Phase separation on microtubules: from droplet formation to cellular function?
Vladimir A. Volkov, Anna Akhmanova
Abstract
Liquid–liquid phase separation (LLPS) is a common phenomenon observed for microtubule-binding proteins expressed recombinantly in vitro, or overexpressed in cells.LLPS of microtubule-binding proteins in vitro can be driven by very different types of interactions, involving intrinsically disordered regions and folded domains.Binding to microtubules can promote formation of protein condensates.Condensates of tubulin-binding proteins can potentially promote microtubule nucleation and accelerate microtubule elongation.Condensate formation by the same or homologous proteins strongly depends on the species, cell type, or cell cycle phase.Conclusive evidence that LLPS occurs at physiological conditions in cells is often missing. LLPS (see Glossary) is a process of demixing of two immiscible or semi-miscible liquids, often illustrated through the ‘vinegar in oil’ analogy. LLPS has been proposed to be the mechanism behind the formation of membraneless cell compartments, often termed ‘biomolecular condensates’, including nucleoli, P-bodies, and stress granules, where specific macromolecules such as proteins or nucleic acids are concentrated (reviewed in [1.Banani S.F. et al.Biomolecular condensates: organizers of cellular biochemistry.Nat. Rev. Mol. Cell Biol. 2017; 18: 285-298Crossref PubMed Scopus (2457) Google Scholar,2.Hyman A.A. et al.Liquid-liquid phase separation in biology.Annu. Rev. Cell Dev. Biol. 2014; 30: 39-58Crossref PubMed Google Scholar]). In this review, we will focus on microtubule-binding proteins and protein complexes and discuss the evidence that they form non-stoichiometric condensates through LLPS. We will use the term LLPS broadly, as is currently common in cell biology literature, although it is clear that the term is often used to describe phases that are not simple liquids [3.Mittag T. Pappu R.V. A conceptual framework for understanding phase separation and addressing open questions and challenges.Mol. Cell. 2022; 82: 2201-2214Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar]. What interactions drive proteins into the condensed phase? Homo- and heterotypic protein–protein interactions can depend on the binding between folded domains, between a folded domain and a linear motif, or the interactions between intrinsically disordered protein regions (IDRs) [4.Musacchio A. On the role of phase separation in the biogenesis of membraneless compartments.EMBO J. 2022; 41e109952Crossref PubMed Scopus (38) Google Scholar,5.Dignon G.L. et al.Biomolecular phase separation: from molecular driving forces to macroscopic properties.Annu. Rev. Phys. Chem. 2020; 71: 53-75Crossref PubMed Scopus (182) Google Scholar] (Figure 1A ). Many proteins that exhibit LLPS in vitro carry IDRs; presence of IDRs correlates with the ability of these proteins to phase-separate [6.Vernon R.M. Forman-Kay J.D. First-generation predictors of biological protein phase separation.Curr. Opin. Struct. Biol. 2019; 58: 88-96Crossref PubMed Scopus (71) Google Scholar]. IDRs often display poor sequence conservation, leading to the idea that formation of mesoscale compartments by LLPS is driven by low-affinity interactions between IDRs and relies on physical properties of amino acids rather than sequence-encoded specific, high-affinity interactions [7.Hyman A.A. Brangwynne C.P. Beyond stereospecificity: liquids and mesoscale organization of cytoplasm.Dev. Cell. 2011; 21: 14-16Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar,8.Wang J. et al.A molecular grammar governing the driving forces for phase separation of prion-like RNA binding proteins.Cell. 2018; 174: 688-699Abstract Full Text Full Text PDF PubMed Scopus (855) Google Scholar]. However, in principle, sequence-specific, high affinity interactions involving folded domains might also promote formation of protein condensates [4.Musacchio A. On the role of phase separation in the biogenesis of membraneless compartments.EMBO J. 2022; 41e109952Crossref PubMed Scopus (38) Google Scholar]. An important consequence of LLPS is ‘concentration buffering’: changing the amount of a phase-separating protein leads to the change in the volume of the condensed phase, while the protein’s concentration in the ‘dilute phase’ remains constant (Figure 1A). A simple way to induce protein condensation is therefore to increase its concentration. In vivo, this can be achieved by overexpressing the protein of interest and observing formation of droplets that can fuse over time. When working with purified proteins in vitro, inert ‘crowding agents’ such as polyethylene glycol (PEG) can be added to deplete the solvent volume accessible to proteins and increase their local concentration to promote LLPS. However, the relevance of specific crowding agents in mimicking cellular environment is limited, because cellular components can both promote and inhibit condensate formation and overall can have major effects on phase separation of a particular protein [9.Riback J.A. et al.Composition-dependent thermodynamics of intracellular phase separation.Nature. 2020; 581: 209-214Crossref PubMed Scopus (253) Google Scholar]. These approaches have been routinely used to observe LLPS of numerous proteins, but in fact, studying LLPS of a given protein is only relevant if it happens at the physiological concentration. Given how easily overexpression can result in condensate formation, tight control of expression levels is therefore essential, but, unfortunately, often omitted in studies reporting LLPS (reviewed in [10.McSwiggen D.T. et al.Evaluating phase separation in live cells: diagnosis, and Dev. 2019; PubMed Scopus Google Scholar]). as the for the LLPS mechanism to the condensed phase and with the to this is by of protein or by However, for can be easily with of protein and can be by interactions from such as binding to a In protein droplets are not with the and are as or In these it be to their formation occurs through LLPS or the concentration of in the phase be very to observe with the condensed used to LLPS from are of types of interactions of complexes to J. 21: PubMed Scopus Google Scholar] and can LLPS of proteins both in vitro and in vivo, while on these are et a to the properties of 2017; Google Scholar] and is very et used to phase and Biol. Chem. 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