Liquid-liquid phase separation: Orchestrating cell signaling through time and space

Abstract

Cell signaling is a complex process. The faithful transduction of information into specific cellular actions depends on the synergistic effects of many regulatory molecules, nurtured by their strict spatiotemporal regulation. Over the years, we have gained copious insights into the subcellular architecture supporting this spatiotemporal control, including the roles of membrane-bound organelles and various signaling nanodomains. Recently, liquid-liquid phase separation (LLPS) has been recognized as another potentially ubiquitous framework for organizing signaling molecules with high specificity and precise spatiotemporal control in cells. Here, we review the pervasive role of LLPS in signal transduction, highlighting several key pathways that intersect with LLPS, including examples in which LLPS is controlled by signaling events. We also examine how LLPS orchestrates signaling by compartmentalizing signaling molecules, amplifying signals non-linearly, and moderating signaling dynamics. We focus on the specific molecules that drive LLPS and highlight the known functional and pathological consequences of LLPS in each pathway.

Keywords: Compartmentation; biomolecular condensates; membraneless organelle.

Figure 1.. LLPS regulates cGAS signaling.

(i) Cytosolic LLPS of cGAS is driven by several factors, including multivalent charge-charge interactions with dsDNA. These properties drive cGAS to undergo an abrupt phase transition to form condensates with dsDNA at a low threshold concentration. (ii) LLPS compartmentalizes the cGAS signaling machinery, forming a core cGAS/DNA condensate that is surrounded by a phase-separated outer shell containing TREX1, a dsDNA-degrading enzyme that antagonizes cGAS signaling. Depletion of dsDNA from the outer shell helps suppress TREX1 function and promote cGAS-mediated DNA sensing. The concentrated environment within the cGAS/DNA core dramatically enhances cGAS activity by increasing DNA-binding efficiency. (iii) cGAS self-associates via its core catalytic domain to form dimers along dsDNA strands, which activates cGAS to catalyze the conversion of GTP and ATP into cGAMP and induce additional downstream signaling.

Figure 2.. Amplification of immune signaling by LLPS.

(i) Following T-cell receptor (TCR) activation, phosphorylated LAT (pLAT) scaffolds LLPS of Grb2 and Sos1 at the plasma membrane, which is driven by extensive multivalent interactions. (ii) The Nck-N-WASP-Arp2/3 complex similarly undergoes LLPS upon T-cell activation via multivalent interactions that are scaffolded by either pLAT or pNephrin (shown). (iii) Both Sos1 and Arp2/3 are activated at the plasma membrane via slow, multi-step processes where molecules with long dwell times exhibit disproportionally higher activation rates. Under diffuse conditions (grey curve), Sos1 or Arp2/3 molecules have low plasma membrane dwell times. In contrast, the multivalent interaction networks formed through LLPS increase the plasma membrane dwell time of Sos1 and Arp2/3 molecules, allowing them to readily overcome these kinetic activation barriers and achieve activation of downstream (i) Ras/MAPK signaling or (ii) actin polymerization.

Figure 3.. LLPS organizes synaptic signaling proteins.

(i) Electrical and chemical signals are communicated from one neuron to another via synapses formed between (ii) an axon terminal from a presynaptic neuron and a dendritic spine from a postsynaptic neuron. (iii) The postsynaptic density, a protein-rich structure that sits immediately below the plasma membrane in dendritic spines, plays a crucial role in spine function and was recently shown to form via LLPS. (iv) Multivalent binding between the scaffold PSD95 and SynGAP, as well as numerous other components including guanylate kinase-associated protein (GKAP), Shank, and Homer, create a unique molecular interaction network to organize clusters of cell-surface receptors (e.g., N-methyl-D-aspartate receptor; NMDAR) and ion channels, recruit signaling enzymes, and drive cytoskeletal remodeling to modulate synaptic strength.

Figure 4.. Compartmentation of the Wnt pathway by LLPS.

(i) Under basal conditions, the Wnt signaling pathway is kept inactive via the degradosome, which promotes the highly efficient phosphorylation of β-catenin to ultimately trigger its proteasomal degradation. (ii) The degradosome is formed via LLPS driven by multivalent interactions between the scaffold proteins Axin and APC, which recruit β-catenin, along with kinases such as GSKβ, as client proteins. (iii) When Wnt binds the cell-surface receptors Frizzled and LRP5/6, Axin gets recruited to Dishevelled oligomers at the plasma membrane, triggering LLPS of the Wnt signalosome (iv). Inhibition of GSK3β within the signalosome (not shown) allows β-catenin to accumulate in the cytosol and subsequently translocate into the nucleus (v), where β-catenin itself undergoes LLPS at transcriptional enhancers to induce target gene expression.

Figure 5.. Phosphorylation regulates Hippo pathway LLPS.

(i) Under growth-suppressing conditions, LLPS of the transcriptional regulator TAZ is blocked by the upstream kinases Mst1/2 and LATS1/2, which induce TAZ phosphorylation and degradation in the cytosol. (ii) Under growth-stimulating conditions, however, Mst1/2 and LATS1/2 are inactive, allowing TAZ to enter the nucleus (iii) and undergo phase separation via multivalent self-association driven by its WW and coiled-coil (CC) domains, as well as intrinsic disorder within its TEAD-binding (TB) and transactivation (TA) domains (iv). TAZ condensates recruit TEAD proteins and numerous other transcription factors, efficiently inducing target gene transcription.

Figure 6.. LLPS of PKA RIα buffers cAMP and drives pathway compartmentation.

(i) LLPS of the PKA regulatory subunit RIα is dynamically regulated by cAMP. Binding of cAMP to RIα induces a conformational change that releases the PKA catalytic (C) subunit from its interaction with the RIα inhibitory sequence (IS), exposing the disordered RIα linker region. (ii) RIα biomolecular condensates play a key role in compartmentalizing cAMP to ensure signaling specificity. RIα condensates dynamically sequester and trap cAMP, lowering cytosolic free cAMP concentrations, while PDEs catalytically degrade excess free cAMP into AMP. Combined, these two processes enable PDEs to establish cAMP-free nanodomains that set the boundaries of cAMP signaling compartments.