Biology subjects

Swaffer, M.

Publications and source records attributed to Swaffer, M..

3 recordsLinked to original sources

bioRxiv2025

A Fkh1/2 binding site array in the WHI5 promoter drives sub-scaling transcription

Cells typically regulate their size within a relatively tight range by coupling growth to the cell division cycle using a dedicated set of molecular mechanisms. In budding yeast, cells are born with a similar amount of the G1/S inhibitor protein Whi5 that is then diluted by growth throughout G1. As cells grow, Whi5 concentration decreases and cells become more likely to enter the cell cycle. Cells are born in G1 with similar amounts of Whi5 because of the size-independent (sub-scaling) expression of WHI5 mRNA during S/G2/M phases and the equal partitioning of Whi5 protein at division. While the latter is known to be achieved by association with chromatin before anaphase, the mechanism for the former is poorly understood. Through systematic mutations of the WHI5 promoter, we discovered that WHI5s core promoter region located -126 to -75 base pairs upstream of the start codon is responsible for sub-scaling expression. This sequence contains a repeating array of binding sites for the transcription factors Fkh1 and Fkh2. Mutation of any of these sites, deletion of either FKH1 or FKH2, or preventing Fkh1 or Fkh2 dimerization weakens the sub-scaling of WHI5 transcription. Taken together with structural predictions and a mathematical model of cooperative Fkh-DNA binding, we conclude that WHI5s sub-scaling transcription is regulated by a Fkh1/2 heteropolymer binding an array of sites in its core promoter.

cell biology↗

bioRxiv2024

Intracellular diffusion in the cytoplasm increases with cell size in fission yeast

Diffusion in the cytoplasm can greatly impact cellular processes, yet regulation of macromolecular diffusion remains poorly understood. There is increasing evidence that cell size affects the density and macromolecular composition of the cytoplasm. Here, we studied whether cell size affects diffusion at the scale of macromolecules tens of microns in diameter. We analyzed the diffusive motions of intracellular genetically-encoded multimeric 40 nm nanoparticles (cytGEMs) in the cytoplasm of the fission yeast Schizosaccharomyces pombe. Using cell size mutants, we showed that cytGEMs diffusion coefficients decreased in smaller cells and increased in larger cells. This increase in diffusion in large cells may be due to a decrease in the DNA-to-Cytoplasm ratio, as diffusion was not affected in large multinucleate cytokinesis mutants. In investigating the underlying causes of altered cytGEMs diffusion, we found that the proteomes of large and small cells exhibited size-specific changes, including the sub-scaling of ribosomal proteins in large cells. Comparison with a similar dataset from human cells revealed that features of size-dependent proteome remodeling were conserved. These studies demonstrate that cell size is an important parameter in determining the biophysical properties and the composition of the cytoplasm.

cell biology↗

bioRxiv2024

Genome concentration limits cell growth and modulates proteome composition in Escherichia coli

Defining the cellular factors that drive growth rate and proteome composition is essential for understanding and manipulating cellular systems. In bacteria, ribosome concentration is known to be a constraining factor of cell growth rate, while gene concentration is usually assumed not to be limiting. Here, using single-molecule tracking, quantitative single-cell microscopy, and modeling, we show that genome dilution in Escherichia coli cells arrested for DNA replication limits total RNA polymerase activity within physiological cell sizes across tested nutrient conditions. This rapid-onset limitation on bulk transcription results in sub-linear scaling of total active ribosomes with cell size and sub-exponential growth. Such downstream effects on bulk translation and cell growth are near-immediately detectable in a nutrient-rich medium, but delayed in nutrient-poor conditions, presumably due to cellular buffering activities. RNA sequencing and tandem-mass-tag mass spectrometry experiments further reveal that genome dilution remodels the relative abundance of mRNAs and proteins with cell size at a global level. Altogether, our findings indicate that chromosome concentration is a limiting factor of transcription and a global modulator of the transcriptome and proteome composition in E. coli. Experiments in Caulobacter crescentus and comparison with eukaryotic cell studies identify broadly conserved DNA concentration-dependent scaling principles of gene expression.

cell biology↗