Liquid–liquid phase separation in cellular signaling systems

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Highlights

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    Protein liquid-liquid phase separation has implications for cellular signaling.

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    Multivalent interactions give liquid, non-membrane-contained phase-separated droplets.

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    Phase separation can be 3D or “2D” if involving a membrane bound-component.

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    Phase separation can amplify signals and yield switch-like or emergent properties.

Liquid–liquid demixing or phase separation of protein with RNA is now recognized to be a key part of the mechanism for assembly of ribonucleoprotein granules. Cellular signaling also appears to employ phase separation as a mechanism for amplification or control of signal transduction both within the cytoplasm and at the membrane. The concept of receptor clustering, identified more than 3 decades ago, is now being examined through the lens of phase separation leading to new insights. Intrinsically disordered proteins or regions are central to these processes owing to their flexibility and accessibility for dynamic protein–protein interactions and post-translational modifications. We review some recent examples, examine the mechanisms driving phase separation and delineate the implications for signal transduction systems.

Introduction

Assembly of multi-component protein complexes is accepted as an important process for signal transduction [1, 2, 3]. More recently some signaling complexes have been demonstrated to be highly dynamic with the properties of a separate protein-rich liquid phase. Liquid–liquid demixing or phase separation of proteins has been identified as a mechanism for formation of membraneless compartments, especially ribonucleoprotein granules [4, 5••, 6, 7, 8•], but is also emerging as an important concept in signaling. Examples include phase separation of Dishevelled (Dvl) in the Wnt signaling pathway [9, 10, 11], which is important in development, and phase separation of nephrin/Nck/N-WASP proteins[12••, 13••], which function in the assembly of actin filaments [14, 15]. Phase separation has been demonstrated to rely on multivalent protein interactions [12••, 13••] and often involves intrinsically disordered proteins (IDPs) or proteins that contain intrinsically disordered regions (IDRs). The frequent occurrence of multiple modular domains in signaling proteins [2] and IDRs in signaling hubs [16, 17, 18, 19, 20] makes it likely that these phase-separation mechanisms are common in signaling. Three-dimensional membraneless compartments in the cytoplasm are also called membraneless organelles, puncta or droplets, alluding to their characteristics. Membraneless refers to these droplets not being membrane delimited or contained. However, the phase-separated proteins can be linked to a membrane through a membrane-bound protein component, leading to a pseudo two-dimensional phase separation at the membrane surface. Such two-dimensional phase separation at the membrane is closely tied to and may be the driving force behind receptor clustering. Phase separation allows for the creation of a microenvironment with emergent properties that may be useful for sequestering substrates or enzymes or creating specialized reaction environments [5••, 21•, 22, 23]. Phase separation also provides an added layer of control in cell signaling and a mechanism for switch-like behavior and for integrating multiple inputs [11, 12••].

Section snippets

Dvl: an early example of phase separation in signal transduction

Dvl is an intracellular protein that transmits Wnt signals to cytoplasmic targets in response to binding of extracellular Wnt by the Frizzled receptor [10, 24]. Dvl or Dvl2, a mammalian form of Dvl, either overexpressed or endogenous, can form cellular puncta [25, 26, 27]. These puncta were initially assumed to represent Dvl protein associated with cytoplasmic membrane-bound vesicles. However, extensive testing of this hypothesis demonstrated that Dvl2 puncta were not associated with a membrane

The role of multivalency, affinity and concentration in phase separation

What properties enable some proteins to form separate liquid phases? The first requirement is likely an ability to engage in multiple interactions. Mechanistic studies employing pairs of synthetic constructs, with one member of the pair containing a variable number of SH3 domains and the other member a variable number of proline-rich motifs (PRM), support a requirement for multivalent interactions [12••, 13••]. The ability of these protein pairs to phase separate was shown to depend strongly on

Two-dimensional phase separation or clustering

The multivalent interactions in the preceding example are derived from the cytoplasmic proteins Nck and N-WASP, which promote assembly of actin filaments together with the membrane-bound adhesion receptor nephrin and the Arp2/3 complex [13••, 33]. Nephrin has three tyrosines on a long disordered tail; upon phosphorylation these can bind to Nck's single SH2 domain [14]. Nck also has three SH3 domains that bind to PRMs in N-WASP (Figure 2). The multivalent interactions between nephrin, Nck and

A role for intrinsically disordered proteins in phase separation

Disordered regions are very common in proteins that phase separate or cluster near membranes [22]. Examples include the disordered regions of CFTR (R region and C-terminus), Ddx4 [5••], Fus [50], the carboxy terminal domain of RNA polymerase II [50, 51], TAF15 [52], DYRK3 [53], hnRNPA1 [8], LAF-1 [29], the nephrin tail and the N-WASP PRM containing regions [54]. The majority of these proteins have disordered regions of greater than 100 residues in length. Disordered proteins have

Conclusions: implications of phase separation for signal transduction

Given the importance of phase separation for RNA processing bodies [22] and evidence of phase separation in signaling puncta below activated receptors [21•], we envision that signals are propagated in various ways in the cell utilizing phase-separated protein states. Signaling controls cellular ion concentrations, expression levels of various proteins and activation of kinases and other enzymes that can post-translationally modify proteins forming the matrix of cellular bodies and puncta, all

Conflict of interest statement

Nothing declared.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

This work was supported by grants to J.D.F.-K. from the Canadian Institutes for Health Research (MOP 114985), Canadian Cancer Society Research Institute (703477) and Cystic Fibrosis Canada (3175). J.D.F.-K. holds a Tier 1 Canada Research Chair in Intrinsically Disordered Proteins.

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