Biomolecular condensates as drivers of membrane trafficking and remodelling.

Membrane remodelling is essential for the trafficking of macromolecules throughout the cell, a process that regulates various aspects of cellular health and pathology. Recent studies implicate the role of biomolecular condensates in regulating multiple steps of the membrane trafficking pathway including but not limited to the organization of the trafficking machinery, dynamic remodeling of membranes, spatial and functional regulation, and response to cellular signals. The implicated proteins contain key structural elements, most notably prion-like domains within intrinsically disordered regions that are necessary for biomolecular condensate formation at fusion sites in processes like endocytic assembly, autophagy, organelle biosynthesis and synaptic vesicle fusion. Experimental and theoretical advances in the field continue to demonstrate that protein condensates can perform mechanical work, the implications of which can be extrapolated to diverse areas of membrane biology.

Peroxisome biogenesis initiated by protein phase separation.

Peroxisomes are organelles that carry out ß-oxidation of fatty acids and amino acids. Both rare and prevalent diseases are caused by their dysfunction1. Among disease-causing variant genes are those required for protein transport into peroxisomes. The peroxisomal protein import machinery, which also shares similarities with chloroplasts2, is unique in transporting folded and large, up to 10 nm in diameter, protein complexes into peroxisomes3. Current models postulate a large pore formed by transmembrane proteins4; however, so far, no pore structure has been observed. In the budding yeast Saccharomyces cerevisiae, the minimum transport machinery includes the membrane proteins Pex13 and Pex14 and the cargo-protein-binding transport receptor, Pex5. Here we show that Pex13 undergoes liquid-liquid phase separation (LLPS) with Pex5-cargo. Intrinsically disordered regions in Pex13 and Pex5 resemble those found in nuclear pore complex proteins. Peroxisomal protein import depends on both the number and pattern of aromatic residues in these intrinsically disordered regions, consistent with their roles as 'stickers' in associative polymer models of LLPS5,6. Finally, imaging fluorescence cross-correlation spectroscopy shows that cargo import correlates with transient focusing of GFP-Pex13 and GFP-Pex14 on the peroxisome membrane. Pex13 and Pex14 form foci in distinct time frames, suggesting that they may form channels at different saturating concentrations of Pex5-cargo. Our findings lead us to suggest a model in which LLPS of Pex5-cargo with Pex13 and Pex14 results in transient protein transport channels7.

Adaptive partitioning of a gene locus to the nuclear envelope in Saccharomyces cerevisiae is driven by polymer-polymer phase separation.

Partitioning of active gene loci to the nuclear envelope (NE) is a mechanism by which organisms increase the speed of adaptation and metabolic robustness to fluctuating resources in the environment. In the yeast Saccharomyces cerevisiae, adaptation to nutrient depletion or other stresses, manifests as relocalization of active gene loci from nucleoplasm to the NE, resulting in more efficient transport and translation of mRNA. The mechanism by which this partitioning occurs remains a mystery. Here, we demonstrate that the yeast inositol depletion-responsive gene locus INO1 partitions to the nuclear envelope, driven by local histone acetylation-induced polymer-polymer phase separation from the nucleoplasmic phase. This demixing is consistent with recent evidence for chromatin phase separation by acetylation-mediated dissolution of multivalent histone association and fits a physical model where increased bending stiffness of acetylated chromatin polymer causes its phase separation from de-acetylated chromatin. Increased chromatin spring stiffness could explain nucleation of transcriptional machinery at active gene loci.

The modular cell gets connected.

Integrative molecular cell biology can be used to interpret networks beyond modules.

Endocytic proteins with prion-like domains form viscoelastic condensates that enable membrane remodeling.

Membrane invagination and vesicle formation are key steps in endocytosis and cellular trafficking. Here, we show that endocytic coat proteins with prion-like domains (PLDs) form hemispherical puncta in the budding yeast, Saccharomyces cerevisiae These puncta have the hallmarks of biomolecular condensates and organize proteins at the membrane for actin-dependent endocytosis. They also enable membrane remodeling to drive actin-independent endocytosis. The puncta, which we refer to as endocytic condensates, form and dissolve reversibly in response to changes in temperature and solution conditions. We find that endocytic condensates are organized around dynamic protein-protein interaction networks, which involve interactions among PLDs with high glutamine contents. The endocytic coat protein Sla1 is at the hub of the protein-protein interaction network. Using active rheology, we inferred the material properties of endocytic condensates. These experiments show that endocytic condensates are akin to viscoelastic materials. We use these characterizations to estimate the interfacial tension between endocytic condensates and their surroundings. We then adapt the physics of contact mechanics, specifically modifications of Hertz theory, to develop a quantitative framework for describing how interfacial tensions among condensates, the membrane, and the cytosol can deform the plasma membrane to enable actin-independent endocytosis.

Structure-based Design of a Specific, Homogeneous Luminescence Enzyme Reporter Assay for SARS-CoV-2.

Recombinant antibodies (Abs) against the SARS-CoV-2 virus hold promise for treatment of COVID-19 and high sensitivity and specific diagnostic assays. Here, we report engineering principles and realization of a Protein-fragment Complementation Assay (PCA) detector of SARS-CoV-2 antigen by coupling two Abs to complementary N- and C-terminal fragments of the reporter enzyme Gaussia luciferase (Gluc). Both Abs display comparably high affinities for distinct epitopes of viral Spike (S)-protein trimers. Gluc activity is reconstituted when the Abs are simultaneously bound to S-protein bringing the Ab-fused N- and C-terminal fragments close enough together (8 nm) to fold. We thus achieve high specificity both by requirement of simultaneous binding of the two Abs to the S-protein and also, in a steric configuration in which the two Gluc complementary fragments can fold and thus reconstitute catalytic activity. Gluc activity can also be reconstituted with virus-like particles that express surface S-protein with detectable signal over background within 5 min of incubation. Design principles presented here can be readily applied to develop reporters to virtually any protein with sufficient available structural details. Thus, our results present a general framework to develop reporter assays for COVID-19, and the strategy can be readily deployed in response to existing and future pathogenic threats and other diseases.

Genetics' Piece of the PI: Inferring the Origin of Complex Traits and Diseases from Proteome-Wide Protein-Protein Interaction Dynamics.

How do common and rare genetic polymorphisms contribute to quantitative traits or disease risk and progression? Multiple human traits have been extensively characterized at the genomic level, revealing their complex genetic architecture. However, it is difficult to resolve the mechanisms by which specific variants contribute to a phenotype. Recently, analyses of variant effects on molecular traits have uncovered intermediate mechanisms that link sequence variation to phenotypic changes. Yet, these methods only capture a fraction of genetic contributions to phenotype. Here, in reviewing the field, it is proposed that complex traits can be understood by characterizing the dynamics of biochemical networks within living cells, and that the effects of genetic variation can be captured on these networks by using protein-protein interaction (PPI) methodologies. This synergy between PPI methodologies and the genetics of complex traits opens new avenues to investigate the molecular etiology of human diseases and to facilitate their prevention or treatment.

Induced and spontaneous colitis mouse models reveal complex interactions between IL-10 and IL-12/IL-23 pathways.

Crohn's disease (CD) and ulcerative colitis (UC) are the two major forms of inflammatory bowel disease (IBD). These idiopathic and chronic diseases result from inflammation of the gastrointestinal tract and are mainly mediated by the immune system. Genome wide association studies link genes of the IL-12 and IL-23 biology to both CD and UC susceptibility. IL-12 and IL-23 cytokines share a functional subunit, p40, and their respective receptors also share a functional subunit, IL-12Rß1. However, clinical trials targeting p40, and thus inhibiting both IL-12 and IL-23 pathways, provided mitigated effects on IBD, suggesting context dependent effects for each cytokine. In addition to IL-12 and IL-23, genetic deficiencies in IL-10 also result in severe IBD pathology. We generated various mouse models to determine how IL-12 or IL-23 interacts with IL-10 in IBD pathology. Whereas defects in both IL-10 and IL-12R do not impact the severity of the Dextran Sulfate Sodium (DSS)-induced colitis, combined deficiencies in both IL-10 and IL-23R aggravate the disease. In contrast to DSS-induced colitis, defects in IL-12R and IL-23R both protect from the spontaneous colitis observed in IL10-/- mice. Together, these studies exemplify the complexity of genetic and environmental interactions for identifying biological pathways predictive of pathological inflammatory processes.

Predicted amino acid motif repeats in proteins potentially encode extensive multivalent macromolecular assemblies in the human proteome.

There are emerging interests in understanding higher order assemblies of biopolymers within and between cells, such as protein-protein and protein-RNA biomolecular condensates. These biomolecular condensates are thought to assemble/disassemble via multivalent interactions, including those mediated particularly by unique repeated amino acid motifs (URM). We asked how common are proteins with such URMs, their incidence and abundance, by exhaustively enumerating repeating motifs of length 3-10 in the human proteome. We found that URMs are very common and widely distributed across the human proteome. Moreover, the number of repetitions and intervals between them do not correlate with their lengths, which suggests that the number of repeats among proteins in the proteome is independent of length, contrary to the notion that short motifs are more abundant then long motifs. Finally, we describe two examples of URMs in proteins known to form higher order biopolymer assemblies: multi-PDZ domain-containing proteins and the FUS family of RNA binding proteins. For the FUS family, we predicted a known sequence 'grammar', specific motifs and interval sequence compositions that are essential to phase separation and material properties of condensates formed by this family of proteins. In PDZ domain-containing proteins we found a novel repeated motif that was surprisingly both within and between individual PDZ domains. We speculate that these motifs could be binding sites for multivalent interactions, a residual result of the mechanism by which PDZ-domain duplications occurred or that the linker sequences between PDZ domains may encode cryptic PDZ domains.

Probing Inhomogeneous Diffusion in the Microenvironments of Phase-Separated Polymers under Confinement.

Biomolecular condensates formed by liquid-liquid phase separation of proteins and nucleic acids have been recently discovered to be prevalent in biology. These dynamic condensates behave like biochemical reaction vessels, but little is known about their structural organization and biophysical properties, which are likely related to condensate size. Thus, it is critical that we study them on scales found in vivo. However, previous in vitro studies of condensate assembly and physical properties have involved condensates up to 1000 times larger than those found in vivo. Here, we apply confinement microscopy to visualize condensates and control their sizes by creating appropriate confinement length scales relevant to the cell environment. We observe anomalous diffusion of probe particles embedded within confined condensates, as well as heterogeneous dynamics in condensates formed from PEG/dextran and in ribonucleoprotein complexes of RNA and the RNA-binding protein Dhh1. We propose that the observed non-Gaussian dynamics indicate a hopping diffusion mechanism inside condensates. We also observe that, for dextran-rich condensates, but not for ribonucleo condensates, probe particle diffusion depends on condensate size.

Author Correction: Genome-wide SWAp-Tag yeast libraries for proteome exploration.

The version of Supplementary Table 1 originally published online with this article contained incorrect localization annotations for one plate. This error has been corrected in the online Supplementary Information.

Tuberculosis and impaired IL-23-dependent IFN-γ immunity in humans homozygous for a common TYK2 missense variant.

Inherited IL-12Rß1 and TYK2 deficiencies impair both IL-12- and IL-23-dependent IFN-γ immunity and are rare monogenic causes of tuberculosis, each found in less than 1/600,000 individuals. We show that homozygosity for the common TYK2 P1104A allele, which is found in about 1/600 Europeans and between 1/1000 and 1/10,000 individuals in regions other than East Asia, is more frequent in a cohort of patients with tuberculosis from endemic areas than in ethnicity-adjusted controls (P = 8.37 × 10-8; odds ratio, 89.31; 95% CI, 14.7 to 1725). Moreover, the frequency of P1104A in Europeans has decreased, from about 9% to 4.2%, over the past 4000 years, consistent with purging of this variant by endemic tuberculosis. Surprisingly, we also show that TYK2 P1104A impairs cellular responses to IL-23, but not to IFN-α, IL-10, or even IL-12, which, like IL-23, induces IFN-γ via activation of TYK2 and JAK2. Moreover, TYK2 P1104A is properly docked on cytokine receptors and can be phosphorylated by the proximal JAK, but lacks catalytic activity. Last, we show that the catalytic activity of TYK2 is essential for IL-23, but not IL-12, responses in cells expressing wild-type JAK2. In contrast, the catalytic activity of JAK2 is redundant for both IL-12 and IL-23 responses, because the catalytically inactive P1057A JAK2, which is also docked and phosphorylated, rescues signaling in cells expressing wild-type TYK2. In conclusion, homozygosity for the catalytically inactive P1104A missense variant of TYK2 selectively disrupts the induction of IFN-γ by IL-23 and is a common monogenic etiology of tuberculosis.

Changes of Cell Biochemical States Are Revealed in Protein Homomeric Complex Dynamics.

We report here a simple and global strategy to map out gene functions and target pathways of drugs, toxins, or other small molecules based on "homomer dynamics" protein-fragment complementation assays (hdPCA). hdPCA measures changes in self-association (homomerization) of over 3,500 yeast proteins in yeast grown under different conditions. hdPCA complements genetic interaction measurements while eliminating the confounding effects of gene ablation. We demonstrate that hdPCA accurately predicts the effects of two longevity and health span-affecting drugs, the immunosuppressant rapamycin and the type 2 diabetes drug metformin, on cellular pathways. We also discovered an unsuspected global cellular response to metformin that resembles iron deficiency and includes a change in protein-bound iron levels. This discovery opens a new avenue to investigate molecular mechanisms for the prevention or treatment of diabetes, cancers, and other chronic diseases of aging.

Genome-wide SWAp-Tag yeast libraries for proteome exploration.

Yeast libraries revolutionized the systematic study of cell biology. To extensively increase the number of such libraries, we used our previously devised SWAp-Tag (SWAT) approach to construct a genome-wide library of ~5,500 strains carrying the SWAT NOP1promoter-GFP module at the N terminus of proteins. In addition, we created six diverse libraries that restored the native regulation, created an overexpression library with a Cherry tag, or enabled protein complementation assays from two fragments of an enzyme or fluorophore. We developed methods utilizing these SWAT collections to systematically characterize the yeast proteome for protein abundance, localization, topology, and interactions.

Mechanics, Structure and Function of Biopolymer Condensates.

The spontaneous nature of biopolymer phase separation in cells entails that the resulting condensates can be thermodynamic machines, which, in the process of condensing, can take on distinct forms themselves and deform neighboring cellular structures. We introduce here general notions of material and mechanical properties of protein condensates with an emphasis on how molecular arrangements and intermolecular interaction within condensates determine their ability to do work on their surroundings. We further propose functional implications of these concepts to cellular and subcellular morphology and biogenesis.