From microbial colonies and biofilms to epithelial tissues and solid tumors, cells proliferating in confined spaces exert mechanical pressure on their surroundings while their growth rate declines. This growth-induced pressure (GIP) couples mass production, volume expansion, and intracellular physical state, yet the rate-limiting step linking mechanical confinement to reduced protein production remains unresolved. The relative contribution of canonical stress signaling versus direct physicochemical effects of macromolecular crowding is likewise unclear.
This thesis addresses these questions in Saccharomyces cerevisiae by combining microfluidic confinement, single-molecule imaging, spike-in absolute RNA-seq, quantitative proteomics, and live-cell biophysical readouts. Two PDMS platforms were developed for this work: a narrow self-closing chip preserving spatially uniform nutrient supply for imaging, and a pressure-recovery chip enabling bulk biochemical sampling under defined GIP. Independent measurement of every step of the expression cascade identifies transcription as the dominant rate-limiting node. At 0.5 MPa, per-cell mRNA and global transcription flux fall to about 40% of the unconfined value, while translation and degradation change weakly or compensate, so that protein abundance is maintained.
At the molecular level, Pol II elongation rate and bulk Pol II–chromatin engagement are preserved, but cooperative Gal4 binding and within-burst exchange at the promoter are selectively weakened. Initiation-proximal TF exchange thus emerges as a pressure-sensitive step. Spike-in-corrected RNA-seq combined with WGCNA reveals a graded repression of the growth program with selective induction of storage-carbohydrate and glycerol modules, rather than broad activation of a Hog1-, Msn2/4-, or Gcn4-dependent stress response. Unexpectedly, neither Sfp1 nor Tod6 shows a detectable nuclear-cytoplasmic relocalization under sub-MPa GIP, even though both reporters respond normally to rapamycin and osmotic shock, indicating that the chronic Ribi/RP repression is not transmitted through canonical Sfp1 or Tod6 nuclear export in this regime. Physically, intracellular dry mass density increases approximately linearly with pressure, ATP and pH remain stable up to about 0.6 MPa, and GEM tracking shows that nucleoplasmic mesoscale diffusion is more tightly coupled to growth rate than cytoplasmic diffusion.
GIP therefore does not act as an acute stress signal but drives a coordinated downshift of the growth program. Pressure is sensed at transcription initiation, reshapes a Ribi/RP-centered growth gene program through routes that go beyond the canonical growth-regulatory pathways, and is accompanied by a denser, more constrained nucleoplasm.