Essential lipids enrich membrane-associated condensates to rescue synaptic morpho-functional deficits in a mouse model of autism.

Synaptic proteins form intracellular condensates with their scaffolds, but it is unknown whether and how essential lipids transform dynamic cytosolic condensates into stable, functional macromolecular assemblies at the membrane. We show that docosahexaenoic acid (DHA), independent of canonical fatty acid receptor 4 signaling, facilitates the re-localization of cytosolic "full-droplet" condensates composed of the key synaptic elements PSD95 and Kv1.2 to the plasma membrane as "half-droplets.

Interaction of CFTR Modulators with Mammalian Membrane Mimetics: The Role of Cholesterol.

Lumacaftor and Ivacaftor are two FDA-approved medications currently used to treat cystic fibrosis (CF), a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel located in epithelial cell membranes; however, the detailed mechanism(s) of their action remains to be elucidated. Both drugs, termed modulators, bind CFTR at a protein-lipid interface, yet Lumacaftor acts at the endoplasmic reticulum (ER), while Ivacaftor acts at the plasma membrane (PM). A major difference among biological membranes is their level of cholesterol (viz.

Protein interactions, calcium, phosphorylation, and cholesterol modulate CFTR cluster formation on membranes.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel whose dysfunction leads to intracellular accumulation of chloride ions, dehydration of cell surfaces, and subsequent damage to airway and ductal organs. Beyond its function as a chloride channel, interactions between CFTR, epithelium sodium channel, and solute carrier (SLC) transporter family membrane proteins and cytoplasmic proteins, including calmodulin and Na+/H+ exchanger regulatory factor-1 (NHERF-1), coregulate ion homeostasis. CFTR has also been observed to form mesoscale membrane clusters.

Measurement of solubility product reveals the interplay of oligomerization and self-association for defining condensate formation.

Cellular condensates often consist of 10s to 100s of distinct interacting molecular species. Because of the complexity of these interactions, predicting the point at which they will undergo phase separation is daunting. Using experiments and computation, we therefore studied a simple model system consisting of polySH3 and polyPRM designed for pentavalent heterotypic binding.

Measurement of solubility product in a model condensate reveals the interplay of small oligomerization and self-association.

Cellular condensates often consist of 10s to 100s of distinct interacting molecular species. Because of the complexity of these interactions, predicting the point at which they will undergo phase separation into discrete compartments is daunting. Using experiments and computation, we therefore studied a simple model system consisting of 2 proteins, polySH3 and polyPRM, designed for pentavalent heterotypic binding.

Coupling of protein condensates to ordered lipid domains determines functional membrane organization.

During T cell activation, the transmembrane adaptor protein LAT (linker for activation of T cells) forms biomolecular condensates with Grb2 and Sos1, facilitating signaling. LAT has also been associated with cholesterol-rich condensed lipid domains; However, the potential coupling between protein condensation and lipid phase separation and its role in organizing T cell signaling were unknown. Here, we report that LAT/Grb2/Sos1 condensates reconstituted on model membranes can induce and template lipid domains, indicating strong coupling between lipid- and protein-based phase separation.

What are the distinguishing features and size requirements of biomolecular condensates and their implications for RNA-containing condensates?

Exciting recent work has highlighted that numerous cellular compartments lack encapsulating lipid bilayers (often called "membraneless organelles"), and that their structure and function are central to the regulation of key biological processes, including transcription, RNA splicing, translation, and more. These structures have been described as "biomolecular condensates" to underscore that biomolecules can be significantly concentrated in them. Many condensates, including RNA granules and processing bodies, are enriched in proteins and nucleic acids.

The role of sigma 1 receptor in organization of endoplasmic reticulum signaling microdomains.

Sigma 1 receptor (S1R) is a 223-amino-acid-long transmembrane endoplasmic reticulum (ER) protein. S1R modulates activity of multiple effector proteins and is a well-established drug target. However, signaling functions of S1R in cells are poorly understood.

Biomolecular condensates in membrane receptor signaling.

Clustering is a prominent feature of receptors at the plasma membrane (PM). It plays an important role in signaling. Liquid-liquid phase separation (LLPS) of proteins is emerging as a novel mechanism underlying the observed clustering.

Dynamin regulates the dynamics and mechanical strength of the actin cytoskeleton as a multifilament actin-bundling protein.

The dynamin GTPase is known to bundle actin filaments, but the underlying molecular mechanism and physiological relevance remain unclear. Our genetic analyses revealed a function of dynamin in propelling invasive membrane protrusions during myoblast fusion in vivo. Using biochemistry, total internal reflection fluorescence microscopy, electron microscopy and cryo-electron tomography, we show that dynamin bundles actin while forming a helical structure.

A composition-dependent molecular clutch between T cell signaling condensates and actin.

During T cell activation, biomolecular condensates form at the immunological synapse (IS) through multivalency-driven phase separation of LAT, Grb2, Sos1, SLP-76, Nck, and WASP. These condensates move radially at the IS, traversing successive radially-oriented and concentric actin networks. To understand this movement, we biochemically reconstituted LAT condensates with actomyosin filaments.

Regulation of Transmembrane Signaling by Phase Separation.

Cell surface transmembrane receptors often form nanometer- to micrometer-scale clusters to initiate signal transduction in response to environmental cues. Extracellular ligand oligomerization, domain-domain interactions, and binding to multivalent proteins all contribute to cluster formation. Here we review the current understanding of mechanisms driving cluster formation in a series of representative receptor systems: glycosylated receptors, immune receptors, cell adhesion receptors, Wnt receptors, and receptor tyrosine kinases.

Stoichiometry controls activity of phase-separated clusters of actin signaling proteins.

Biomolecular condensates concentrate macromolecules into foci without a surrounding membrane. Many condensates appear to form through multivalent interactions that drive liquid-liquid phase separation (LLPS). LLPS increases the specific activity of actin regulatory proteins toward actin assembly by the Arp2/3 complex.

Who's In and Who's Out-Compositional Control of Biomolecular Condensates.

Biomolecular condensates are two- and three-dimensional compartments in eukaryotic cells that concentrate specific collections of molecules without an encapsulating membrane. Many condensates behave as dynamic liquids and appear to form through liquid-liquid phase separation driven by weak, multivalent interactions between macromolecules. In this review, we discuss current models and data regarding the control of condensate composition, and we describe our current understanding of the composition of representative condensates including PML nuclear bodies, P-bodies, stress granules, the nucleolus, and two-dimensional membrane localized LAT and nephrin clusters.

Allosteric Modulation of Grb2 Recruitment to the Intrinsically Disordered Scaffold Protein, LAT, by Remote Site Phosphorylation.

Tyrosine phosphorylation of membrane receptors and scaffold proteins followed by recruitment of SH2 domain-containing adaptor proteins constitutes a central mechanism of intracellular signal transduction. During early T-cell receptor (TCR) activation, phosphorylation of linker for activation of T cells (LAT) leading to recruitment of adaptor proteins, including Grb2, is one prototypical example. LAT contains multiple modifiable sites, and this multivalency may provide additional layers of regulation, although this is not well understood.

Reconstitution of TCR Signaling Using Supported Lipid Bilayers.

Biochemical reconstitution has served as an important tool for understanding the mechanisms of many cellular processes including DNA replication, transcription, translation, vesicle trafficking, and ubiquitin-mediated proteolysis. Here, we demonstrate that biochemical reconstitution can be applied to studying a complex signaling pathway involving as many as 12 proteins or protein complexes acting at the surface of model membranes. We show that a temporal sequence of events in activated T cells beginning with phosphorylation of the T cell receptor and culminating in the activation of actin polymerization can be replicated in vitro.

Phase separation of signaling molecules promotes T cell receptor signal transduction.

Activation of various cell surface receptors triggers the reorganization of downstream signaling molecules into micrometer- or submicrometer-sized clusters. However, the functional consequences of such clustering have been unclear. We biochemically reconstituted a 12-component signaling pathway on model membranes, beginning with T cell receptor (TCR) activation and ending with actin assembly.

There is more than one way to model an elephant. Experiment-driven modeling of the actin cytoskeleton.

Mathematical modeling has established its value for investigating the interplay of biochemical and mechanical mechanisms underlying actin-based motility. Because of the complex nature of actin dynamics and its regulation, many of these models are phenomenological or conceptual, providing a general understanding of the physics at play. But the wealth of carefully measured kinetic data on the interactions of many of the players in actin biochemistry cries out for the creation of more detailed and accurate models that could permit investigators to dissect interdependent roles of individual molecular components.

Stoichiometry of Nck-dependent actin polymerization in living cells.

Regulation of actin dynamics through the Nck/N-WASp (neural Wiskott-Aldrich syndrome protein)/Arp2/3 pathway is essential for organogenesis, cell invasiveness, and pathogen infection. Although many of the proteins involved in this pathway are known, the detailed mechanism by which it functions remains undetermined. To examine the signaling mechanism, we used a two-pronged strategy involving computational modeling and quantitative experimentation.

Microarray gene expression profiling using core biopsies of renal neoplasia.

We investigate the feasibility of using microarray gene expression profiling technology to analyze core biopsies of renal tumors for classification of tumor histology. Core biopsies were obtained ex-vivo from 7 renal tumors-comprised of four histological subtypes-following radical nephrectomy using 18-gauge biopsy needles. RNA was isolated from these samples and, in the case of biopsy samples, amplified by in vitro transcription.