Intracellular dynamics is highly complex, and includes diffusion of poly-dispersed objects in a non-homogeneous, out-of-equilibrium medium. Assuming non-equilibrium steady-state, we developed a framework that relates non-equilibrium fluctuations to diffusion, and generalized entropy in cells. We employed imaging of live Jurkat T cells, and showed that active cells have higher diffusion parameters (Kα
and α) and entropy relative to the same cells after ATP depletion. Kα
and α were related in ATP-depleted cells while this relation was not apparent in untreated cells, probably due to non-equilibrium applied work. Next we evaluated the effect of intracellular diffusion and entropy on the cell content homogeneity, which was displayed by the extent of its liquid–liquid phase separation (LLPS). Correlations between intracellular diffusion parameters, entropy and cell homogeneity could be demonstrated only in active cells while these correlations disappeared after ATP depletion. We conclude that non-equilibrium contributions to diffusivity and entropy by ATP-dependent mechanical work allow cells to control their content homogeneity and LLPS state. Such understanding may enable better intervention in extreme LLPS conditions associated with various cell malignancies and degenerative diseases.
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