Mammalian gene expression variability is explained by underlying cell state.
ABSTRACT: Gene expression variability in mammalian systems plays an important role in physiological and pathophysiological conditions. This variability can come from differential regulation related to cell state (extrinsic) and allele-specific transcriptional bursting (intrinsic). Yet, the relative contribution of these two distinct sources is unknown. Here, we exploit the qualitative difference in the patterns of covariance between these two sources to quantify their relative contributions to expression variance in mammalian cells. Using multiplexed error robust RNA fluorescent in situ hybridization (MERFISH), we measured the multivariate gene expression distribution of 150 genes related to Ca2+ signaling coupled with the dynamic Ca2+ response of live cells to ATP. We show that after controlling for cellular phenotypic states such as size, cell cycle stage, and Ca2+ response to ATP, the remaining variability is effectively at the Poisson limit for most genes. These findings demonstrate that the majority of expression variability results from cell state differences and that the contribution of transcriptional bursting is relatively minimal.
Project description:Pregnancy-derived uterine artery endothelial cells (P-UAEC) express P2Y2 receptors and at high cell density show sustained and synchronous [Ca2+]i burst responses in response to ATP Bursts in turn require coupling of transient receptor potential canonical type3 channel (TRPC3) and inositol 1,4,5-triphosphate receptor type 2 (IP3R2), which is upregulated in P-UAEC in a manner dependent on connexin 43 (Cx43) gap junctions. While there is no known direct interaction of TRPC3 with Cx43, early descriptions of TRPC3 function showed it may also be influenced by altered membrane potential (Vm). Herein, we ask if enhanced TRPC3 Ca2+ bursting due to enhanced Cx43 coupling may be coupled via dynamic alterations in Vm in P-UAEC, as reported in some (HUVEC) but not all endothelial cells. Following basic electrical characterization of UAEC, we employed a high sensitivity cell imaging system to simultaneously monitor cell Vm and [Ca2+]i in real time in continuous monolayers of UAEC Our findings show that while acute and sustained phase [Ca2+]i bursting occur dose-dependently in response to ATP, Vm is not coregulated with any periodicity related to [Ca2+]i bursting. Only a small but significant progressive change in Vm is seen, and this is more closely related to overall mobilization of Ca2+. Surprisingly, this is also most apparent in NP-UAEC > P-UAEC In contrast [Ca2+]i bursting is more synchronous in P-UAEC and even achieves [Ca2+]i waves passing through the P-UAEC monolayer. The relevance of these findings to mechanisms of pregnancy adaptation and its failure in hypertensive pregnancy are discussed.
Project description:Oxytocin is an important neuromodulator in the mammalian brain that increases information salience and circuit plasticity, but its signaling mechanisms and circuit effect are not fully understood. Here we report robust oxytocinergic modulation of intrinsic properties and circuit operations in hippocampal area CA2, a region of emerging importance for hippocampal function and social behavior. Upon oxytocin receptor activation, CA2 pyramidal cells depolarize and fire bursts of action potentials, a consequence of phospholipase C signaling to modify two separate voltage-dependent ionic processes. A reduction of potassium current carried by KCNQ-based M channels depolarizes the cell; protein kinase C activity attenuates spike rate of rise and overshoot, dampening after-hyperpolarizations. These actions, in concert with activation of fast-spiking interneurons, promote repetitive firing and CA2 bursting; bursting then governs short-term plasticity of CA2 synaptic transmission onto CA1 and, thus, efficacy of information transfer in the hippocampal network.
Project description:MOTIVATION:Multiplexed error-robust fluorescence in-situ hybridization (MERFISH) is a recent technology to obtain spatially resolved gene or transcript expression profiles in single cells for hundreds to thousands of genes in parallel. So far, no statistical framework to analyze MERFISH data is available. RESULTS:We present a Bayesian model for single cell transcript expression analysis on MERFISH data. We show that the model successfully captures uncertainty in MERFISH data and eliminates systematic biases that can occur in raw RNA molecule counts obtained with MERFISH. Our model accurately estimates transcript expression and additionally provides the full probability distribution and credible intervals for each transcript. We further show how this enables MERFISH to scale towards the whole genome while being able to control the uncertainty in obtained results. AVAILABILITY AND IMPLEMENTATION:The presented model is implemented on top of Rust-Bio (Köster, 2016) and available open-source as MERFISHtools (https://merfishtools.github.io). It can be easily installed via Bioconda (Grüning et al., 2018). The entire analysis performed in this paper is provided as a fully reproducible Snakemake (Köster and Rahmann, 2012) workflow via Zenodo (https://doi.org/10.5281/zenodo.752340). SUPPLEMENTARY INFORMATION:Supplementary data are available at Bioinformatics online.
Project description:Protein levels differ considerably between otherwise identical cells, and these differences significantly affect biological function and phenotype. Previous work implicated various noise mechanisms that drive variability in protein copy numbers across an isogenic cell population. For example, transcriptional bursting of mRNAs has been shown to be a major source of noise in the expression of many genes. Additional expression variability, referred to as extrinsic noise, arises from intercellular variations in mRNA transcription and protein translation rates attributed to cell-to-cell differences in cell size, abundance of ribosomes, etc. We propose a method to determine the magnitude of different noise sources in a given gene of interest. The method relies on blocking transcription and measuring changes in protein copy number variability over time. Our results show that this signal has sufficient information to quantify both the extent of extrinsic noise and transcription bursting in gene expression. Moreover, if the mean mRNA count is known, then the relative contributions of transcription versus translation rate fluctuations to extrinsic noise can also be determined. In summary, our study provides an easy-to-implement method for characterizing noisy protein expression that complements existing techniques for studying stochastic dynamics of genetic circuits.
Project description:Spatially and temporally coordinated variations of the cytosolic free calcium concentration ([Ca2+]c) play a crucial role in a variety of tissues. In the developing sensory epithelium of the mammalian cochlea, elevation of extracellular adenosine trisphosphate concentration ([ATP]e) triggers [Ca2+]c oscillations and propagation of intercellular inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ waves. What remains uncertain is the relative contribution of gap junction channels and connexin hemichannels to these fundamental mechanisms, defects in which impair hearing acquisition. Another related open question is whether [Ca2+]c oscillations require oscillations of the cytosolic IP3 concentration ([IP3]c) in this system. To address these issues, we performed Ca2+ imaging experiments in the lesser epithelial ridge of the mouse cochlea around postnatal day 5 and constructed a computational model in quantitative adherence to experimental data. Our results indicate that [Ca2+]c oscillations are governed by Hopf-type bifurcations within the experimental range of [ATP]e and do not require [IP3]c oscillations. The model replicates accurately the spatial extent and propagation speed of intercellular Ca2+ waves and predicts that ATP-induced ATP release is the primary mechanism underlying intercellular propagation of Ca2+ signals. The model also uncovers a discontinuous transition from propagating regimes (intercellular Ca2+ wave speed > 11 ?m?s-1) to propagation failure (speed = 0), which occurs upon lowering the maximal ATP release rate below a minimal threshold value. The approach presented here overcomes major limitations due to lack of specific connexin channel inhibitors and can be extended to other coupled cellular systems.
Project description:Coexpression of the beta subunit (KV,Cabeta) with the alpha subunit of mammalian large conductance Ca2+- activated K+ (BK) channels greatly increases the apparent Ca2+ sensitivity of the channel. Using single-channel analysis to investigate the mechanism for this increase, we found that the beta subunit increased open probability (Po) by increasing burst duration 20-100-fold, while having little effect on the durations of the gaps (closed intervals) between bursts or on the numbers of detected open and closed states entered during gating. The effect of the beta subunit was not equivalent to raising intracellular Ca2+ in the absence of the beta subunit, suggesting that the beta subunit does not act by increasing all the Ca2+ binding rates proportionally. The beta subunit also inhibited transitions to subconductance levels. It is the retention of the BK channel in the bursting states by the beta subunit that increases the apparent Ca2+ sensitivity of the channel. In the presence of the beta subunit, each burst of openings is greatly amplified in duration through increases in both the numbers of openings per burst and in the mean open times. Native BK channels from cultured rat skeletal muscle were found to have bursting kinetics similar to channels expressed from alpha subunits alone.
Project description:Astrocytes are a major cell type in the mammalian brain. They are not electrically excitable, but generate prominent Ca2+ signals related to a wide variety of critical functions. The mechanisms driving these Ca2+ events remain incompletely understood. In this study, we integrate Ca2+ imaging, quantitative data analysis, and mechanistic computational modeling to study the spatial and temporal heterogeneity of cortical astrocyte Ca2+ transients evoked by focal application of ATP in mouse brain slices. Based on experimental results, we tune a single-compartment mathematical model of IP3-dependent Ca2+ responses in astrocytes and use that model to study response heterogeneity. Using information from the experimental data and the underlying bifurcation structure of our mathematical model, we categorize all astrocyte Ca2+ responses into four general types based on their temporal characteristics: Single-Peak, Multi-Peak, Plateau, and Long-Lasting responses. We find that the distribution of experimentally-recorded response types depends on the location within an astrocyte, with somatic responses dominated by Single-Peak (SP) responses and large and small processes generating more Multi-Peak responses. On the other hand, response kinetics differ more between cells and trials than with location within a given astrocyte. We use the computational model to elucidate possible sources of Ca2+ response variability: (1) temporal dynamics of IP3, and (2) relative flux rates through Ca2+ channels and pumps. Our model also predicts the effects of blocking Ca2+ channels/pumps; for example, blocking store-operated Ca2+ (SOC) channels in the model eliminates Plateau and Long-Lasting responses (consistent with previous experimental observations). Finally, we propose that observed differences in response type distributions between astrocyte somas and processes can be attributed to systematic differences in IP3 rise durations and Ca2+ flux rates.
Project description:In the accompanying article, we compared main functional properties of the three mammalian inositol 1,4,5-trisphosphate receptors (InsP3R) isoforms. In this article we focused on modulation of mammalian InsP3R isoforms by cytosolic Ca2+. We found that: 1), when recorded in the presence of 2 microM InsP3 and 0.5 mM ATP all three mammalian InsP3R isoforms display bell-shaped Ca2+ dependence in physiological range of Ca2+ concentrations (pCa 8-5); 2), in the same experimental conditions InsP3R3 is most sensitive to modulation by Ca2+ (peak at 107 nM Ca2+), followed by InsP3R2 (peak at 154 nM Ca2+), and then by InsP3R1 (peak at 257 nM Ca2+); 3), increase in ATP concentration to 5 mM had no significant effect of Ca2+ dependence of InsP3R1 and InsP3R2; 4), increase in ATP concentration to 5 mM converted Ca2+ dependence of InsP3R3 from "narrow" shape to "square" shape; 5), ATP-induced change in the shape of InsP3R3 Ca2+ dependence was mainly due to an >200-fold reduction in the apparent affinity of the Ca2+-inhibitory site; 6), the apparent Ca2+ affinity of the Ca2+ sensor region (Cas) determined in biochemical experiments is equal to 0.23 microM Ca2+ for RT1-Cas, 0.16 microM Ca2+ for RT2-Cas, and 0.10 microM Ca2+ for RT3-Cas; and 7), Ca2+ sensitivity of InsP3R1 and InsP3R3 isoforms recorded in the presence of 2 microM InsP3 and 0.5 mM ATP or 2 microM InsP3 and 5 mM ATP can be exchanged by swapping their Cas regions. Obtained results provide novel information about functional properties of mammalian InsP3R isoforms and support the importance of the Ca2+ sensor region (Cas) in determining the sensitivity of InsP3R isoforms to modulation by Ca2+.
Project description:Heterogeneity in the expression levels of mammalian genes is large even in clonal populations and has phenotypic consequences. Alternative splicing is a fundamental aspect of gene expression, yet its contribution to heterogeneity is unknown. Here, we use single-molecule imaging to characterize the cell-to-cell variability in mRNA isoform ratios for two endogenous genes, CAPRIN1 and MKNK2. We show that isoform variability in non-transformed, diploid cells is remarkably close to the minimum possible given the stochastic nature of individual splicing events, while variability in HeLa cells is considerably higher. Analysis of the potential sources of isoform ratio heterogeneity indicates that a difference in the control over splicing factor activity is one origin of this increase. Our imaging approach also visualizes non-alternatively spliced mRNA and active transcription sites, and yields spatial information regarding the relationship between splicing and transcription. Together, our work demonstrates that mammalian cells minimize fluctuations in mRNA isoform ratios by tightly regulating the splicing machinery.
Project description:The pancreatic islet is a functional microorgan involved in maintaining normoglycemia through regulated secretion of insulin and other hormones. Extracellular glucose stimulates insulin secretion from islet beta cells through an increase in redox state, which can be measured by NAD(P)H autofluorescence. Glucose concentrations over approximately 7 mM generate synchronous oscillations in beta cell intracellular Ca2+ concentration ([Ca2+]i), which lead to pulsatile insulin secretion. Prevailing models assume that the pancreatic islet acts as a functional syncytium, and the whole islet [Ca2+]i response has been modeled in terms of islet bursting and pacemaker models. To test these models, we developed a microfluidic device capable of partially stimulating an islet, while allowing observation of the NAD(P)H and [Ca2+]i responses. We show that beta cell [Ca2+]i oscillations occur only within regions stimulated with more than approximately 6.6 mM glucose. Furthermore, we show that tolbutamide, an antagonist of the ATP-sensitive K+ channel, allows these oscillations to travel farther into the nonstimulated regions of the islet. Our approach shows that the extent of Ca2+ propagation across the islet depends on a delicate interaction between the degree of coupling and the extent of ATP-sensitive K+-channel activation and illustrates an experimental paradigm that will have utility for many other biological systems.