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. 2022 Mar 1;41(5):e109952.
doi: 10.15252/embj.2021109952. Epub 2022 Feb 2.

On the role of phase separation in the biogenesis of membraneless compartments

Andrea Musacchio  1
Affiliations

On the role of phase separation in the biogenesis of membraneless compartments

Andrea Musacchio. EMBO J. .

Abstract

Molecular mechanistic biology has ushered us into the world of life's building blocks, revealing their interactions in macromolecular complexes and inspiring strategies for detailed functional interrogations. The biogenesis of membraneless cellular compartments, functional mesoscale subcellular locales devoid of strong internal order and delimiting membranes, is among mechanistic biology's most demanding current challenges. A developing paradigm, biomolecular phase separation, emphasizes solvation of the building blocks through low-affinity, weakly adhesive unspecific interactions as the driver of biogenesis of membraneless compartments. Here, I discuss the molecular underpinnings of the phase separation paradigm and demonstrate that validating its assumptions is much more challenging than hitherto appreciated. I also discuss that highly specific interactions, rather than unspecific ones, appear to be the main driver of biogenesis of subcellular compartments, while phase separation may be harnessed locally in selected instances to generate material properties tailored for specific functions, as exemplified by nucleocytoplasmic transport.

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Figures

Figure 1
Figure 1. Examples of site‐specific interactions and their combination in MVPs
(A–E) Cartoon diagrams of various protein domains and proteins discussed in the text (yellow), with their cognate ligands shown in stick. (A) Src homology 3 (SH3) domain with proline‐rich motif (PRM); (B) Src homology 2 domain with tyrosine‐phosphorylated peptide; (C) Tudor domain with peptide from histone H3 trimethylated on lysine 36; (D) Importin‐β with Gly‐Leu‐Gly‐Phe (GLGF) peptide; (E) Enlargement of area boxed in D. The protein data bank (PDB) codes are indicated; (F) Domain organization of the NCK and N‐WASP proteins, and sequence of the proline‐rich region of N‐WASP (Uniprot, human sequence); and (G) Artificial multivalent constructs used by Li et al (2012).
Figure 2
Figure 2. Examples of IDPs and low‐affinity, non‐site‐specific interactions
(A) Domain organization of three IDPs and specific sequence stretches from each of three low‐complexity regions of human FUS; (B) Examples of low‐affinity interactions believed to drive homotypic PS of IDPs; (C) Droplets of FUS, DDX4, and LAF‐1 (indicated as “mesoscale”) are proposed to arise from multiple nanoscale interactions shown in B; (D) Sticker‐and‐spacer model for interactions of MVPs (left) and of IDPs (right). Gln and Tyr (Q and Y, respectively) are considered stickers that could interact through dipoles and stacking. The entire figure is an adaptation of Fig 2 from Brangwynne et al (2015).
Figure 3
Figure 3. Hierarchy of compartment assembly
Compartments are usually hierarchical. Nephrin, as an element of membrane clusters, is a transmembrane protein whose intracellular domain undergoes regulated phosphorylation. As primary scaffold, it acts as a binding site for the NCK client through its SH2 domain. N‐WASP binds NCK through phosphorylation. Regulators of actin polymerization may be further recruited to clusters. During mitosis, the CPC kinase complex is recruited to specific phosphorylated residues of histone H2A and H3 that are enriched in the centromere region. It is therefore a client of the centromere, although it has been suggested to act as scaffold in PS. The kinetochore assembles on the histone H3 variant CENP‐A, which is part of a specialized centromeric histone complex. Constitutive centromere‐associated network (CCAN) subunits are recruited through interactions with CENP‐A. The Knl1‐Mis12‐Ndc80 (KMN) complex is recruited to CCAN through established SSIs. Not shown are tertiary clients like the spindle checkpoint proteins BUB1 and MAD2 discussed in the text. In Cajal bodies, specific RNAs transcripts, for instance, of histones, but also spliceosome subunits, act as scaffolds for the recruitment of a variety of downstream proteins (Kaiser et al, ; Shevtsov & Dundr, 2011). The recruitment hierarchy remains poorly understood. Other nuclear bodies, like the histone body or the nucleolus, also require transcription (see main text for details).
Figure 4
Figure 4. Liquid like does not exclude SSIs
(A) KNL1, an IDP at kinetochores, contains multiple sequence‐related phosphorylation sites for the recruitment of the BUB1/BUB3 complex, where BUB3 is a phospho–amino acid adaptor. KNL1 docks to CCAN in the core kinetochore. A FRAP experiment on BUB1/BUB3 and on a core kinetochore subunit would lead to fundamentally different conclusions on the nature of the compartment, as the core subunit do not exchange and would not recover, whereas BUB1/BUB3 would exchange in seconds; (B) Hand‐drawn curves representing the recovery behavior shown in A; (C) Two imaginary directly interacting MVPs in neighboring phase‐separated droplets (1 and 2, where the small differences in color are meant to recognize the origin of molecules in the original droplets) could easily mix if the interaction times of the individual modules allowed relatively rapid exchange.
Figure 5
Figure 5. Interactions in an imaginary compartment
(A) Compartment X concentrates several components, three of which are shown in blue, brown, and green colors together with their turnover times; (B) An assembly hierarchy might have been identified while investigating the interactions in Compartment X; (C) Upper left: Can a primary client with wild‐type sequence be recruited to a scaffold with mutations in a binding site for the primary client? If so, the binding interaction is at least necessary for the recruitment. Upper right: Grafting of binding site of primary client for scaffold allows recruitment of unrelated macromolecule. Minimal binding sites indicate sufficiency. Bottom left: Iterative analysis down the interaction hierarchy. Bottom right: A mutant client that fails to be recruited to X but undergoes PS in vitro like the wild‐type counterpart indicates PS in vitro not predictive.
Figure 6
Figure 6. PS in the cavity of the nuclear pore complex
(A) The Scaffold‐Nups subunits of the NPC (not shown individually) build a complex circular structure with a central cavity. The FG‐Nups (only a subset of which are shown) interact with the Scaffold‐Nups through SSIs. This allows to position their IDRs in the cavity of the pore, where they may form a hydrogel; (B) Domain organization of human NUP98, one of the human FG‐Nups. A short segment of the FG/GLFG sequence is shown; (C) An inert cargo molecule of sufficiently large size will be unable to cross the NPC cavity; (D) The cargo needs to bind to an import/export receptor like importin‐β, which binds the FG repeats, locally “melting” the meshwork, and therefore dissolving in it.
Figure 7
Figure 7. Complexity of a multivalent system
(A) A single SH3/proline‐rich motif module may have relatively low affinity; (B) Multiple modules on the two A and B ligands bind each other more tightly. The complex forms first as the concentration of A and B is increased. Further increasing the concentration leads to assemble clustered complexes in which multiple multivalent ligands interact at the same time in a network. At high concentrations, this system undergoes PS in vitro, demonstrating that PS is perfectly compatible with the SSIs that this system is based on. (C) The nephrin/NCK/N‐WASP system does not configure general PS because the SSIs elicited by phosphorylation of nephrin at three different sites are required for recruiting downstream components and membrane clustering. Clustering occurs when bound NCK promotes further recruitment of N‐WASP, which crosslinks the NCK molecules near P‐nephrin. Thus, binding interactions are required at all stages of assembly of this membrane‐bound signaling compartment; (D) Putative mutations at the SH3/PRM interface prevent formation of the simple AB complex as well as of the clustered A2B2 complex, indicating SSIs are necessary for both; (E) Changing stoichiometries in clustered compartments changes the degree of clustering, which may lead to graded functional responses. This strategy does not probe PS but rather binding saturation; (F) Part 2 of the test aims to detect PS within a compartment held together by SSIs. Ideally, PS should be measured under conditions of equal saturation of the binding interactions between all components. The solubility‐determining linkers between interacting “stickers” may determine whether a density transition has taken place or not. Functional output should be compared for the two depicted scenarios.
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