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Evaluating Phase Separation in Live Cells: Diagnosis, Caveats, and Functional Consequences

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Evaluating Phase Separation in Live Cells: Diagnosis, Caveats, and Functional Consequences Downloaded from genesdev.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press PERSPECTIVE Evaluating phase separation in live cells: diagnosis, caveats, and functional consequences David T. McSwiggen,1,2 Mustafa Mir,1 Xavier Darzacq,1,2 and Robert Tjian1,3 1Department of Molecular and Cell Biology, University of California Berkeley, California 94720, USA; 2California Institute of Regenerative Medicine Center of Excellence, University of California Berkeley, California 94720, USA; 3Howard Hughes Medical Institute, University of California Berkeley, California 94720, USA The idea that liquid–liquid phase separation (LLPS) may sure that these assemblies occur, and furthermore, that be a general mechanism by which molecules in the com- they do so on timescales relevant to their biological func- plex cellular milieu may self-organize has generated much tion. Prototypical examples of cellular organization are excitement and fervor in the cell biology community. the membrane-bound organelles, but it has long been ap- While this concept is not new, its rise to preeminence preciated that many compartments exist in the cell with- has resulted in renewed interest in the mechanisms that out an enclosing membrane (Montgomery 1898; Wilson shape and drive diverse cellular self-assembly processes 1899). from gene expression to cell division to stress responses. In the past decade, a fresh perspective on membraneless In vitro biochemical data have been instrumental in deriv- compartments—now often referred to as biomolecular ing some of the fundamental principles and molecular condensates (Banani et al. 2017)—has led to a resurgence grammar by which biological molecules may phase sepa- in the idea that a majority of these compartments may ex- rate, and the molecular basis of these interactions. Defin- ist as separate liquid phases (Courchaine et al. 2016). itive evidence is lacking as to whether the same principles There has been a renaissance in understanding how liq- apply in the physiological environment inside living cells. uid–liquid phase separation (LLPS) might function in com- In this Perspective, we analyze the evidence supporting partment formation and maintenance (Hyman et al. 2014; phase separation in vivo across multiple cellular process- Banani et al. 2017). Perhaps the most often cited example es. We find that the evidence for in vivo LLPS is often phe- is the nucleolus, where a convergence of studies examin- nomenological and inadequate to discriminate between ing its liquid-like behavior (Brangwynne et al. 2011), sup- phase separation and other possible mechanisms. More- ported with biochemical (Feric et al. 2016; Mitrea et al. over, the causal relationship and functional consequences 2016) and in vivo experiments (Berry et al. 2015; Weber of LLPS in vivo are even more elusive. We underscore the and Brangwynne 2015), collectively support a model importance of performing quantitative measurements on where the nucleolus behaves as a separate liquid phase proteins in their endogenous state and physiological abun- within the nucleus. Inspired by these and other early dance, as well as make recommendations for experiments examples of compartments with liquid-like properties that may yield more conclusive results. (Brangwynne et al. 2009), there has been a surge of publi- cations revisiting the formation of well-known cellular compartments through the lens of LLPS. Far from being Fundamentally, a cell is a collection of molecules com- the peculiarity it once was, phase separation now has partmentalized in a manner to modulate biochemical re- become, for many, the default explanation to rationalize actions that support diverse cellular activities. The the remarkable way in which a cell achieves various types challenges faced by a cell in managing these biochemical of compartmentalization, prompting significant debate processes scales with organismal complexity. In eukary- within the scientific community (Mir et al. 2019). otes, where some cellular tasks can require the coordinat- Much of the debate around LLPS condensates arises ed activity of tens to hundreds of individual molecular because it is unclear how strong the evidence for in vivo components, elaborate mechanisms have evolved to en- LLPS is, particularly when LLPS is invoked so broadly across many cellular contexts. The current focus on LLPS as a mechanism may come at the expense of under- [Keywords: fluorescence recovery after photobleaching; condensate; standing alternative mechanisms by which a high local liquid–liquid phase separation; phase separation] Corresponding authors: [email protected], [email protected] Article published online ahead of print. Article and publication date are online at http://www.genesdev.org/cgi/doi/10.1101/gad.331520.119. Free- © 2019 McSwiggen et al. This article, published in Genes & Develop- ly available online through the Genes & Development Open Access ment, is available under a Creative Commons License (Attribution 4.0 In- option. ternational), as described at http://creativecommons.org/licenses/by/4.0/. GENES & DEVELOPMENT 33:1–16 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/19; www.genesdev.org 1 Downloaded from genesdev.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press McSwiggen et al. concentration of factors can be achieved in the absence of volumes occupied by the two phases (Fig. 1). A simplistic a membrane. For example, while nucleoli exhibit many example of this is the nucleation and growth of water properties consistent with LLPS, the formation of nucleoli droplets on a cold glass. Accumulating evidence suggests and many other nuclear bodies have previously been ex- the potential for LLPS to occur widely with biological plained by alternative mechanisms (Mao et al. 2011a,b; macromolecules as well, and it has been shown that cer- Shevtsov and Dundr 2011). In a recent study we found tain classes of proteins—as well as RNA and other biolog- that Herpes Simplex Virus replication compartments ical polymers—readily undergo LLPS in vitro (Jain and derive their ability to concentrate cellular factors through Vale 2017; Wang et al. 2018). transient nonspecific binding to the viral DNA in a man- The topic of phase separation in biology has been exten- ner distinct from liquid–liquid phase separation (McSwig- sively reviewed elsewhere, and the reader is encouraged to gen et al. 2019). Despite this mechanistic distinction, refer to these reviews for a more thorough explanation of these replication compartments display many of the hall- the forces that drive liquid–liquid demixing (Hyman marks that are often deemed sufficient to claim that such et al. 2014; Brangwynne et al. 2015; Banani et al. 2017; a compartment is formed via LLPS (McSwiggen et al. Boeynaems et al. 2018). Much of what we know now has 2019). foundations in early works on polymer physics (Overbeek Our data on replication compartments, as well as other and Voorn 1957) and has been advanced by efforts to recent studies from our group (Mir et al. 2017, 2018; improve crystallographic methods for which phase separa- Chong et al. 2018) demonstrate that there are multiple tion was used as a means of increasing a protein’s concen- routes to establish regions with high local concentrations tration without it crashing out of solution (Lomakin et al. of specific factors inside the cell. These studies prompted 1996; Asherie 2004; Vekilov 2010). Other types of phase us to critically reexamine the current evidence for LLPS in transition have also been proposed to occur in cells. vivo. The appeal for invoking phase separation is under- For example, it has been proposed that some proteins standable, as it presents a way to rationalize—and at least may transition into gel-like structures (Kato et al. 2012; superficially explain—certain behaviors of cellular com- Kwon et al. 2013) or liquid-crystalline structures (Rog partments. However, in light of various recent studies et al. 2017), again drawing models from lessons learned and upon further analysis, we find that the evidence for in polymer physics and materials science for inspiration. LLPS occurring in the cell is often far from conclusive. Physical models exist to explain liquid demixing (Loma- This is not to imply that LLPS cannot function in biolog- kin et al. 1996; Velasco et al. 1998), and for purified com- ical contexts, but rather to highlight how the tests com- ponents like proteins or nucleic acids, there exist rigorous monly used in probing LLPS are insufficient to rule out standards by which one may determine whether a given other mechanistic interpretations. system is undergoing liquid–liquid demixing. Modulating In this Perspective, we summarize the evidence used to the concentration of a polymer, the ionic strength of the diagnose liquid–liquid phase separation in vivo. Recently, buffer, the temperature of the system, and intra- or inter- others have similarly urged caution in overinterpreting in polymer interactions can all quantifiably change the pro- vivo experiments to test LLPS (Alberti et al. 2019), but the pensity of the polymer to demix (Lomakin et al. 1996; issues in this field run deeper than the authors discuss. Velasco et al. 1998; Vekilov 2010; Brangwynne 2013). Fol- This Perspective is, to our knowledge, the first to system- lowing this model, beautiful in vitro experiments have atically and holistically consider the evidence presented been performed demonstrating the ability of LLPS sys- by this emerging field. We first provide a summary of tems to exhibit exclusivity (Nott et al. 2015; Banani the state of evidence for LLPS condensates across multiple et al. 2016; Feric et al. 2016); to form and dissolve on the contexts, and address important considerations for this basis of post-translational modifications (Li et al. 2012; evidence. Second, we address the evidence for the func- Lu et al. 2018) and to exhibit changes in viscosity and to tional consequences of LLPS in the underlying biological “ripen” or harden over time (Patel et al. 2015; Wegmann processes being studied. Finally, we urge the application et al.
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