Nanometer condensate organization in live cells derived from partitioning measurements

Biomolecules self-organize into membrane-less organelles known as condensates that compartmentalize essential biochemical processes, such as ribosome biogenesis in the nucleolus1-3. Molecular dynamics within condensates are governed by chemical preferences and interaction networks that can imbue nanoscale structure4-7. Such organization is typically inferred from ensemble-averaged measurements, such as scattering and electron microscopy, which reveal molecular arrangements8-14. However, the complexity of cells obscures the interpretability of these techniques, limiting insight into condensate internal structure and roles in macromolecular assembly and transport. Here, we develop an approach to quantify the average microenvironment surrounding specific proteins within condensates in live cells, using thermodynamic principles to interpret the partitioning of designed protein probes. Using this approach, we find that condensates in cells, including the nucleolus, stress granule, and nuclear pore, exhibit spatial inhomogeneity, aligning with emerging views of condensates as networked fluids5,6,15-18. Within the nucleolus, we link spatial inhomogeneity to ribosome biogenesis, which progressively loosens the average local meshwork, facilitating transport of assembled ribosomal subunits. Within the nuclear pore, we find that transporters experience a weaker local meshwork than nucleoporins, consistent with the selective phase model19,20. Together, our approach uncovers a distinct mode of biomolecular control arising from nanoscale structure, which we term microenvironment coupling, whereby internal interaction landscapes shape transport to enable regulation and proofreading.