Cell Biology Befriends Soft Matter Physics Phase Separation Creates Complex Condensates in Eukaryotic Cells

technology feature Cell biology befriends soft matter physics Phase separation creates complex condensates in eukaryotic cells. To study these mysterious droplets, many disciplines come together. Vivien Marx n many cartoons, eukaryotic cells have When the right contacts are made, phase a tidy look. A taut membrane surrounds separation occurs. Ia placid, often pastel-hued lake with well-circumscribed organelles such as Methods multitude ribosomes and a few bits and bobs. It’s a In the young condensate field, Amy helpful simplification of the jam-packed Gladfelter of the University of North cytoplasm. In some areas, there’s even more Carolina at Chapel Hill says her lab intense milling about1–4. To get a sense of intertwines molecular-biology-based these milieus, head to the kitchen with approaches, modeling, and in vitro Tony Hyman of the Max Planck Institute reconstitution experiments using simplified of Molecular Cell Biology and Genetics systems. “You can’t build models that have and Christoph Weber and Frank Jülicher of all the parameters that exist in a cell,” she the Max Planck Institute for the Physics of says. But researchers need to consider that Complex Systems, who note5: “For instance, in vitro work or overexpressing proteins when you make vinaigrette and leave it, you will not do complete justice to condensates come back annoyed to find that the oil and in situ. “Anything will phase separate vinegar have demixed into two different in a test tube,” she says, highlighting phases: an oil phase and a vinegar phase.” Just Eukaryotic cells contain mysterious, malleable, experiments in which crowding agents as oil and vinegar can separate into two stable liquid-like condensates in which there is intense such as polyethylene glycol are used to phases, such liquid–liquid phase separation activity. Credit: Y. Drozdova/Alamy Stock Vector. induce phase separation. Another widely can take place in the cytoplasm. This used method involves measurement of creates condensates, which are malleable, fluorescence recovery after photobleaching. liquid-like areas where proteins, peptides giving way to rigorous approaches and tools Such FRAP-ing, “that’s just not telling you and RNAs congregate that might otherwise to study “native biological condensates” and that much.” Better measurements are needed be on opposite sides of Lake Cytoplasm. manipulate them, he says. “Methods are still to, for example, characterize viscosity P-bodies and stress granules are some of pretty underdeveloped in this field,” he says. in a condensate, she says. New types of the condensates in the cytoplasm. Nucleoli Given that molecular forces and biophysical density probes will be useful, as will dyes and Cajal bodies are some of those in the factors drive condensate formation and that fluoresce to reflect how crowded a nucleus. Unlike organelles, condensates have maintenance, advances need to draw on condensate is. Her lab applies rotor dyes to no membrane, but there is a “non-membrane several disciplines. Cell and molecular assess how much ‘tumbling’ is taking place boundary,” says Hyman, with, for example, biologists, proteomics labs and those in in a condensate. One organism Gladfelter density and concentration unlike those of polymer physics are intrigued by condensates. studies is the fungus Ashbya, which has the surrounding cytoplasm or nucleoplasm. Brangwynne, then a postdoctoral fellow multinucleated cells, as do other organisms Condensates are a little like flash mobs, says in the Hyman lab, discovered that P granules and human muscle cells, too. In her Woods Hyman. When the music is on, people come phase-separate in the posterior of the Hole summer lab, she began collaborating together, and when the music stops, they embryo as part of germ cell development in on condensates with Brangwynne, Hyman, disperse. Condensates are dense locales. A Caenorhabditis elegans1. During a research Michael Rosen of the University of Texas eukaryotic cell typically contains billions stint at the Marine Biological Laboratory Southwestern Medical Center and others. of proteins. A condensate might be one in Woods Hole, Massachusetts, and using In Ashbya’s non-membrane-bound areas, to two micrometers in diameter and hold quantitative particle tracking, he and one she came across Whi3, an RNA-binding around one million proteins. It’s akin, he of the students in the physiology course says, to situating the world’s population in tagged P granules and showed how they greater Los Angeles. And everyone in this move, condense and coalesce to bring RNAs megalopolis stays on the move. and proteins together. Hyman, Jülicher Components in a condensate tumble and colleagues were excited about the around one another, touch briefly and move finding, says Brangwynne. “Such phase on, which leads to a dynamic of interactions, transitions may represent a fundamental says Princeton University researcher Clifford physicochemical mechanism for structuring Brangwynne. A wide range of biochemical the cytoplasm,” the authors noted. P activities take place in condensates, such as granules may look granular, but they are chromatin organization or stress responses. actually phase-separated liquid droplets. Early papers in the condensate field were What is known, says Hyman, is that the cell’s more qualitative and descriptive, but that is proteins interact and ‘sample’ one another. NATURE METHODS | VOL 17 | JUNE 2020 | 567–570 | www.nature.com/naturemethods 567 technology feature protein. Her team in collaboration with Sua versus intrinsic disorder have given way to the Myong, now at Johns Hopkins University, concept of multivalency, says Pappu, which used techniques such as single-molecule the model represents. Adapting concepts fluorescence resonance energy transfer from polymer science, the model considers (FRET) and found that Whi3 binding segments along a protein’s chain of amino changes the conformational dynamics of the acids to be more or less prone to interactions: RNAs it targets. RNAs and the RNA–protein some behave like ‘stickers’, others as ‘spacers’. interactions shape condensate formation “The balancing of sticker-to-spacer ratio and maintenance and give condensates becomes quite important,” he says. With distinct identities. Condensates will differ only stickers, a protein can end up a goopy in their Whi3 levels and location. Some mess, which can be a hallmark of disease. condensates form at the cell tips where Phase separation, says Pappu, may play a growth is taking place. Gladfelter explores role in Huntington’s disease. A mutation in molecular mechanisms of condensate the huntingtin gene leads to a polyglutamine dynamics to understand how sequestering repeat that contributes to fibrillary tangles a biochemical reaction in a condensate has in cells. Working on huntingtin led him advantages for the cell. Since condensates to condensates, says Pappu. Using online are so dynamic, classic structural biology Optogenetics methods can be used to study the resources, scientists can find out how sticky approaches to studying proteins and protein biophysics of phase separation. Shown here, the individual residues are, but these resources complexes don’t address all of a condensate’s nucleus of a living human cell at different times lack context for the particular sequence one complexity, says Hyman. Mass spectrometry after optogenetic activation with blue light. is looking at, says Pappu. This motivates him can help labs establish the metabolites in a Credit: D. Bracha, C. Brangwynne, Princeton U. to contribute to the community’s efforts to condensate. And when condensates can be build “a rigorous grammar” of condensates purified and they stay intact, says Gladfelter, to help predict, from sequence, how proteins mass spec helps to identify proteins, It’s a way to get a quantitative readout of will interact. which has worked with stress granules, the intrinsically disordered proteins that This prediction is informed by for example. For the RNAs, labs can use might be driving phase separation in cells. experiments such as single-molecule FRET RNA sequencing. For the condensates she Brangwynne is particularly fond of Corelet measurements that capture resonance studies, however, “we have not been able to because it helps query how oligomerized an transfer efficiencies in disordered proteins. purify the condensates and do that.” Nuclear environment has to be for a condensate to Pappu’s then postdoctoral fellow Kiersten magnetic resonance spectroscopy (NMR) form. “It lets us map these phase diagrams,” Ruff worked out how to give FRET has been applied to some proteins relevant he says. Corelet involves a self-assembled measurements a molecular context with to phase separation. She points to the wealth GFP-tagged particle, with a 24-mer ferritin all-atom simulations and machine-learning of tools from the Brangwynne lab and says, core in which each core is fused to an approaches to characterize protein “I personally don’t think any one thing optogenetic domain. As the particles ‘touch’ ensembles. Calculated FRET efficiencies is sufficient.” The method optoDroplet6, proteins with internally disordered regions, are calibrated with experimental ones. developed in the Brangwynne lab, uses light more or fewer proteins attach, depending on What results is calculated conformational to trigger protein association and selectively conditions. Corelet is like having an “entropy heterogeneity of proteins in condensates. control a condensate. Researchers can knob” on cells, says Brangwynne. Just as They

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