RNA transcript abundance at the single-cell level is highly heterogeneous, even among genetically uniform cells grown under identical conditions. Such variability was originally regarded as noise arising from stochastic gene expression (1), however, it has been shown more recently that the morphological and population-context features of a single cell can often be used to accurately predict the abundance of transcripts of a given gene. This suggests that transcript abundance is minimally stochastic, and closely coordinated with the cellular phenotype. The mechanistic basis of such deterministic ‘single-cell’ gene regulation is largely unexplored.
One major driver of cell-to-cell variability in transcript abundance is cell-size variation. For most genes, larger cells contain more transcripts. While such ‘scaling’ of RNA content with cell size is intuitive, the molecular mechanisms responsible are unknown (3). To begin to address this, we have developed a high-throughput imaging assay that reveals the precise contributions of cell-cycle stage and cell volume to global RNA production rates in human cells. Furthermore, we have used this assay to uncover genetic perturbations that disrupt the relationship between cell size and transcription.
High-throughput imaging of single-cells has also revealed that, for certain genes, transcript abundance is more closely coordinated with cell surface area than cell volume (2). To investigate causal links between cell morphology and gene expression, we have used micropatterns to directly manipulate the cell surface area to volume relationship. Using these assays, we show that ~30% of the transcriptome is rapidly modulated in response to constraining cell spreading. Genes whose expression is regulated according to cell surface area typically encode plasma membrane proteins, or cytoplasmic proteins involved in transmitting membrane-derived signals. This suggests that scaling of transcript abundance with surface area may contribute to density homeostasis for membrane proteins, and also to coordination of cytoplasmic protein concentrations with membrane abundance.