Actomyosin Fluctuations Couple Temperature to Intracellular Transport and ELPV Phase Separation.

How living cells transduce thermal perturbations into altered intracellular organization remains a fundamental biophysical question. Here, using single-particle tracking of quantum dot (QD) diffusion and endocytic vesicle transport, we show that temperature-enhanced transport is not a simple passive solvent effect; instead, heating enhances active cytoplasmic dynamics mediated by myosin-II-dependent actomyosin activity. While active fluctuations enhance random QD diffusion, they also accelerate active vesicular transport by reducing its mechanical resistance. Using thermoresponsive elastin-like polypeptide (ELPV) phase separation as a physical-chemical reporter, we demonstrate that myosin-II-dependent active fluctuations modulate phase separation. Near the transition temperature, the enhanced droplet formation after myosin-II inhibition suggests that active fluctuations oppose ELPV nucleation, whereas at higher temperatures, deeper within the two-phase regime, thermodynamic driving forces dominate. This work directly links nonequilibrium cytoplasmic mechanics with the physical chemistry of intracellular phase boundaries.