Two-dimensional molecular condensation in cell signaling and mechanosensing.

Membraneless organelles (MLO) regulate diverse biological processes in a spatiotemporally controlled manner spanning from inside to outside of the cells. The plasma membrane (PM) at the cell surface serves as a central platform for forming multi-component signaling hubs that sense mechanical and chemical cues during physiological and pathological conditions. During signal transduction, the assembly and formation of membrane-bound MLO are dynamically tunable depending on the physicochemical properties of the surrounding environment and partitioning biomolecules. Biomechanical properties of MLO-associated membrane structures can control the microenvironment for biomolecular interactions and assembly. Lipid-protein complex interactions determine the catalytic region's assembly pattern and assembly rate and, thereby, the amplitude of activities. In this review, we will focus on how cell surface microenvironments, including membrane curvature, surface topology and tension, lipid-phase separation, and adhesion force, guide the assembly of PM-associated MLO for cell signal transductions.

Molecular interactions contributing to FUS SYGQ LC-RGG phase separation and co-partitioning with RNA polymerase II heptads.

The RNA-binding protein FUS (Fused in Sarcoma) mediates phase separation in biomolecular condensates and functions in transcription by clustering with RNA polymerase II. Specific contact residues and interaction modes formed by FUS and the C-terminal heptad repeats of RNA polymerase II (CTD) have been suggested but not probed directly. Here we show how RGG domains contribute to phase separation with the FUS N-terminal low-complexity domain (SYGQ LC) and RNA polymerase II CTD. Using NMR spectroscopy and molecular simulations, we demonstrate that many residue types, not solely arginine-tyrosine pairs, form condensed-phase contacts via several interaction modes including, but not only sp2-π and cation-π interactions. In phases also containing RNA polymerase II CTD, many residue types form contacts, including both cation-π and hydrogen-bonding interactions formed by the conserved human CTD lysines. Hence, our data suggest a surprisingly broad array of residue types and modes explain co-phase separation of FUS and RNA polymerase II.

Scaffolding protein CcmM directs multiprotein phase separation in ß-carboxysome biogenesis.

Carboxysomes in cyanobacteria enclose the enzymes Rubisco and carbonic anhydrase to optimize photosynthetic carbon fixation. Understanding carboxysome assembly has implications in agricultural biotechnology. Here we analyzed the role of the scaffolding protein CcmM of the ß-cyanobacterium Synechococcus elongatus PCC 7942 in sequestrating the hexadecameric Rubisco and the tetrameric carbonic anhydrase, CcaA. We find that the trimeric CcmM, consisting of γCAL oligomerization domains and linked small subunit-like (SSUL) modules, plays a central role in mediation of pre-carboxysome condensate formation through multivalent, cooperative interactions. The γCAL domains interact with the C-terminal tails of the CcaA subunits and additionally mediate a head-to-head association of CcmM trimers. Interestingly, SSUL modules, besides their known function in recruiting Rubisco, also participate in intermolecular interactions with the γCAL domains, providing further valency for network formation. Our findings reveal the mechanism by which CcmM functions as a central organizer of the pre-carboxysome multiprotein matrix, concentrating the core components Rubisco and CcaA before ß-carboxysome shell formation.

Phase separation drives the self-assembly of mitochondrial nucleoids for transcriptional modulation.

Mitochondria, the only semiautonomous organelles in mammalian cells, possess a circular, double-stranded genome termed mitochondrial DNA (mtDNA). While nuclear genomic DNA compaction, chromatin compartmentalization and transcription are known to be regulated by phase separation, how the mitochondrial nucleoid, a highly compacted spherical suborganelle, is assembled and functions is unknown. Here we assembled mitochondrial nucleoids in vitro and show that mitochondrial transcription factor A (TFAM) undergoes phase separation with mtDNA to drive nucleoid self-assembly. Moreover, nucleoid droplet formation promotes recruitment of the transcription machinery via a special, co-phase separation that concentrates transcription initiation, elongation and termination factors, and retains substrates to facilitate mtDNA transcription. We propose a model of mitochondrial nucleoid self-assembly driven by phase separation, and a pattern of co-phase separation involved in mitochondrial transcriptional regulation, which orchestrates the roles of TFAM in both mitochondrial nucleoid organization and transcription.

Accurate model of liquid-liquid phase behavior of intrinsically disordered proteins from optimization of single-chain properties.

Many intrinsically disordered proteins (IDPs) may undergo liquid-liquid phase separation (LLPS) and participate in the formation of membraneless organelles in the cell, thereby contributing to the regulation and compartmentalization of intracellular biochemical reactions. The phase behavior of IDPs is sequence dependent, and its investigation through molecular simulations requires protein models that combine computational efficiency with an accurate description of intramolecular and intermolecular interactions. We developed a general coarse-grained model of IDPs, with residue-level detail, based on an extensive set of experimental data on single-chain properties. Ensemble-averaged experimental observables are predicted from molecular simulations, and a data-driven parameter-learning procedure is used to identify the residue-specific model parameters that minimize the discrepancy between predictions and experiments. The model accurately reproduces the experimentally observed conformational propensities of a set of IDPs. Through two-body as well as large-scale molecular simulations, we show that the optimization of the intramolecular interactions results in improved predictions of protein self-association and LLPS.

Phase separation in the outer membrane of Escherichia coli.

Gram-negative bacteria are surrounded by a protective outer membrane (OM) with phospholipids in its inner leaflet and lipopolysaccharides (LPS) in its outer leaflet. The OM is also populated with many ß-barrel outer-membrane proteins (OMPs), some of which have been shown to cluster into supramolecular assemblies. However, it remains unknown how abundant OMPs are organized across the entire bacterial surface and how this relates to the lipids in the membrane. Here, we reveal how the OM is organized from molecular to cellular length scales, using atomic force microscopy to visualize the OM of live bacteria, including engineered Escherichia coli strains and complemented by specific labeling of abundant OMPs. We find that a predominant OMP in the E. coli OM, the porin OmpF, forms a near-static network across the surface, which is interspersed with barren patches of LPS that grow and merge with other patches during cell elongation. Embedded within the porin network is OmpA, which forms noncovalent interactions to the underlying cell wall. When the OM is destabilized by mislocalization of phospholipids to the outer leaflet, a new phase appears, correlating with bacterial sensitivity to harsh environments. We conclude that the OM is a mosaic of phase-separated LPS-rich and OMP-rich regions, the maintenance of which is essential to the integrity of the membrane and hence to the lifestyle of a gram-negative bacterium.

Local thermodynamics govern formation and dissolution of Caenorhabditis elegans P granule condensates.

Membraneless compartments, also known as condensates, provide chemically distinct environments and thus spatially organize the cell. A well-studied example of condensates is P granules in the roundworm Caenorhabditis elegans that play an important role in the development of the germline. P granules are RNA-rich protein condensates that share the key properties of liquid droplets such as a spherical shape, the ability to fuse, and fast diffusion of their molecular components. An outstanding question is to what extent phase separation at thermodynamic equilibrium is appropriate to describe the formation of condensates in an active cellular environment. To address this question, we investigate the response of P granule condensates in living cells to temperature changes. We observe that P granules dissolve upon increasing the temperature and recondense upon lowering the temperature in a reversible manner. Strikingly, this temperature response can be captured by in vivo phase diagrams that are well described by a Flory-Huggins model at thermodynamic equilibrium. This finding is surprising due to active processes in a living cell. To address the impact of such active processes on intracellular phase separation, we discuss temperature heterogeneities. We show that, for typical estimates of the density of active processes, temperature represents a well-defined variable and that mesoscopic volume elements are at local thermodynamic equilibrium. Our findings provide strong evidence that P granule assembly and disassembly are governed by phase separation based on local thermal equilibria where the nonequilibrium nature of the cytoplasm is manifested on larger scales.

Quantitative photoconversion analysis of internal molecular dynamics in stress granules and other membraneless organelles in live cells.

Photoconversion enables real-time labeling of protein sub-populations inside living cells, which can then be tracked with submicrometer resolution. Here, we detail the protocol of comparing protein dynamics inside membraneless organelles in live HEK293T cells using a CRISPR-Cas9 PABPC1-Dendra2 marker of stress granules. Measuring internal dynamics of membraneless organelles provides insight into their functional state, physical properties, and composition. Photoconversion has the advantage over other imaging techniques in that it is less phototoxic and allows for dual color tracking of proteins. For complete details on the use and execution of this protocol, please refer to Amen and Kaganovich (2020).