We describe the computational tools for network characterization using single-cell data and discuss potential confounding factors that may affect single-cell analyses.
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Highlights
•Signaling networks can be highly heterogeneous across cells in a tissue.•Various technologies allow analyzing signaling networks at single-cell resolution.•The advantages and limitations of each single-cell approach are summarized.•Confounding factors in single-cell signaling network analysis are discussed.
Signaling networks process intra- and extracellular information to modulate the functions of a cell. Deregulation of signaling networks results in abnormal cellular physiological states and often drives diseases. Network responses to a stimulus or a drug treatment can be highly heterogeneous across cells in a tissue because of many sources of cellular genetic and non-genetic variance. Signaling network heterogeneity is the key to many biological processes, such as cell differentiation and drug resistance. Only recently, the emergence of multiplexed single-cell measurement technologies has made it possible to evaluate this heterogeneity. In this review, we categorize currently established single-cell signaling network profiling approaches by their methodology, coverage, and application, and we discuss the advantages and limitations of each type of technology. We also describe the available computational tools for network characterization using single-cell data and discuss potential confounding factors that need to be considered in single-cell signaling network analyses.
We investigate a new form of collective dynamics displayed by Thiovulum majus, one of the fastest-swimming bacteria known. Cells spontaneously organize on a surface into a visually striking ...two-dimensional hexagonal lattice of rotating cells. As each constituent cell rotates its flagella, it creates a tornadolike flow that pulls neighboring cells towards and around it. As cells rotate against their neighbors, they exert forces on one another, causing the crystal to rotate and cells to reorganize. We show how these dynamics arise from hydrodynamic and steric interactions between cells. We derive the equations of motion for a crystal, show that this model explains several aspects of the observed dynamics, and discuss the stability of these active crystals.
We investigate swimming and chemotactic behaviors of the polarly flagellated marine bacteria Vibrio alginolyticus in an aqueous medium. Our observations show that V. alginolyticus execute a cyclic, ...three-step (forward, reverse, and flick) swimming pattern that is distinctively different from the run-tumble pattern adopted by Escherichia coli. Specifically, the bacterium backtracks its forward swimming path when the motor reverses. However, upon resuming forward swimming, the flagellum flicks and a new swimming direction is selected at random. In a chemically homogeneous medium (no attractant or repellent), the consecutive forward tf and backward tb swimming times are uncorrelated. Interestingly, although tf and tb are not distributed in a Poissonian fashion, their difference Δt = |tf - tb| is. Near a point source of attractant, on the other hand, tf and tb are found to be strongly correlated, and Δt obeys a bimodal distribution. These observations indicate that V. alginolyticus exploit the time-reversal symmetry of forward and backward swimming by using the time difference to regulate their chemotactic behavior. By adopting the three-step cycle, cells of V. alginolyticus are able to quickly respond to a chemical gradient as well as to localize near a point source of attractant.
Recent studies have shown that cell cycle and cell volume are confounding factors when studying biological phenomena in single cells. Here we present a combined experimental and computational method, ...CellCycleTRACER, to account for these factors in mass cytometry data. CellCycleTRACER is applied to mass cytometry data collected on three different cell types during a TNFα stimulation time-course. CellCycleTRACER reveals signaling relationships and cell heterogeneity that were otherwise masked.
Cells react to their microenvironment by integrating external stimuli into phenotypic decisions via an intracellular signaling network. To analyze the interplay of environment, local neighborhood, ...and internal cell state effects on phenotypic variability, we developed an experimental approach that enables multiplexed mass cytometric imaging analysis of up to 240 pooled spheroid microtissues. We quantified the contributions of environment, neighborhood, and intracellular state to marker variability in single cells of the spheroids. A linear model explained on average more than half of the variability of 34 markers across four cell lines and six growth conditions. The contributions of cell‐intrinsic and environmental factors to marker variability are hierarchically interdependent, a finding that we propose has general implications for systems‐level studies of single‐cell phenotypic variability. By the overexpression of 51 signaling protein constructs in subsets of cells, we also identified proteins that have cell‐intrinsic and cell‐extrinsic effects. Our study deconvolves factors influencing cellular phenotype in a 3D tissue and provides a scalable experimental system, analytical principles, and rich multiplexed imaging datasets for future studies.
SYNOPSIS
A barcoding‐based, high‐throughput approach enables multiplexed imaging of 3D spheroid microtissues. Quantitative single‐cell analyses show interdependence of global environment, local neighborhood, and internal cell state in determining cellular phenotype.
A novel barcoding‐based, high‐throughput approach enables multiplexed mass cytometric imaging of 3D microtissues.
A linear model quantifies environment, neighborhood, and internal cell state dependencies of marker expression.
A strong interdependence is identified between environmental and internal cell state predictors of cellular marker expression.
Systematic overexpression of signaling proteins within cells of 3D microtissues revealed non‐cell autonomous signaling.
A barcoding‐based, high‐throughput approach enables multiplexed imaging of 3D spheroid microtissues. Quantitative single‐cell analyses show interdependence of global environment, local neighborhood, and internal cell state in determining cellular phenotype.
To build comprehensive models of cellular states and interactions in normal and diseased tissue, genetic and proteomic information must be extracted with single-cell and spatial resolution. Here, we ...extended imaging mass cytometry to enable multiplexed detection of mRNA and proteins in tissues. Three mRNA target species were detected by RNAscope-based metal in situ hybridization with simultaneous antibody detection of 16 proteins. Analysis of 70 breast cancer samples showed that HER2 and CK19 mRNA and protein levels are moderately correlated on the single-cell level, but that only HER2, and not CK19, has strong mRNA-to-protein correlation on the cell population level. The chemoattractant CXCL10 was expressed in stromal cell clusters, and the frequency of CXCL10-expressing cells correlated with T cell presence. Our flexible and expandable method will allow an increase in the information content retrieved from patient samples for biomedical purposes, enable detailed studies of tumor biology, and serve as a tool to bridge comprehensive genomic and proteomic tissue analysis.
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•Imaging mass cytometry enables multiplexed RNA and protein detection in situ•mRNA measurement in IMC enables detection of as little as 6–14 mRNA copies per cell•Among patients, mRNA-to-protein ratios vary for CK19 but not for HER2•CXCL10-expressing cells form patches and are associated with T cell abundance
Here, we extend imaging mass cytometry to enable multiplexed detection of mRNA and protein in single cells in tissue sections. We show rigorous validation of the method and apply it to 70 samples from breast cancer patients. We investigate single-cell and population-based RNA-to-protein correlations in tissue and identify rare chemokine-expressing cells in the stroma that are associated with T cell abundance.
Marine bacterium Vibrio alginolyticus uses a single polar flagellum to navigate in an aqueous environment. Similar to Escherichia coli cells, the polar flagellar motor has two states; when the motor ...is counter-clockwise, the cell swims forward and when the motor is clockwise, the cell swims backward. V. alginolyticus also incorporates a direction randomization step at the start of the forward swimming interval by flicking its flagellum. To gain an understanding on how the polar flagellar motor switch is regulated, distributions of the forward Δf and backward Δb intervals are investigated herein. We found that the steady-state probability density functions, P(Δf) and P(Δb), of freely swimming bacteria are strongly peaked at a finite time, suggesting that the motor switch is not Poissonian. The short-time inhibition is sufficiently strong and long lasting, i.e., several hundred milliseconds for both intervals, which is readily observed and characterized. Treating motor reversal dynamics as a first-passage problem, which results from conformation fluctuations of the motor switch, we calculated P(Δf) and P(Δb) and found good agreement with the measurements.
Three-point bending test, compression test and tensile test can detect the mechanical properties of the whole layer of skull, but cannot detect the mechanical properties of the inner plate, the ...diploe and the outer plate of the skull. In this study, nanoindentation technology was applied to detect mechanical properties of micro-materials of the skull, and differences in micro-mechanical properties of the inner, diploe and outer plates of the skull and cranial suture of human carcasses at different ages were analyzed. The differences in hardness (HIT) and modulus of elasticity (E) were statistically significant among different age groups (P < 0.01). In terms of structure, the E of diploe was higher than that of other structures, while HIT had no significant statistical difference. In terms of location, both HIT and E showed that left frontal (LF) was significantly higher than coronal suture (CS). The above results were consistent with the multi-factor ANOVAs. In addition, the multi-factor ANOVAs further explained the interaction of HIT and E with age, location and structure. It was believed that the nanoindentation technique could be used to analyze laws of micromechanical properties of different structures of human cadaveric skull and cranial suture.
Bacteria use different motility patterns to navigate and explore natural habitats. However, how these motility patterns are selected, and what their benefits may be, are not understood. In this ...article, we analyze the effect of motility patterns on a cell’s ability to migrate in a chemical gradient and to localize at the top of the gradient, the two most important characteristics of bacterial chemotaxis. We will focus on two motility patterns, run-tumble and run-reverse-flick, that are observed and characterized in enteric bacterium Escherichia coli and marine bacterium Vibrio alginolyticus, respectively. To make an objective comparison, master equations are developed on the basis of microscopic motions of the bacteria. An unexpected yet significant result is that by adopting the run-reverse-flick motility pattern, a bacterium can reduce its diffusivity without compromising its drift in the chemical gradient. This finding is biologically important as it suggests that the motility pattern can improve a microorganism’s ability to sequester nutrients in a competitive environment.
Using self-trapped Escherichia coli bacteria that have intact flagellar bundles on glass surfaces, we study statistical fluctuations of cell-body rotation in a steady (unstimulated) state. These ...fluctuations underline direction randomization of bacterial swimming trajectories and plays a fundamental role in bacterial chemotaxis. A parallel study is also conducted using a classical rotation assay in which cell-body rotation is driven by a single flagellar motor. These investigations allow us to draw the important conclusion that during periods of counterclockwise motor rotation, which contributes to a run, all flagellar motors are strongly correlated, but during the clockwise period, which contributes to a tumble, individual motors are uncorrelated in long times. Our observation is consistent with the physical picture that formation and maintenance of a coherent flagellar bundle is provided by a single dominant flagellum in the bundle.
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