Scale-dependent physical constraints on active intracellular fluctuations

Abstract

Living cells exhibit nonequilibrium dynamics that shape intracellular processes across length scales, from nanoscale molecular assembly to the organization of macroscopic organelles. While dynamics at micrometer scales are known to be constrained by the actin meshwork at low frequencies, the physical principles governing active fluctuations at the nanoscale remain elusive. Here, we present an analytical framework integrating fluorescence correlation spectroscopy with nonequilibrium modeling to delineate the physical scaling of intracellular mechanics. Applying this framework to fibroblasts, we demonstrate that, in contrast to larger components, nanoscale active fluctuations remain prominent at high frequencies and are predominantly driven by local nonmuscle myosin II activity, establishing a distinct functional hierarchy in intracellular mechanics: local active forces promote rapid spatial exploration for nanoscale molecules, whereas macroscopic actin constraints ensure the structural stability required for larger molecular complexes and organelles. To integrate these scale-dependent behaviors within a single physical framework, we formulated a model that captures the transition of active fluctuations across length scales, revealing that the physical properties of the cytoplasm are governed by the balance between active driving forces and passive structural constraints. Furthermore, applying this model to cellular senescence reveals a reduction in nonequilibrium complexity associated with cytoskeletal rigidification. Thus, our findings bridge the dimensional gap between local molecular kinetics and macroscopic constraints, providing a fundamental physical basis for understanding the hierarchical organization of intracellular dynamics.