Hierarchical Heterogeneities in Spatiotemporal Dynamics of the Cytoplasm

The physical properties of the cytoplasm fundamentally constrain the dynamics and fidelity of cellular processes. Endogenous fluctuations carry signatures of these properties, yet how their collective dynamics across mesoscopic spatiotemporal scales relate to cytoplasmic heterogeneity and organization remains poorly understood. Here, we use differential dynamic microscopy (DDM) to quantify these fluctuations in eukaryotic cytoplasms. In Xenopus laevis egg extracts, we identify signatures of subdiffusive fractional Brownian motion (fBm) and non-Gaussian displacement distributions, consistent with a crowded, heterogeneous environment. We introduce an empirical model combining fBm with an inverse Gaussian diffusivity distribution, enabling robust estimation of fractional diffusivities and scaling exponents across conditions. Denser cytoplasm exhibits lower fractional diffusivity and scaling exponent, while energy addition induces non-stationary dynamics. Stabilization of microtubule networks introduces a secondary timescale, rationalized by a two-state fBm model separating cytosolic and network-associated contributions. In HeLa cells, the framework reveals comparable scaling exponents but lower diffusivities than in egg extract. Upon microtubule depolymerization, the dynamics become slower yet more Brownian-like. This highlights dual roles of the cytoskeleton as confinement but also as fluidizer of the cytoplasm. These results establish label-free DDM as an interpretable, collective, multiscale readout of how composition, activity, and cytoskeletal organization shape hierarchical cytoplasmic dynamics.