Dynamics and Molecular Interactions of GPI-Anchored CD59.

CD59 is a GPI-anchored cell surface receptor that serves as a gatekeeper to controlling pore formation. It is the only membrane-bound inhibitor of the complement membrane attack complex (MAC), an immune pore that can damage human cells. While CD59 blocks MAC pores, the receptor is co-opted by bacterial pore-forming proteins to target human cells. Recent structures of CD59 in complexes with binding partners showed dramatic differences in the orientation of its ectodomain relative to the membrane. Here, we show how GPI-anchored CD59 can satisfy this diversity in binding modes. We present a PyLipID analysis of coarse-grain molecular dynamics simulations of a CD59-inhibited MAC to reveal residues of complement proteins (C6:Y285, C6:R407 C6:K412, C7:F224, C8ß:F202, C8ß:K326) that likely interact with lipids. Using modules of the MDAnalysis package to investigate atomistic simulations of GPI-anchored CD59, we discover properties of CD59 that encode the flexibility necessary to bind both complement proteins and bacterial virulence factors.

Ceramide structure dictates glycosphingolipid nanodomain assembly and function.

Gangliosides in the outer leaflet of the plasma membrane of eukaryotic cells are essential for many cellular functions and pathogenic interactions. How gangliosides are dynamically organized and how they respond to ligand binding is poorly understood. Using fluorescence anisotropy imaging of synthetic, fluorescently labeled GM1 gangliosides incorporated into the plasma membrane of living cells, we found that GM1 with a fully saturated C16:0 acyl chain, but not with unsaturated C16:1 acyl chain, is actively clustered into nanodomains, which depends on membrane cholesterol, phosphatidylserine and actin. The binding of cholera toxin B-subunit (CTxB) leads to enlarged membrane domains for both C16:0 and C16:1, owing to binding of multiple GM1 under a toxin, and clustering of CTxB. The structure of the ceramide acyl chain still affects these domains, as co-clustering with the glycosylphosphatidylinositol (GPI)-anchored protein CD59 occurs only when GM1 contains the fully saturated C16:0 acyl chain, and not C16:1. Thus, different ceramide species of GM1 gangliosides dictate their assembly into nanodomains and affect nanodomain structure and function, which likely underlies many endogenous cellular processes.

PCTR1 improves pulmonary edema fluid clearance through activating the sodium channel and lymphatic drainage in lipopolysaccharide-induced ARDS.

Acute respiratory distress syndrome (ARDS) is a lethal clinical syndrome characterized by damage of the epithelial barriers and accumulation of pulmonary edema fluid. Protectin conjugates in tissue regeneration 1 (PCTR1), an endogenously produced lipid mediator, are believed to exert anti-inflammatory and pro-resolution effects. PCTR1 (1 µg/kg) was injected at 8 hr after lipopolysaccharide (LPS; 14 mg/kg) administration, and the rate of pulmonary fluid clearance was measured in live rats at 1 hr after PCTR1 treatment. The primary type II alveolar epithelial cells were cultured with PCTR1 (10 nmol/ml) and LPS (1 µg/ml) for 8 hr. PCTR1 effectively improved pulmonary fluid clearance and ameliorated morphological damage and reduced inflammation of lung tissue, as well as improved the survival rate in the LPS-induced acute lung injury (ALI) model. Moreover, PCTR1 markedly increased sodium channel expression as well as Na, K-ATPase expression and activity in vivo and in vitro. In addition, PCTR1i also upregulated the expression of LYVE-1 in vivo. Besides that, BOC-2, HK7, and LY294002 blocked the promoted effect of PCTR1 on pulmonary fluid clearance. Taken together, PCTR1 upregulates sodium channels' expression via activating the ALX/cAMP/P-Akt/Nedd4-2 pathway and increases Na, K-ATPase expression and activity to promote alveolar fluid clearance. Moreover, PCTR1 also promotes the expression of LYVE-1 to recover the lymphatic drainage resulting in the increase of lung interstitial fluid clearance. In summary, these results highlight a novel systematic mechanism for PCTR1 in pulmonary edema fluid clearance after ALI/ARDS, suggesting its potential role in a therapeutic approach for ALI/ARDS.

Relating GPI-Anchored Ly6 Proteins uPAR and CD59 to Viral Infection.

The Ly6 (lymphocyte antigen-6)/uPAR (urokinase-type plasminogen activator receptor) superfamily protein is a group of molecules that share limited sequence homology but conserved three-fingered structures. Despite diverse cellular functions, such as in regulating host immunity, cell adhesion, and migration, the physiological roles of these factors in vivo remain poorly characterized. Notably, increasing research has focused on the interplays between Ly6/uPAR proteins and viral pathogens, the results of which have provided new insight into viral entry and virus-host interactions. While LY6E (lymphocyte antigen 6 family member E), one key member of the Ly6E/uPAR-family proteins, has been extensively studied, other members have not been well characterized. Here, we summarize current knowledge of Ly6/uPAR proteins related to viral infection, with a focus on uPAR and CD59. Our goal is to provide an up-to-date view of the Ly6/uPAR-family proteins and associated virus-host interaction and viral pathogenesis.

Characterization of a CD59 in orange-spotted grouper (Epinephelus coioides).

CD59, a multifunctional glycoprotein, not only plays a regulatory role in complement cascades, but also participates in modulation of teleostean immunity. In this study, full length sequence of EcCD59 was obtained, comprising a 5'UTR of 163 bp, an ORF of 354 bp and a 3'UTR of 559 bp. EcCD59 gene encoded a polypeptide of 117 amino acids. Tissue-specific analysis revealed that the highest expression of EcCD59 mRNA was observed in muscle. Vibrio alginolyticus challenge can significantly increase EcCD59 mRNA expression in liver, kidney and spleen. EcCD59 distribution was detected by a combined approach using GFP-overexpression, immunofluorescence and ELISA assay, indicating that EcCD59 may be predominantly aggregated in cellular membrane. Both EcCD59 and EcCD59delGPI can directly bind to V. alginolyticus and decrease the in vitro growth of V. alginolyticus. Additionally, vibrio injection experiment indicated that the binding of EcCD59 or EcCD59delGPI to V. alginolyticus can restrict its growth rate in vivo. In this study, we found that EcCD59 may be involved in immune defense against vibrio infection in a complement-independent manner.

CHARMM-GUI Membrane Builder for Complex Biological Membrane Simulations with Glycolipids and Lipoglycans.

Glycolipids (such as glycoglycerolipids, glycosphingolipids, and glycosylphosphatidylinositol) and lipoglycans (such as lipopolysaccharides (LPS), lipooligosaccharides (LOS), mycobacterial lipoarabinomannan, and mycoplasma lipoglycans) are typically found on the surface of cell membranes and play crucial roles in various cellular functions. Characterizing their structure and dynamics at the molecular level is essential to understand their biological roles, but systematic generation of glycolipid and lipoglycan structures is challenging because of great variations in lipid structures and glycan sequences (i.e., carbohydrate types and their linkages). To facilitate the generation of all-atom glycolipid/LPS/LOS structures, we have developed Glycolipid Modeler and LPS Modeler in CHARMM-GUI ( http://www.charmm-gui.org ), a web-based interface that simplifies building of complex biological simulation systems. In addition, we have incorporated these modules into Membrane Builder so that users can readily build a complex symmetric or asymmetric biological membrane system with various glycolipids and LPS/LOS. These tools are expected to be useful in innovative and novel glycolipid/LPS/LOS modeling and simulation research by easing tedious and intricate steps in modeling complex biological systems and shall provide insight into structures, dynamics, and underlying mechanisms of complex glycolipid-/LPS-/LOS-containing biological membrane systems.

Unraveling of Lipid Raft Organization in Cell Plasma Membranes by Single-Molecule Imaging of Ganglioside Probes.

Ganglioside s are involved in a variety of physiological roles and particularly in the formation and function of lipid rafts in cell membranes. However, the dynamic behaviors of gangliosides have not been investigated in living cells owing to the lack of fluorescent probes that behave like their parental molecules. This has recently been resolved by developing new fluorescent ganglioside analogues that act similarly to their parental molecules, synthesized by only chemical methods. We performed single fluorescent-molecule imaging and revealed that ganglioside probes dynamically enter and exit rafts containing CD59, a glycosylphosphatidylinositol (GPI)-anchored protein, both before and after stimulation. The residency time of our ganglioside probes in CD59 oligomers was 48 ms after stimulation. The residency times in CD59 homodimer and monomer rafts were 40 and 12 ms, respectively. These results reveal the first direct evidence that GPI-anchored receptors and gangliosides interact in a cholesterol-dependent manner. Furthermore, they demonstrate that gangliosides continually move in and out of rafts that contain CD59 in an extremely dynamic manner and at a much higher frequency than expected. In this chapter, we review methods for the development and single-molecule imaging of new fluorescent ganglioside analogues and discuss how raft domains are formed, both before and after receptor engagement.

Cell-Derived Viral Genes Evolve under Stronger Purifying Selection in Rhadinoviruses.

Like many other large double-stranded DNA (dsDNA) viruses, herpesviruses are known to capture host genes to evade host defenses. Little is known about the detailed natural history of such genes, nor do we fully understand their evolutionary dynamics. A major obstacle is that they are often highly divergent, maintaining very low sequence similarity to host homologs. Here we use the herpesvirus genus Rhadinovirus as a model system to develop an analytical approach that combines complementary evolutionary and bioinformatic techniques, offering results that are both detailed and robust for a range of genes. Using a systematic phylogenetic strategy, we identify the original host lineage of viral genes with high confidence. We show that although host immunomodulatory genes evolve rapidly compared to other host genes, they undergo a clear increase in purifying selection once captured by a virus. To characterize this shift in detail, we developed a novel technique to identify changes in selection pressure that can be attributable to particular domains. These findings will inform us on how viruses develop strategies to evade the immune system, and our synthesis of techniques can be reapplied to other viruses or biological systems with similar analytical challenges.IMPORTANCE Viruses and hosts have been shown to capture genes from one another as part of the evolutionary arms race. Such genes offer a natural experiment on the effects of evolutionary pressure, since the same gene exists in vastly different selective environments. However, sequences of viral homologs often bear little similarity to the original sequence, complicating the reconstruction of their shared evolutionary history with host counterparts. In this study, we use a genus of herpesviruses as a model system to comprehensively investigate the evolution of host-derived viral genes, using a synthesis of genomics, phylogenetics, selection analysis, and nucleotide and amino acid modeling.

Determination of the Membrane Environment of CD59 in Living Cells.

The organization and dynamics of proteins and lipids in the plasma membrane, and their role in membrane functionality, have been subject of a long-lasting debate. Specifically, it is unclear to what extent membrane proteins are affected by their immediate lipid environment and vice versa. Studies on model membranes and plasma membrane vesicles indicated preferences of proteins for lipid phases characterized by different acyl chain order; however, whether such phases do indeed exist in live cells is still not known. Here, we refine a previously developed micropatterning approach combined with single molecule tracking to quantify the influence of the glycosylphosphatidylinositol-anchored (GPI-anchored) protein CD59 on its molecular environment directly in the live cell plasma membrane. We find that locally enriched and immobilized CD59 presents obstacles to the diffusion of fluorescently labeled lipids with a different phase-partitioning behavior independent of cell cholesterol levels and type of lipid. Our results give no evidence for either specific binding of the lipids to CD59 or the existence of nanoscopic ordered membrane regions associated with CD59.

PDn-3 DPA Pathway Regulates Human Monocyte Differentiation and Macrophage Function.

Macrophages are central in orchestrating the clearance of apoptotic cells and cellular debris during inflammation, with the mechanism(s) regulating this process remaining of interest. Herein, we found that the n-3 docosapentaenoic acid-derived protectin (PDn-3 DPA) biosynthetic pathway regulated the differentiation of human monocytes, altering macrophage phenotype, efferocytosis, and bacterial phagocytosis. Using lipid mediator profiling, human primary cells and recombinant enzymes we found that human 15-lipoxygenases initiate the PDn-3 DPA pathway catalyzing the formation of an allylic epoxide. The complete stereochemistry of this epoxide was determined using stereocontrolled total organic synthesis as 16S,17S-epoxy-7Z,10Z,12E,14E,19Z-docosapentaenoic acid (16S,17S-ePDn-3 DPA). This intermediate was enzymatically converted by epoxide hydrolases to PD1n-3 DPA and PD2n-3 DPA, with epoxide hydrolase 2 converting 16S,17S-ePDn-3 DPA to PD2n-3 DPA in human monocytes. Taken together these results establish the PDn-3 DPA biosynthetic pathway in human monocytes and macrophages and its role in regulating macrophage resolution responses.

Structural Analysis of CD59 of Chinese Tree Shrew: A New Reference Molecule for Human Immune System Specific CD59 Drug Discovery.

BACKGROUND: Chinese tree shrews (Tupaia belangeri chinensis) bear several characteristics that are considered to be very crucial for utilizing in animal experimental models in biomedical research. Subsequent to the identification of key aspects and signaling pathways in nervous and immune systems, it is revealed that tree shrews acquire common as well as unique characteristics, and hence offer a genetic basis for employing them as a prospective model for biomedical research. CD59 glycoprotein, commonly referred to as MAC-inhibitory protein (MAC-IP), membrane inhibitor of reactive lysis (MIRL), or protectin, is encoded by the CD59 gene in human beings. It is the member of the LY6/uPAR/alpha-neurotoxin protein family. OBJECTIVES: With this initial point, the objective of this study was to determine a comparative composite based structure of CD59 of Chinese tree shrew. The additional objective of this study was to examine the distribution of negatively and positively charged amino acid over molecular modeled structure, distribution of secondary structural elements, hydrophobicity molecular surface analysis and electrostatic potential analysis with the assistance of several bioinformatical analytical tools. METHODS: CD59 Amino acid sequence of Chinese tree shrew was collected from the online database system of National Centre for Biotechnology Information. SignalP 4.0 online server was employed for detection of signal peptide instance within the protein sequence of CD59. Molecular model structure of CD59 protein was generated by the Iterative Threading ASSEmbly Refinement (I-TASSER) suite. The confirmation for three-dimensional structural model was evaluated by structure validation tools. Location of negatively and positively charged amino acid over molecular modeled structure, distribution of secondary structural elements, and hydrophobicity molecular surface analysis was performed with the help of Chimera tool. Electrostatic potential analysis was carried out with the adaptive Poisson-Boltzmann solver package. Subsequently validated model was used for the functionally critical amino acids and active site prediction. The functionally critical amino acids and ligand- binding site (LBS) of the proteins (modeled) were determined using the COACH program. RESULT: Analysis of Ramachandran plot for Chinese tree shrew depicted that overall, 100% of the residues in homology model were observed in allowed and favored regions, sequentially leading to the validation of the standard of generated protein structural model. In case of CD59 of Chinese tree shrew, the total score of G-factor was found to be -0.66 that was generally larger than the acceptable value. This approach suggests the significance and acceptability of the modeled structure of CD59 of Chinese tree shrew. The molecular model data in cooperation to other relevant post model analysis data put forward molecular insight into protecting activity of CD59 protein molecule of Chinese tree shrew. CONCLUSION: In the present study, we have proposed the first molecular model structure of uncharted CD59 of Chinese tree shrew by significantly utilizing the comparative composite modeling approach. Therefore, the development of a structural model of the CD59 protein was carried out and analyzed further for deducing molecular enrichment technique. The collaborative effort of molecular model and other relevant data of post model analysis carry forward molecular understanding to protecting activity of CD59 functions towards better insight of features of this natural lead compound.

Development of new ganglioside probes and unraveling of raft domain structure by single-molecule imaging.

Gangliosides are involved in a variety of biological roles and are a component of lipid rafts found in cell plasma membranes (PMs). Gangliosides are especially abundant in neuronal PMs and are essential to their physiological functions. However, the dynamic behaviors of gangliosides have not been investigated in living cells due to a lack of fluorescent probes that behave like their parental molecules. We have recently developed, using an entirely chemical method, four new ganglioside probes (GM1, GM2, GM3, and GD1b) that act similarly to their parental molecules in terms of raft partitioning and binding affinity. Using single fluorescent-molecule imaging, we have found that ganglioside probes dynamically enter and leave rafts featuring CD59, a GPI-anchored protein. This occurs both before and after stimulation. The residency time of our ganglioside probes in rafts with CD59 oligomers was 48ms, after stimulation. The residency times in CD59 homodimer and monomer rafts were 40ms and 12ms, respectively. In this review, we introduce an entirely chemical-based ganglioside analog synthesis method and describe its application in single-molecule imaging and for the study of the dynamic behavior of gangliosides in cell PMs. Finally, we discuss how raft domains are formed, both before and after receptor engagement. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa.

Differences in proteomic profiles of milk fat globule membrane in yak and cow milk.

Milk fat globule membrane (MFGM) is an important milk component which is rich in bioactive proteins. In this work, the isobaric tags for relative and absolute quantitation (iTRAQ) proteomic approach was used to investigate the differences in the MFGM proteins between yak and cow milk. Over 450 proteins were identified between the yak and cow MFGM. The MFGM proteins with significant differences were compared based on the relative abundance. Proteins such as Glycosylation-dependent cell adhesion molecule 1 (GlyCAM1), CD59 molecule and lactadherin, were identified having a much higher abundance (4.6-10.1 fold) in yak MFGM than cow MFGM. These proteins are thought to have biological functions such as the antimicrobial and antitumor effects. This may be due to the need that yak produces high nutritive milk including high levels of bioactive compounds in order to resist the extreme high altitude environment.

Disentangling the roles of cholesterol and CD59 in intermedilysin pore formation.

The plasma membrane provides an essential barrier, shielding a cell from the pressures of its external environment. Pore-forming proteins, deployed by both hosts and pathogens alike, breach this barrier to lyse target cells. Intermedilysin is a cholesterol-dependent cytolysin that requires the human immune receptor CD59, in addition to cholesterol, to form giant ß-barrel pores in host membranes. Here we integrate biochemical assays with electron microscopy and atomic force microscopy to distinguish the roles of these two receptors in mediating structural transitions of pore formation. CD59 is required for the specific coordination of intermedilysin (ILY) monomers and for triggering collapse of an oligomeric prepore. Movement of Domain 2 with respect to Domain 3 of ILY is essential for forming a late prepore intermediate that releases CD59, while the role of cholesterol may be limited to insertion of the transmembrane segments. Together these data define a structural timeline for ILY pore formation and suggest a mechanism that is relevant to understanding other pore-forming toxins that also require CD59.

Structural Basis for Receptor Recognition by the Human CD59-Responsive Cholesterol-Dependent Cytolysins.

Cholesterol-dependent cytolysins (CDCs) are a family of pore-forming toxins that punch holes in the outer membrane of eukaryotic cells. Cholesterol serves as the receptor, but a subclass of CDCs first binds to human CD59. Here we describe the crystal structures of vaginolysin and intermedilysin complexed to CD59. These studies, together with small-angle X-ray scattering, reveal that CD59 binds to each at different, though overlapping, sites, consistent with molecular dynamics simulations and binding studies. The CDC consensus undecapeptide motif, which for the CD59-responsive CDCs has a proline instead of a tryptophan in the motif, adopts a strikingly different conformation between the structures; our data suggest that the proline acts as a selectivity switch to ensure CD59-dependent CDCs bind their protein receptor first in preference to cholesterol. The structural data suggest a detailed model of how these water-soluble toxins assemble as prepores on the cell surface.

Immune Protection of Retroviral Vectors Upon Molecular Painting with the Complement Regulatory Protein CD59.

Glycosylphosphatidylinositol anchoring is a type of post-translational modification that allows proteins to be presented on the exterior side of the cell membrane. Purified glycosylphosphatidylinositol-anchored protein can spontaneously re-insert into lipid bilayer membranes in a process termed Molecular Painting. Here, we demonstrate the possibility of inserting purified, recombinant CD59 into virus particles produced from a murine retroviral producer cell line. CD59 is a regulator of the complement system that helps protect healthy cells from the lytic activity of the complement cascade. In this study, we could show that Molecular Painting confers protection from complement activity upon murine retroviral vector particles. Indeed, increased infectivity of CD59-modified virus particles was observed upon challenge with human serum, indicating that Molecular Painting is suitable for modulating the immune system in gene therapy or vaccination applications.

Raft-based interactions of gangliosides with a GPI-anchored receptor.

Gangliosides, glycosphingolipids containing one or more sialic acid(s) in the glyco-chain, are involved in various important physiological and pathological processes in the plasma membrane. However, their exact functions are poorly understood, primarily because of the scarcity of suitable fluorescent ganglioside analogs. Here, we developed methods for systematically synthesizing analogs that behave like their native counterparts in regard to partitioning into raft-related membrane domains or preparations. Single-fluorescent-molecule imaging in the live-cell plasma membrane revealed the clear but transient colocalization and codiffusion of fluorescent ganglioside analogs with a fluorescently labeled glycosylphosphatidylinisotol (GPI)-anchored protein, human CD59, with lifetimes of 12 ms for CD59 monomers, 40 ms for CD59's transient homodimer rafts in quiescent cells, and 48 ms for engaged-CD59-cluster rafts, in cholesterol- and GPI-anchoring-dependent manners. The ganglioside molecules were always mobile in quiescent cells. These results show that gangliosides continually and dynamically exchange between raft domains and the bulk domain, indicating that raft domains are dynamic entities.

Adeno-associated virus mediated delivery of an engineered protein that combines the complement inhibitory properties of CD46, CD55 and CD59.

BACKGROUND: A variety of disorders are associated with the activation of complement. CD46, CD55 and CD59 are the major membrane associated regulators of complement on human cells. Previously, we have found that independent expression of CD55, CD46 or CD59 through gene transfer protects murine tissues against human complement mediated attack. In the present study, we investigated the potential of combining the complement regulatory properties of CD46, CD55 and CD59 into single gene products expressed from an adeno-associated virus (AAV) vector in a soluble non-membrane anchored form. METHODS: Minigenes encoding the complement regulatory domains from CD46, CD55 and CD59 (SACT) or CD55 and CD59 (DTAC) were cloned into an AAV vector. The specific regulatory activity of each component of SACT and DTAC was measured in vitro. The recombinant AAV vectors were injected into the peritoneum of mice and the efficacy of the transgene products for being able to protect murine liver vasculature against human complement, specifically the membrane attack complex (MAC), was measured. RESULTS: SACT and DTAC exhibited properties similar to CD46, CD55 and CD59 or CD55 and CD59, respectively, in vitro. AAV mediated delivery of SACT or DTAC protected murine liver vasculature from human MAC deposition by 63.2% and 56.7%, respectively. CONCLUSIONS: When delivered to mice in vivo via an AAV vector, SACT and DTAC are capable of limiting human complement mediated damage. SACT and DTAC merit further study as potential therapies for complement mediated disorders when delivered via a gene therapy approach.

Differential mobility separation of leukotrienes and protectins.

Differential mobility spectrometry (DMS) is capable of separating stereoisomeric molecular ions based on their mobility in an oscillating electrical field with an asymmetric waveform. Thus, it is an "orthogonal" technique to chromatography and (tandem) mass spectrometry. Bioactive lipids, particularly of the eicosanoid and docosanoid class feature numerous stereoisomers, which exhibit a highly specific structure-activity relationship. Moreover, the geometry of these compounds also reflects their biochemical origin. Therefore, the unambiguous characterization of related isomers of the eicosanoid and docosanoid classes is of fundamental importance to the understanding of their origin and function in many biological processes. Here we show, that SelexION DMS technology coupled to µLC-MS/MS is capable of differentiating at least five closely related leukotrienes partially coeluting and (almost) unresolvable using LC-MS/MS only. We applied the developed method to the separation of LTB4 and its coeluting isomer 5S,12S-diHETE in murine peritoneal exudate cells, showing that LTB4 is present only after zymosan A injection while its isomer 5S,12S-diHETE is produced after saline (PBS) administration. Additionally, we show that the SelexION technology can also be applied to the separation of PD1 and PDX (10S,17S-diHDHA), two isomeric protectins.

GPI-anchored proteins do not reside in ordered domains in the live cell plasma membrane.

The organization of proteins and lipids in the plasma membrane has been the subject of a long-lasting debate. Membrane rafts of higher lipid chain order were proposed to mediate protein interactions, but have thus far not been directly observed. Here we use protein micropatterning combined with single-molecule tracking to put current models to the test: we rearranged lipid-anchored raft proteins (glycosylphosphatidylinositol(GPI)-anchored-mGFP) directly in the live cell plasma membrane and measured the effect on the local membrane environment. Intriguingly, this treatment does neither nucleate the formation of an ordered membrane phase nor result in any enrichment of nanoscopic-ordered domains within the micropatterned regions. In contrast, we find that immobilized mGFP-GPIs behave as inert obstacles to the diffusion of other membrane constituents without influencing their membrane environment over distances beyond their physical size. Our results indicate that phase partitioning is not a fundamental element of protein organization in the plasma membrane.