Determinants that enable disordered protein assembly into discrete condensed phases.

Cells harbour numerous mesoscale membraneless compartments that house specific biochemical processes and perform distinct cellular functions. These protein- and RNA-rich bodies are thought to form through multivalent interactions among proteins and nucleic acids, resulting in demixing via liquid-liquid phase separation. Proteins harbouring intrinsically disordered regions (IDRs) predominate in membraneless organelles. However, it is not known whether IDR sequence alone can dictate the formation of distinct condensed phases. We identified a pair of IDRs capable of forming spatially distinct condensates when expressed in cells. When reconstituted in vitro, these model proteins do not co-partition, suggesting condensation specificity is encoded directly in the polypeptide sequences. Through computational modelling and mutagenesis, we identified the amino acids and chain properties governing homotypic and heterotypic interactions that direct selective condensation. These results form the basis of physicochemical principles that may direct subcellular organization of IDRs into specific condensates and reveal an IDR code that can guide construction of orthogonal membraneless compartments.

Determinants of Disordered Protein Co-Assembly Into Discrete Condensed Phases.

Cells harbor numerous mesoscale membraneless compartments that house specific biochemical processes and perform distinct cellular functions. These protein and RNA-rich bodies are thought to form through multivalent interactions among proteins and nucleic acids resulting in demixing via liquid-liquid phase separation (LLPS). Proteins harboring intrinsically disordered regions (IDRs) predominate in membraneless organelles. However, it is not known whether IDR sequence alone can dictate the formation of distinct condensed phases. We identified a pair of IDRs capable of forming spatially distinct condensates when expressed in cells. When reconstituted in vitro, these model proteins do not co-partition, suggesting condensation specificity is encoded directly in the polypeptide sequences. Through computational modeling and mutagenesis, we identified the amino acids and chain properties governing homotypic and heterotypic interactions that direct selective condensation. These results form the basis of physicochemical principles that may direct subcellular organization of IDRs into specific condensates and reveal an IDR code that can guide construction of orthogonal membraneless compartments.

Protein Condensate Formation via Controlled Multimerization of Intrinsically Disordered Sequences.

Many proteins harboring low complexity or intrinsically disordered sequences (IDRs) are capable of undergoing liquid-liquid phase separation to form mesoscale condensates that function as biochemical niches with the ability to concentrate or sequester macromolecules and regulate cellular activity. Engineered disordered proteins have been used to generate programmable synthetic membraneless organelles in cells. Phase separation is governed by the strength of interactions among polypeptides with multivalency enhancing phase separation at lower concentrations. Previously, we and others demonstrated enzymatic control of IDR valency from multivalent precursors to dissolve condensed phases. Here, we develop noncovalent strategies to multimerize an individual IDR, the RGG domain of LAF-1, using protein interaction domains to regulate condensate formation in vitro and in living cells. First, we characterize modular dimerization of RGG domains at either terminus using cognate high-affinity coiled-coil pairs to form stable condensates in vitro. Second, we demonstrate temporal control over phase separation of RGG domains fused to FRB and FKBP in the presence of dimerizer. Further, using a photocaged dimerizer, we achieve optically induced condensation both in cell-sized emulsions and within live cells. Collectively, these modular tools allow multiple strategies to promote phase separation of a common core IDR for tunable control of condensate assembly.

Incorporation and Assembly of a Light-Emitting Enzymatic Reaction into Model Protein Condensates.

Eukaryotic cells partition enzymes and other cellular components into distinct subcellular compartments to generate specialized biochemical niches. A subclass of these compartments form in the absence of lipid membranes, via liquid-liquid phase separation of proteins to form biomolecular condensates or "membraneless organelles" such as nucleoli, stress granules, and P-bodies. Because of their propensity to form compartments from simple starting materials, membraneless organelles are an attractive target for engineering new functionalities in both living cells and protocells. In this work, we demonstrate incorporation of a novel enzymatic activity in protein coacervates with the light-generating enzyme, NanoLuc, to produce bioluminescence. Using condensates comprised of the disordered RGG domain of Caenorhabditis elegans LAF-1, we functionalized condensates with enzymatic activity in vitro and show that enzyme localization to coacervates enhances assembly and activity of split enzymes. To build condensates that function as light-emitting reactors, we designed a NanoLuc enzyme flanked by RGG domains. The resulting condensates concentrated NanoLuc by 10-fold over bulk solution and displayed significantly increased reaction rates. We further show that condensate viscosity impacts light emission due to diffusion-limited behavior. Because our model condensates have low viscosities, we predict NanoLuc diffusion-limited behavior in most other condensates and thus propose the condensate-Nanoluc system as a potential strategy for high-throughput screening of condensate targeting drugs. By splitting the NanoLuc enzyme into its constituent components, we demonstrate that NanoLuc activity can be reconstituted via co-condensation. In addition, we demonstrate control of the spatial localization of the enzyme within condensates by targettng NanoLuc to the surface of in vitro condensates. Collectively, this work demonstrates that membraneless organelles can be endowed with localized enzymatic activity and that this activity can be spatially and temporally controlled via biochemical reconstitution and design of protein surfactants.

Designer membraneless organelles sequester native factors for control of cell behavior.

Subcellular compartmentalization of macromolecules increases flux and prevents inhibitory interactions to control biochemical reactions. Inspired by this functionality, we sought to build designer compartments that function as hubs to regulate the flow of information through cellular control systems. We report a synthetic membraneless organelle platform to control endogenous cellular activities through sequestration and insulation of native proteins. We engineer and express a disordered protein scaffold to assemble micron-size condensates and recruit endogenous clients via genomic tagging with high-affinity dimerization motifs. By relocalizing up to 90% of targeted enzymes to synthetic condensates, we efficiently control cellular behaviors, including proliferation, division and cytoskeletal organization. Further, we demonstrate multiple strategies for controlled cargo release from condensates to switch cells between functional states. These synthetic organelles offer a powerful and generalizable approach to modularly control cell decision-making in a variety of model systems with broad applications for cellular engineering.

A septin-Hof1 scaffold at the yeast bud neck binds and organizes actin cables.

Cellular actin arrays are often highly organized, with characteristic patterns critical to their in vivo functions, yet the mechanisms for establishing these higher order geometries remain poorly understood. In Saccharomyces cerevisiae, formin-polymerized actin cables are spatially organized and aligned along the mother-bud axis to facilitate polarized vesicle traffic. Here, we show that the bud neck-associated F-BAR protein Hof1, independent of its functions in regulating the formin Bnr1, binds to actin filaments and organizes actin cables in vivo. Hof1 bundles actin filaments and links them to septins in vitro. F-actin binding is mediated by the "linker" domain of Hof1, and its deletion leads to cable organization defects in vivo. Using superresolution imaging, we show that Hof1 and septins are patterned at the bud neck into evenly spaced axial pillars (∼200 nm apart), from which actin cables emerge and grow into the mother cell. These results suggest that Hof1, while bound to septins at the bud neck, not only regulates Bnr1 activity, but also binds to actin cables and aligns them along the mother-bud axis. More broadly, these findings provide a strong example of how an actin regulatory protein can be spatially patterned at the cell cortex to govern actin network geometry.

Genetically inspired in vitro reconstitution of Saccharomyces cerevisiae actin cables from seven purified proteins.

A major goal of synthetic biology is to define the minimal cellular machinery required to assemble a biological structure in its simplest form. Here, we focused on Saccharomyces cerevisiae actin cables, which provide polarized tracks for intracellular transport and maintain defined lengths while continuously undergoing rapid assembly and turnover. Guided by the genetic requirements for proper cable assembly and dynamics, we show that seven evolutionarily conserved S. cerevisiae proteins (actin, formin, profilin, tropomyosin, capping protein, cofilin, and AIP1) are sufficient to reconstitute the formation of cables that undergo polarized turnover and maintain steady-state lengths similar to actin cables in vivo. Further, the removal of individual proteins from this simple in vitro reconstitution system leads to cable defects that closely approximate in vivo cable phenotypes caused by disrupting the corresponding genes. Thus, a limited set of molecular components is capable of self-organizing into dynamic, micron-scale actin structures with features similar to cables in living cells.

Integrated control of formin-mediated actin assembly by a stationary inhibitor and a mobile activator.

Formins are essential actin assembly factors whose activities are controlled by a diverse array of binding partners. Until now, most formin ligands have been studied on an individual basis, leaving open the question of how multiple inputs are integrated to regulate formins in vivo. Here, we show that the F-BAR domain of Saccharomyces cerevisiae Hof1 interacts with the FH2 domain of the formin Bnr1 and blocks actin nucleation. Electron microscopy of the Hof1-Bnr1 complex reveals a novel dumbbell-shaped structure, with the tips of the F-BAR holding two FH2 dimers apart. Deletion of Hof1's F-BAR domain in vivo results in disorganized actin cables and secretory defects. The formin-binding protein Bud6 strongly alleviates Hof1 inhibition in vitro, and bud6Δ suppresses hof1Δ defects in vivo. Whereas Hof1 stably resides at the bud neck, we show that Bud6 is delivered to the neck on secretory vesicles. We propose that Hof1 and Bud6 functions are intertwined as a stationary inhibitor and a mobile activator, respectively.

The budding yeast Polo-like kinase localizes to distinct populations at centrosomes during mitosis.

The budding yeast Polo-like kinase Cdc5 is a key regulator of many mitotic events. Cdc5 coordinates its functions spatially and temporally by changing its localization during the cell cycle: Cdc5 is imported into the nucleus in G2 phase and released to the cytoplasm in anaphase, where it accumulates at the bud neck. Cdc5 also localizes to the spindle pole bodies (SPBs) from S phase until the end of mitosis. Whether Cdc5 changes its SPB population during the cell cycle is not known. We find that Cdc5 localizes to distinct SPB subpopulations, depending on the mitotic stage. Cdc5 localizes to the nuclear side of the SPBs during metaphase and early anaphase and to the cytoplasmic surface of the SPBs during late anaphase. Cdc14 is necessary to relocalize Cdc5 from the nuclear SPB plaque. Accumulation of Cdc5 at the daughter SPB in late anaphase is controlled by Bfa1. We also show that Cdc5 and Bfa1 are found in spatially distinct locations at the SPBs during G2/M arrest after DNA damage. Collectively our data reveal that Cdc5 is a dynamic component of the SPBs during mitosis and provide new insight into its regulation during both late mitotic events and DNA damage-induced G2/M arrest.