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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.

Cells compartmentalize their intracellular environment to orchestrate countless simultaneous biochemical processes. Many intracellular tasks rely on membrane-less organelles, multicomponent condensates that assemble by liquid–liquid phase separation. A decade of intensive research has provided a basic understanding of the biomolecular driving forces underlying the form and function of such organelles. Here we review the technologies enabling these developments, along with approaches to designing spatiotemporally actuated organelles based on multivalent low-affinity interactions. With these recent advances, it is now becoming possible both to modulate the properties of native condensates and to engineer entirely new structures, with the potential for widespread biomedical and biotechnological applications. Engineering and manipulating phase-separated liquid organelles is the latest frontier in the quest to mimic and interrogate living systems at the molecular level.

The coalescence of basic biochemical reactions into compartments is a major hallmark of a living cell. Using surface-bound DNA and a transcription reaction, we investigate the conditions for boundary-free compartmentalization. The DNA self-organizes into a dense and ordered phase with coding sequences aligned at well-defined distances and orientation relative to the surface, imposing directionality on transcription. Unique to the surface in comparison to dilute homogeneous DNA solution, the reaction slows down early, is inhibited with increased DNA density, is favorable for surface-oriented promoters, and is robust against DNA condensation. We interpret these results to suggest that macromolecules (RNA polymerase and RNA), but not solutes (ions and nucleotides), are partitioned between immobilized DNA and the reservoir. Without any physical barrier, a nonequilibrium directional DNA transaction forms macromolecular gradients that define a compartment, thus offering a boundary-free approach to the assembly of a synthetic cell.

The complexity of intracellular signalling requires both a diversity of molecular players and the sequestration of activity to unique compartments within the cell. Recent findings on the role of liquid–liquid phase separation provide a distinct mechanism for the spatial segregation of proteins to regulate signalling pathway crosstalk. Here, we discover that DACT1 is induced by TGFβ and forms protein condensates in the cytoplasm to repress Wnt signalling. These condensates do not localize to any known organelles but, rather, exist as phase-separated proteinaceous cytoplasmic bodies. The deletion of intrinsically disordered domains within the DACT1 protein eliminates its ability to both form protein condensates and suppress Wnt signalling. Isolation and mass spectrometry analysis of these particles revealed a complex of protein machinery that sequesters casein kinase 2—a Wnt pathway activator. We further demonstrate that DACT1 condensates are maintained in vivo and that DACT1 is critical to breast and prostate cancer bone metastasis. Esposito et al. show that TGF-β-induced DACT1 forms biomolecular condensates that sequester CK2 to repress Wnt signalling and modulate bone metastasis in cancer.

DNA polymers patterned on a biochip can be condensed into designed one-dimensional bundles spanning around 100 μm in length. DNA can be programmed to assemble into a variety of shapes and patterns on the nanoscale 1 , 2 , 3 , 4 , 5 and can act as a template for hybrid nanostructures 6 such as conducting wires 7 , 8 , 9 , protein arrays 8 and field-effect transistors 10 , 11 . Current DNA nanostructures are typically in the sub-micrometre range, limited by the sequence space and length of the assembled strands. Here we show that on a patterned biochip 12 , DNA chains collapse into one-dimensional (1D) fibres that are 20 nm wide and around 70 µm long, each comprising approximately 35 co-aligned chains at its cross-section. Electron beam writing on a photocleavable monolayer was used to immobilize and pattern the DNA molecules, which condense into 1D bundles in the presence of spermidine. DNA condensation can propagate and split at junctions, cross gaps and create domain walls between counterpropagating fronts. This system is inherently adept at solving probabilistic problems and was used to find the possible paths through a maze and to evaluate stochastic switching circuits. This technique could be used to propagate biological or ionic signals 13 in combination with sequence-specific DNA nanotechnology or for gene expression in cell-free DNA compartments 14 .

The complexity of intracellular signalling requires both a diversity of molecular players and the sequestration of activity to unique compartments within the cell. Recent findings on the role of liquid-liquid phase separation provide a distinct mechanism for the spatial segregation of proteins to regulate signalling pathway crosstalk. Here, we discover that DACT1 is induced by TGF[beta] and forms protein condensates in the cytoplasm to repress Wnt signalling. These condensates do not localize to any known organelles but, rather, exist as phase-separated proteinaceous cytoplasmic bodies. The deletion of intrinsically disordered domains within the DACT1 protein eliminates its ability to both form protein condensates and suppress Wnt signalling. Isolation and mass spectrometry analysis of these particles revealed a complex of protein machinery that sequesters casein kinase 2–a Wnt pathway activator. We further demonstrate that DACT1 condensates are maintained in vivo and that DACT1 is critical to breast and prostate cancer bone metastasis.

DNA-transactions in a living cell take place in an intracellular compartment of highly dense DNA. At close proximity between overlapping DNA segments within this segregated polyelectrolyte phase one expects unique collective behavior which does not appear under dilute homogeneous conditions. This shapes the structural organization of a dense DNA environment, and affects enzymatic machinery working within it. Dense phases of DNA in contact with a DNA-free reservoir can be emulated in-vitro by DNA brushes on a surface. We assemble DNA brushes with local concentrations of up to ∼30Mps/μm3, similarly to bacterial nucleoids and eukaryotic nuclei. Brushes provide means to dictate DNA conformation, spatial organization and local density, as well as orientation, position and accessibility to a reservoir of specific sites on the DNA. We investigated the physical-chemistry properties of dense DNA brushes without auxiliary molecules, the partitioning of inert biomolecules in the brush, and biochemical activity such as transcription. We find that osmotic pressure of trapped ions combined with entropic elasticity of individual chain induces major conformational changes, whereas excluded volume interactions are weak and do not induce major collective stretch. We find scaling behavior in agreement with the theory of polyelectrolyte brushes, and identify the osmotic, salted, mushroom, quasi-neutral brushes regimes with mono and divalent ions. Introducing spermidine, a trivalent cation, induces a first order transition with nucleation and growth of condensed domains having collapsed dendritic-like fractal morphology. We find that inert macromolecules in solution, such as DNA, RNA and RNA polymerase, are excluded from the brush depending on its density, suggesting that excluded volume interactions dictate the equilibrium partitioning of macromolecules at high density. Introducing actively transcribing RNA polymerase in the brush modulates this partition by active cycles of transcription, which exhibit strong dependence on DNA density, orientation and position relative to the surface. These data suggest that DNA brushes can form boundaryfree compartments with local concentration gradients and confinement of machinery, and possibly transcription products, in which biochemical activity is segregated under buffered conditions from the reservoir. Our results demonstrate the importance of emulating native DNA characteristics in-vitro for studying and performing cell-free DNA transactions. Furthermore, they suggest that by packaging DNA into a segregated compartment, internal order is imposed on the entire cellular environment, where structural constraints may define destination for spontaneous targeting of biomolecules to form intracellular concentration gradients, which are further enhanced by activity.

Nuclear compartments form via biomolecular phase separation, mediated through multivalent properties of biomolecules concentrated within condensates. Certain compartments are associated with specific chromatin regions, including transcriptional initiation condensates, which are composed of transcription factors and transcriptional machinery, and form at acetylated regions including enhancer and promoter loci. While protein self-interactions, especially within low-complexity and intrinsically disordered regions, are known to mediate condensation, the role of substrate-binding interactions in regulating the formation and function of biomolecular condensates is under-explored. Here, utilizing live-cell experiments in parallel with coarse-grained simulations, we investigate how chromatin interaction of the transcription factor BRD4 modulates its condensate formation. We find that both kinetic and thermodynamic properties of BRD4 condensation are affected by chromatin binding: nucleation rate is sensitive to BRD4-chromatin interactions, providing an explanation for the selective formation of BRD4 condensates at acetylated chromatin regions, and thermodynamically, multivalent acetylated chromatin sites provide a platform for BRD4 clustering below the concentration required for off-chromatin condensation. This provides a molecular and physical explanation of the relationship between nuclear condensates and epigenetically modified chromatin that results in their mutual spatiotemporal regulation, suggesting that epigenetic modulation is an important mechanism by which the cell targets transcriptional condensates to specific chromatin loci.

Summary 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 cell biological and biophysical approaches, the screen reveals an essential role for membrane cholesterol in spike-mediated fusion, which extends to replication-competent SARS-CoV-2 isolates. Our findings 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-cell fusion at ACE2-spike clusters cause pathological syncytia in COVID-19 * Drug screen reveals critical role for membrane lipid composition in fusion * Spike’s unusual membrane-proximal cysteines and aromatics are essential for fusion * Cholesterol tunes relative infectivity of SARS-CoV-2 viral particles Competing Interest Statement C.P.B. is a scientific founder and consultant for Nereid Therapeutics. A.S.H. is a consultant for Dewpoint Therapeutics. Footnotes * ↵14 Lead contact