Macromolecular crowding creates heterogeneous environments of gene expression in picolitre droplets

Abstract

Understanding the dynamics of complex enzymatic reactions in highly crowded small volumes is crucial for the development of synthetic minimal cells. Compartmentalised biochemical reactions in cell-sized containers exhibit a degree of randomness due to the small number of molecules involved. However, it is unknown how the physical environment contributes to the stochastic nature of multistep enzymatic processes. Here, we present a robust method to quantify gene expression noise in vitro using droplet microfluidics. We study the changes in stochasticity in cellfree gene expression of two genes compartmentalised within droplets as a function of DNA copy number and macromolecular crowding. We find that decreased diffusion caused by a crowded environment leads to the spontaneous formation of heterogeneous micro-environments of mRNA as local production rates exceed diffusion rates of macromolecules. This heterogeneity leads to a higher probability of the molecular machinery to stay in the same microenvironment, directly increasing the system's stochasticity.Noise is present in all living cells. It has been studied in prokaryotes and eukaryotes 1 , as well as stem 2,3 and cancer cells 4 , and cells expressing viruses 5 . Gene expression is a key example of a complex stochastic enzymatic process. Careful analysis of variations in mRNA and protein levels has revealed the importance of both amplitude and typical decay time of noise and the ability of cells to exploit or suppress noise in gene expression [6][7][8][9] . Unlike deterministic models of gene expression, which are used to predict dynamics over large populations, stochastic models can correctly predict the dynamics of gene expression at the Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/.Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use

Applications of Microfluidics in Quantitative Biology

“…They found that the DNA copy number and macromolecular crowding were the key factors to the heterogeneous gene expression, which directly increased the system's stochasticity. [82] Moreover, they also used an agarose droplet system to analyze the cellular heterogeneity of cytokine secretion in cancer cells. This system encapsulates cells together with functionalized cytokine-capture beads for subsequent binding, and for following detection of secreted cytokines at single-cell level.…”

Quantitative computational models of molecular self-assembly in systems biology

“…Numerous theoretical and experimental studies have suggested both the need for and the challenge to correcting simulation methods to account for the effects of crowding on assembly processes (e.g., [129, 166]). Examples include the effects on several aspects of DNA replication such as helicase activity and the sensitivity of DNA polymerase to salt [1], on protein-protein binding affinity and specificity [99], on the kinetics and morphology of amyloid self-assembly [115], on the stochasticity of gene expression machinery [76], and on viral capsid assembly [175, 36]. …”

Specific genomic cues regulate Cajal body assembly

“…154 Indeed, crowding effects within picoliter droplets containing DNA and polymerases in vitro enhances the retention of transcriptional machinery and enhanced RNA synthesis at transcription sites. 155 This may be an important contributing factor to the early stages of CB nucleation by enhancing snRNA gene transcription.…”