Organized for action: It is becoming increasingly clear that biomolecular condensates, which are concentrations of macromolecules not surrounded by a membrane, are a key organizational structure in eukaryotic cells (see the Perspective by Martin and Mittag). Now, two papers show how such condensates function in actin assembly or in a Ras signaling pathway. In both cases, the condensates form at the plasma membrane and increase the activity of signaling proteins by increasing their membrane dwell times. Case et al. show that the dwell time is dependent on cluster stoichiometry, so that stoichiometry of regulatory proteins can control actin assembly. Huang et al. demonstrate that the longer dwell time allows kinetic proofreading in receptor-mediated activation of Ras. Science , this issue p. 1093 , p. 1098 ; see also p. 1036

Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important examples of what this technique can yield to biological sciences are the mechanistic insights on protein-protein and protein-nucleic acid interactions. When interactions of proteins are probed at the single-molecule level, the proteins or their substrates are often immobilized on a glass surface, which allows for a long-term observation. This immobilization scheme may introduce unwanted surface artifacts. Therefore, it is essential to passivate the glass surface to make it inert. Surface coating using polyethylene glycol (PEG) stands out for its high performance in preventing proteins from non-specifically interacting with a glass surface. However, the polymer coating procedure is difficult, due to the complication arising from a series of surface treatments and the stringent requirement that a surface needs to be free of any fluorescent molecules at the end of the procedure. Here, we provide a robust protocol with step-by-step instructions. It covers surface cleaning including piranha etching, surface functionalization with amine groups, and finally PEG coating. To obtain a high density of a PEG layer, we introduce a new strategy of treating the surface with PEG molecules over two rounds, which remarkably improves the quality of passivation. We provide representative results as well as practical advice for each critical step so that anyone can achieve the high quality surface passivation.

Phosphorylation reactions, driven by competing kinases and phosphatases, are central elements of cellular signal transduction. We reconstituted a native eukaryotic lipid kinase–phosphatase reaction that drives the interconversion of phosphatidylinositol-4-phosphate [PI(4)P] and phosphatidylinositol-4,5-phosphate [PI(4,5)P₂] on membrane surfaces. This system exhibited bistability and formed spatial composition patterns on supported membranes. In smaller confined regions of membrane, rapid diffusion ensures the system remains spatially homogeneous, but the final outcome—a predominantly PI(4)P or PI(4,5)P₂ membrane composition—was governed by the size of the reaction environment. In larger confined regions, interplay between the reactions, diffusion, and confinement created a variety of differentially patterned states, including polarization. Experiments and kinetic modeling reveal how these geometric confinement effects arise from a mechanism based on stochastic fluctuations in the copy number of membrane-bound kinases and phosphatases. The underlying requirements for such behavior are unexpectedly simple and likely to occur in natural biological signaling systems. Significance Spatial gradients of phosphatidylinositol phosphate (PIP) lipids in the cell membrane regulate a variety of cellular processes, including endocytosis, phagocytosis, and cell migration. The molecules that control these cellular processes must navigate a diverse range of geometric constraints, including membrane sheets, blebs, vesicles, and tubules. Stochastic geometry sensing provides a mechanism for controlling spatial patterning of molecules on cellular membranes, by exploiting differences in molecular copy number and kinetic asymmetries between opposing enzymatic reactions. The generality of the stochastic geometry-sensing mechanism suggests that such effects are likely to occur in cellular signaling systems.