Highlights d Stress granule formation requires RNA-binding nodes with high network connectivity d Capping of nodes by ligands lacking connectivity prevents condensation d Protein disorder and RNA-binding specificity play nonessential, modulatory roles d Competition of RNP networks for connecting nodes controls multiphase organization

Summary Recent studies show that liquid-liquid phase separation plays a key role in the assembly of diverse intracellular structures. However, the biophysical principles by which phase separation can be precisely localized within subregions of the cell are still largely unclear, particularly for low-abundance proteins. Here we introduce an oligomerizing biomimetic system, “Corelets”, and utilize its rapid and quantitative light-controlled tunability to map full intracellular phase diagrams, which dictate the concentrations at which phase separation occurs, and the mode of phase separation. Surprisingly, both experiments and simulations show that while intracellular concentrations may be insufficient for global phase separation, sequestering protein ligands to slowly diffusing nucleation centers can move the cell into a different region of the phase diagram, resulting in localized phase separation. This diffusive capture mechanism liberates the cell from the constraints of global protein abundance and is likely exploited to pattern condensates associated with diverse biological processes.

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Many enveloped viruses induce multinucleated cells (syncytia), reflective of membrane fusion events caused by the same machinery that underlies viral entry. These syncytia are thought to facilitate replication and evasion of the host immune response. Here, we report that co-culture of human cells expressing the receptor ACE2 with cells expressing SARS-CoV-2 spike, results in synapse-like intercellular contacts that initiate cell-cell fusion, producing syncytia resembling those we identify in lungs of COVID-19 patients. To assess the mechanism of spike/ACE2-driven membrane fusion, we developed a microscopy-based, cell-cell fusion assay to screen ~6000 drugs and >30 spike variants. Together with quantitative cell biology approaches, the screen reveals an essential role for biophysical aspects of the membrane, particularly cholesterol-rich regions, in spike-mediated fusion, which extends to replication-competent SARS-CoV-2 isolates. Our findings potentially provide a molecular basis for positive outcomes reported in COVID-19 patients taking statins and suggest new strategies for therapeutics targeting the membrane of SARS-CoV-2 and other fusogenic viruses.

Cell-free gene expression in localized DNA brushes on a biochip has been shown to depend on gene density and orientation, suggesting that brushes form compartments with partitioned conditions. At high density, the interplay of DNA entropic elasticity, electrostatics, and excluded volume interactions leads to collective conformations that affect the function of DNA-associated proteins. Hence, measuring the collective interactions in dense DNA, free of proteins, is essential for understanding crowded cellular environments and for the design of cell-free synthetic biochips. Here, we assembled dense DNA polymer brushes on a biochip along a density gradient and directly measured the collective extension of DNA using evanescent fluorescence. DNA of 1 kbp in a brush undergoes major conformational changes, from a relaxed random coil to a stretched configuration, following a universal function of density to ionic strength ratio with scaling exponent of 1/3. DNA extends because of the swelling force induced by the osmotic pressure of ions, which are trapped in the brush to maintain local charge neutrality, in competition with the restoring force of DNA entropic elasticity. The measurements reveal in DNA crossover between regimes of osmotic, salted, mushroom, and quasineutral brush. It is surprising to note that, at physiological ionic strength, DNA density does not induce collective stretch despite significant chain overlap, which implies that excluded volume interactions in DNA are weak.DNA biophysics | synthetic biology D ouble-helix DNA polymers exhibit relaxed random-walk configurations at lengths beyond the persistence scale l p = 50 nm, occupying volume to maximize their entropy. Unfolding DNA entropic degrees of freedom to full contour-length stretch requires large forces of 500 k B T/l p (50 pN) using a force-extension apparatus (1, 2). However, the transition of DNA into an ordered stretched state can also result from an entropy increase in a coupled system when chains experience significant overlap. Such is the case of polymer brushes where individual polymers stretch to minimize the free energy of the brush (3). For charged polymers, such as DNA, the collective extension also increases the mixing entropy of the ions that are trapped within the brush to maintain local charge neutrality (4-7).In the past two decades, DNA brushes have become useful in a range of applications such as next-generation sequencing (8, 9), hybridization arrays (10-14), protein biosynthesis compartments (15-18), and coated particle assembly (19,20). The utility of the diverse DNA-based reactions carried out in such brushes requires an in-depth understanding of their basic materials properties. To date, the focus has been on short DNA brushes (∼100 bp) (21, 22) having negligible polymer degrees of freedom. The compression of a few kilobase-pair DNA brushes on beads, as deduced from optical trapping force measurement and Brownian motion analysis (23, 24), was shown to behave as a power of −1/3 with ionic strength. For flexible polyelectrol...