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In plants, the recognition of external and internal stimuli is mediated by plasma membrane-localized receptors, which are heterogeneously distributed and organized in nanodomains. How their formation is regulated and how the specificity of multiprotein receptor complexes is determined, remains unclear. So far, the formation and the maintenance of multiprotein receptor complexes is largely unknown, though it turns out that the spatiotemporal organization and the nanoscale dynamics of membrane proteins is a fundamental aspect of the function of signaling processes. In this work, the function and the involvement of HIR2, a SPFH-domain containing membrane localized protein, which is hypothesized to be involved in the regulation of complex assembly of immune receptor kinases at the plasma membrane, was investigated by genetic and biochemical approaches. The direct interaction of HIR2 with the BIR2 and BIR3 was confirmed by multiple independent interaction experiments. Also, it was demonstrated that HIR2 directly interacts with the co-receptor BAK1, as well as with the PRRs FLS2 and CERK1. Besides the interactions, it remains to be elucidated how HIR2 is involved in PTI signaling. Additionally, HIR2 is suggested to be involved in developmental signaling pathways since it is associated with BRI1, and the hir2-5 mutant exhibits a dwarf growth phenotype, similar to weak bri1 alleles. To study the nanoscale dynamics and cluster formation of plasma membrane localized proteins, single and dual color sptPALM were successfully established for their application in planta and the nanodomain formation was computed by Voronoi tessellation-based segmentation. The analysis revealed a heterogeneous distribution and cluster formation of all studied proteins within the plasma membrane. The receptor kinases FLS2 and BIR3 possessed low lateral mobility within the plasma membrane, which was unaffected by flg22 treatment. In contrast, BAK1 occurred in two different populations with distinguishable lateral mobility behaviors, and flg22 treatment resulted in a reduced mobility of BAK1. Thus, it is hypothesized that BAK1 dynamically associates with different nanodomains and it is not permanently present in a preformed complex with FLS2. With this work, new insights were given (i) into the impact of HIR2 as a putative organizing factor for the assembly of spatially and temporally stable multiprotein complexes at the plasma membrane and (ii) into the spatiotemporal organization of immune receptor kinases and their modulation by ligand perception. Die Dissertation ist gesperrt bis zum 30. November 2024 !!

Lateral organization in the plane of the plasma membrane is an important driver of biological processes. The past dozen years have seen increasing experimental support for the notion that lipid organization plays an important role in modulating this heterogeneity. Various biophysical mechanisms rooted in the concept of liquid-liquid phase separation have been proposed to explain diverse experimental observations of heterogeneity in model and cell membranes, with distinct but overlapping applicability. In this review, we focus on evidence for and consequences of the hypothesis that the plasma membrane is poised near an equilibrium miscibility critical point. Critical phenomena explain certain features of the heterogeneity observed in cells and model systems, but also go beyond heterogeneity to predict other interesting phenomena, including responses to composition perturbations., Comment: 49 pages, 4 Figures

Virulence-associated glycolipids from Mycobacterium tuberculosis (Mtb) act as effector molecules during infection-in addition to proteins. Upon insertion, they alter the host cell's membrane properties modifying the host's functions to aid Mtb survival and disease course. Here we combine tether force experiments and microscopy to reveal previously unknown insights on the potential involvement of the phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) lipid in the Mtb lipid-host interaction landscape. Our data shows that Mtb lipids, having different structural and chemical make-up, distinctly alter a host's PI(4,5)P2 membrane abundance/organization and PI(4,5)P2-actin colocalization, thus impacting the plasma membrane-cytoskeletal adhesion forces. Combined with our previous findings that underscore the role of exogenous Mtb lipids in remodeling host plasma membrane organization and mechanics, this work builds upon a lipid-centric view of tubercular infections. Dynamically changing a host's plasma membrane lipid content - in response to virulent lipids - might represent a so far unexplored mechanism invoked by Mtb to modulate the host cell's adhesive properties to escape immune surveillance. These findings will deepen our collective understanding of the functional role of Mtb lipids in hijacking the host cell processes amenable to pharmacological inhibition.

Our understanding of the plasma membrane structure has undergone a major change since the proposal of the fluid mosaic model of Singer and Nicholson in the 1970s. In this model, the membrane, composed of over thousand lipid and protein species, is organized as a well‐equilibrated two‐dimensional fluid. Here, the distribution of lipids is largely expected to reflect a multicomponent system, and proteins are expected to be surrounded by an annulus of specialized lipid species. With the recognition that a multicomponent lipid membrane is capable of phase segregation, the membrane is expected to appear as patchwork quilt pattern of membrane domains. However, the constituents of a living membrane are far from being well equilibrated. The living cell membrane actively maintains a trans‐bilayer asymmetry of composition, and its constituents are subject to a number of dynamic processes due to synthesis, lipid transfer as well as membrane traffic and turnover. Moreover, membrane constituents engage with the dynamic cytoskeleton of a living cell, and are both passively as well as actively manipulated by this engagement. The extracellular matrix and associated elements also interact with membrane proteins contributing to another layer of interaction. At the nano‐ and mesoscale, the organization of lipids and proteins emerge from these encounters, as well as from protein–protein, protein–lipid, and lipid–lipid interactions in the membrane. New methods to study the organization of membrane components at these scales have also been developed, and provide an opportunity to synthesize a new picture of the living cell surface as an active membrane composite.

International audience; Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are lipid-associated luminal secretory cargoes selectively sorted to the apical surface of the epithelia where they reside and play diverse vital functions. Cholesterol-dependent clustering of GPI-APs in the Golgi is the key step driving their apical sorting and their further plasma membrane organization and activity; however, the specific machinery involved in this Golgi event is still poorly understood. In this study, we show that the formation of GPI-AP homoclusters (made of single GPI-AP species) in the Golgi relies directly on the levels of calcium within cisternae. We further demonstrate that the TGN calcium/manganese pump, SPCA1, which regulates the calcium concentration within the Golgi, and Cab45, a calcium-binding luminal Golgi resident protein, are essential for the formation of GPI-AP homoclusters in the Golgi and for their subsequent apical sorting. Down-regulation of SPCA1 or Cab45 in polarized epithelial cells impairs the oligomerization of GPI-APs in the Golgi complex and leads to their missorting to the basolateral surface. Overall, our data reveal an unexpected role for calcium in the mechanism of GPI-AP apical sorting in polarized epithelial cells and identify the molecular machinery involved in the clustering of GPI-APs in the Golgi.

Cells in tissues experience a plethora of forces that regulate their fate and modulate development and homeostasis. Cells sense mechanical cues through localized mechanoreceptors or by influencing cytoskeletal or plasma membrane organization. Cells translate force and modulate their behavior through a process termed mechanotransduction. Cells tune their tension upon exposure to chronic force by engaging cellular machinery that modulates actin tension, which in turn stimulates matrix remodeling and stiffening and alters cell-cell adhesions until cells achieve a state of tensional homeostasis. Loss of tensional homeostasis can be induced through oncogene activity and/or tissue fibrosis, accompanies tumor progression, and is associated with increased cancer risk. The mechanical stresses that develop in tumors can also foster the mesenchymal-like transdifferentiation of cells to induce a stem-like phenotype that contributes to their aggression, metastatic dissemination, and treatment resistance. Thus, strategies that ameliorate tumor mechanics may comprise an effective strategy to prevent aggressive tumor behavior.

Visualization of the nanoscale organization of cell membranes remains challenging because of the lack of appropriate fluorescent probes. Here, we introduce a new design concept for super-resolution microscopy probes that combines specific membrane targeting, ON/OFF switching, and environment sensing functions. In this design, we propose a functionalization strategy for solvatochromic dye Nile Red that improves its photostability. The dye is grafted to a newly developed membrane-targeting moiety composed of a sulfonate group and an alkyl chain of varied lengths, which ensures high brightness and lipid-order sensitivity of the probes and tunes their binding/unbinding-induced ON/OFF switching required for single-molecule localization microscopy. While the longchain probe with strong membrane binding, NR12A, is suitable for conventional microscopy, the short-chain probe NR4A, due to the reversible binding, enables first nanoscale cartography of the lipid order exclusively at the surface of live cells. The latter probe reveals in plasma membranes the presence of nanoscopic protrusions and invaginations of lower lipid order, suggesting a subtle connection between membrane morphology and lipid organization.

International audience; The plasma membrane (PM) is the biological membrane that separates the interior of all cells from the outside. The PM is constituted of a huge diversity of proteins and lipids. In this review, we will update the diversity of molecular species of lipids found in plant PM. We will further discuss how lipids govern global properties of the plant PM, explaining that plant lipids are unevenly distributed and are able to organize PM in domains. From that observation, it emerges a complex picture showing a spatial and multiscale segregation of PM components. Finally, we will discuss how lipids are key players in the function of PM in plants, with a particular focus on plant-microbe interaction, transport and hormone signaling, abiotic stress responses, plasmodesmata function. The last chapter is dedicated to the methods that the plant membrane biology community needs to develop to get a comprehensive understanding of membrane organization in plants.

Antigen (Ag) crosslinking of immunoglobulin E-receptor (IgE-FcεRI) complexes in mast cells stimulates transmembrane (TM) signaling, requiring phosphorylation of the clustered FcεRI by lipid-anchored Lyn tyrosine kinase. Previous studies showed that this stimulated coupling between Lyn and FcεRI occurs in liquid ordered (Lo)-like nanodomains of the plasma membrane and that Lyn binds directly to cytosolic segments of FcεRI that it initially phosphorylates for amplified activity. Net phosphorylation above a non-functional threshold is achieved in the stimulated state, but not in the resting state, and current evidence supports the hypothesis that this relies on disruption by Ag-crosslinking of a balance between Lyn and tyrosine phosphatase activities. However, the structural interactions that underlie the stimulation process remain poorly defined. This study evaluates the relative contributions and functional importance of different types of interactions leading to supra-threshold phosphorylation of Ag-crosslinked IgE-FcεRI in live rat basophilic leukemia (RBL) mast cells. Our high-precision diffusion measurements by Imaging Fluorescence Correlation Spectroscopy (ImFCS) on multiple structural variants of Lyn and other lipid-anchored probes confirm subtle, stimulated stabilization of the Lo-like nanodomains and concomitant sharpening of segregation from liquid-disordered (Ld)-like regions. With other structural variants we determine that lipid-based interactions are essential for access by Lyn leading to phosphorylation of and protein-based binding to clustered FcεRI. By contrast, TM tyrosine phosphatase, PTPα, is excluded from these regions by steric repulsion of TM segments and preference for Ld-like regions. Overall, we establish a synergy of lipid-based, protein-based, and steric interactions underlying functional TM signaling in mast cells.SIGNIFICANCE STATEMENTLipid organization of the plasma membrane is known to be important for facilitating protein interactions in transmembrane signaling. However, the orchestration of these interactions in live cells has been elusive. We employed ImFCS to systemically investigate the interplay of lipids and proteins during signaling in mast cells, initiated as phosphorylation of Ag-crosslinked IgE-FcεRI by lipid-anchored Lyn kinase. We find lipid-based interactions are first required for protein-based phosphorylation of the clustered FcεRI within Lo-like nanodomains. Transmembrane phosphatases must be excluded from these regions, and we find this is mediated by their preference for Ld-like regions and by steric exclusion from the clustered FcεRI proteins. ImFCS provides quantitative characterization of the functional link between features of plasma membrane organization and transmembrane signaling.

Contains fulltext : 244593.pdf (Publisher’s version ) (Open Access) Membrane protein organization is essential for proper cellular functioning and the result of a dynamic exchange between protein monomers, nanoscale protein clusters, and microscale higher-order structures. This exchange is affected by both lipid bilayer intrinsic factors, such as lipid rafts and tetraspanins, and extrinsic factors, such as cortical actin and galectins. Because membrane organizers act jointly like instruments in a symphony, it is challenging to define the 'key' organizers. Here, we posit, for the first time, definitions of key intrinsic and extrinsic membrane organizers. Tetraspanin nanodomains are key organizers that are often overlooked. We discuss how different key organizers can collaborate, which is important to get a full grasp of plasma membrane biology.

Here, biophysical properties of membranes enriched in three metabolically related sterols are analyzed both in vitro and in vivo. Unlike cholesterol and ergosterol, the common metabolic precursor zymosterol is unable to induce the formation of a liquid ordered (lo) phase in model lipid membranes and can easily accommodate in a gel phase. As a result, Zym has a marginal ability to modulate the passive membrane permeability of lipid vesicles with different compositions, contrary to cholesterol and ergosterol. Using fluorescence-lifetime imaging microscopy of an aminostyryl dye in living mammalian and yeast cells we established a close parallel between sterol-dependent membrane biophysical properties in vivo and in vitro. This approach unraveled fundamental differences in yeast and mammalian plasma membrane organization. It is often suggested that, in eukaryotes, areas that are sterol-enriched are also rich in sphingolipids, constituting highly ordered membrane regions. Our results support that while cholesterol is able to interact with saturated lipids, ergosterol seems to interact preferentially with monounsaturated phosphatidylcholines. Taken together, we show that different eukaryotic kingdoms developed unique solutions for the formation of a sterol-rich plasma membrane, a common evolutionary trait that accounts for sterol structural diversity.

Multiple sclerosis (MS) represents the most common demyelinating disease affecting the central nervous system (CNS) in adults as well as in children. Furthermore, in children, in addition to acquired diseases such as MS, genetically inherited diseases significantly contribute to the incidence of demyelinating disorders. Some genetic defects lead to sphingolipid alterations that are able to elicit neurological symptoms. Sphingolipids are essential for brain development, and their aberrant functionality may thus contribute to demyelinating diseases such as MS. In particular, sphingolipidoses caused by deficits of sphingolipid-metabolizing enzymes, are often associated with demyelination. Sphingolipids are not only structural molecules but also bioactive molecules involved in the regulation of cellular events such as development of the nervous system, myelination and maintenance of myelin stability. Changes in the sphingolipid metabolism deeply affect plasma membrane organization. Thus, changes in myelin sphingolipid composition might crucially contribute to the phenotype of diseases characterized by demyelinalization. Here, we review key features of several sphingolipids such as ceramide/dihydroceramide, sphingosine/dihydrosphingosine, glucosylceramide and, galactosylceramide which act in myelin formation during rat brain development and in human brain demyelination during the pathogenesis of MS, suggesting that this knowledge could be useful in identifying targets for possible therapies.

OBJECTIVES: The emerging obesity epidemic and its co-morbid conditions, such as colorectal cancer, pose a great challenge to global health and healthcare cost. This reinforces the urgent need to elucidate the underlying mechanisms contributing to the promotion of colon cancer in individuals with obesity. Extensive epidemiological studies have demonstrated a consistent and compelling association between obesity and the risk of cancer development and progression. Furthermore, our previous studies demonstrate that obesity induced by high-fat diet results in the expansion of the colonic stem cell pool during cancer initiation. However, the mechanistic link between obesity and the initiation and development of colon cancer remains elusive. Since the biophysical properties of the plasma membrane contribute to cellular signaling, we hypothesized that obesity alters colonic stem cell homeostasis via modulation of the biophysical properties of the plasma membrane, subsequently altering cell signaling events. METHODS: Our studies utilized complementary in vivo and ex vivo mouse models including colonic organoids. Plasma membrane biophysical properties and cholesterol content were determined utilizing the physico-chemical sensitive dye, Di-4-ANEPPDHQ and Filipin III, respectively. Super-resolution stochastic optical reconstruction microscopy (STORM) was utilized to determine the nanoscale clustering of Wnt receptor, LRP6, and epidermal growth factor receptor (EGFR). Effects of nanoclustering on the activation status of downstream signaling targets was subsequently assessed. RESULTS: High-fat diet induced obesity increased plasma membrane free cholesterol and rigidity of colonic crypts and Lgr5(+) cells. The increase in rigidity was associated with an increase in the nanoclustering of key stem cell regulating membrane-bound receptors, LRP6 and EGFR. These biophysical perturbations were associated with changes in stem cell signaling and organoid-forming efficiency, a measure of stemness. CONCLUSIONS: Our results indicate that obesity alters the biophysical properties of the plasma membrane which influences the clustering and downstream signaling of membrane receptors. These findings are noteworthy because they may explain in part how obesity drives colon cancer progression. FUNDING SOURCES: This work was supported by NIH grant R35CA197707.

International audience; The plasma membrane (PM) spatiotemporal organization is one of the major factors controlling cell signaling and whole-cell homeostasis. The PM lipids, including cholesterol, determine the physicochemical properties of the membrane bilayer and thus play a crucial role in all membrane-dependent cellular processes. It is known that lipid content and distribution in the PM are not random, and their transversal and lateral organization is highly controlled. Mainly sphingolipid- and cholesterol-rich lipid nanodomains, historically referred to as rafts, are extremely dynamic “hot spots” of the PM controlling the function of many cell surface proteins and receptors. In the first part of this review, we will focus on the recent advances of PM investigation and the current PM concept. In the second part, we will discuss the importance of several classes of ABC transporters whose substrates are lipids for the PM organization and dynamics. Finally, we will briefly present the significance of lipid ABC transporters for immune responses.

This study highlights that DSF, the quorum sensing signal produced by bacterial phytopathogens, is able to directly modulate plant plasma membrane dynamics by interfering with host lipid profile and thereby, suppress plant immunity responses., Quorum sensing (QS) is a recognized phenomenon that is crucial for regulating population-related behaviors in bacteria. However, the direct specific effect of QS molecules on host biology is largely understudied. In this work, we show that the QS molecule DSF (cis-11-methyl-dodecenoic acid) produced by Xanthomonas campestris pv. campestris can suppress pathogen-associated molecular pattern–triggered immunity (PTI) in Arabidopsis thaliana, mediated by flagellin-induced activation of flagellin receptor FLS2. The DSF-mediated attenuation of innate immunity results from the alteration of FLS2 nanoclusters and endocytic internalization of plasma membrane FLS2. DSF altered the lipid profile of Arabidopsis, with a particular increase in the phytosterol species, which impairs the general endocytosis pathway mediated by clathrin and FLS2 nano-clustering on the plasma membrane. The DSF effect on receptor dynamics and host immune responses could be entirely reversed by sterol removal. Together, our results highlighted the importance of sterol homeostasis to plasma membrane organization and demonstrate a novel mechanism by which pathogenic bacteria use their communicating molecule to manipulate pathogen-associated molecular pattern–triggered host immunity.

Cell shape morphogenesis from spherical to polygonal occurs in epithelial cell formation in metazoan embryogenesis. In syncytial Drosophila embryos, the plasma membrane incompletely surrounds each nucleus and is organized as a polygonal epithelial-like array. Each cortical syncytial division cycle shows circular to polygonal plasma membrane transition along with furrow extension between adjacent nuclei from interphase to metaphase. In this study, we assess the relative contribution of DE-cadherin and Myosin II at the furrow for polygonal shape transition. We show that polygonality initiates during each cortical syncytial division cycle when the furrow extends from 4.75 to 5.75 µm. Polygon plasma membrane organization correlates with increased junctional tension, increased DE-cadherin and decreased Myosin II mobility. DE-cadherin regulates furrow length and polygonality. Decreased Myosin II activity allows for polygonality to occur at a lower length than controls. Increased Myosin II activity leads to loss of lateral furrow formation and complete disruption of polygonal shape transition. Our studies show that DE-cadherin-Myosin II balance regulates an optimal lateral membrane length during each syncytial cycle for polygonal shape transition.

Aberrant lipid metabolism promotes the development of skeletal muscle insulin resistance, but the exact identity of lipid-mediated mechanisms relevant to human obesity remains unclear. A comprehensive lipidomic analysis of primary myocytes from individuals who were insulin-sensitive and lean (LN) or insulin-resistant with obesity (OB) revealed several species of lysophospholipids (lyso-PLs) that were differentially abundant. These changes coincided with greater expression of lysophosphatidylcholine acyltransferase 3 (LPCAT3), an enzyme involved in phospholipid transacylation (Lands cycle). Strikingly, mice with skeletal muscle-specific knockout of LPCAT3 (LPCAT3-MKO) exhibited greater muscle lysophosphatidylcholine/phosphatidylcholine, concomitant with improved skeletal muscle insulin sensitivity. Conversely, skeletal muscle-specific overexpression of LPCAT3 (LPCAT3-MKI) promoted glucose intolerance. The absence of LPCAT3 reduced phospholipid packing of cellular membranes and increased plasma membrane lipid clustering, suggesting that LPCAT3 affects insulin receptor phosphorylation by modulating plasma membrane lipid organization. In conclusion, obesity accelerates the skeletal muscle Lands cycle, whose consequence might induce the disruption of plasma membrane organization that suppresses muscle insulin action.

Aberrant lipid metabolism promotes the development of skeletal muscle insulin resistance, but the exact identity of lipid-mediated mechanisms relevant to human obesity remains unclear. A comprehensive lipidomic analyses of primary myocytes from lean insulin-sensitive (LN) and obese insulin-resistant (OB) individuals revealed several species of lysophospholipids (lyso-PL) that were differentially-abundant. These changes coincided with greater expression of lysophosphatidylcholine acyltransferase 3 (LPCAT3), an enzyme involved in phospholipid transacylation (Lands cycle). Strikingly, mice with skeletal muscle-specific knockout of LPCAT3 (LPCAT3-MKO) exhibited greater muscle lyso-PC/PC, concomitant with greater insulin sensitivityin vivoand insulin-stimulated skeletal muscle glucose uptakeex vivo. Absence of LPCAT3 reduced phospholipid packing of the cellular membranes and increased plasma membrane lipid clustering, suggesting that LPCAT3 affects insulin receptor phosphorylation by modulating plasma membrane lipid organization. In conclusion, obesity accelerates the skeletal muscle Lands cycle, whose consequence might induce the disruption of plasma membrane organization that suppresses muscle insulin action.

The role of lipid metabolism in epithelial stem cell (SC) function and carcinogenesis is poorly understood. The transcription factor Runx1 is known to regulate proliferation in mouse epithelial hair follicle (HF) SCs in vivo and in several mouse and human epithelial cancers. We found a novel subset of in vivo Runx1 HFSC target genes related to lipid metabolism and demonstrated changes in distinct classes of lipids driven by Runx1. Inhibition of lipid-enzymes Scd1 and Soat1 activity synergistically reduces proliferation of mouse skin epithelial cells and of human skin and oral squamous cell carcinoma cultured lines. Varying Runx1 levels induces changes in skin monounsaturated fatty acids (e.g., oleate, a product of Scd1) as shown by our lipidome analysis. Furthermore, varying Runx1 levels, the inhibition of Scd1, or the addition of Scd1-product oleate, individually affects the plasma membrane organization (or fluidity) in mouse keratinocytes. These factors also affect the strength of signal transduction through the membranes for Wnt, a pathway that promotes epithelial (cancer) cell proliferation and HFSC activation. Our working model is that HFSC factor Runx1 modulates the fatty acid production, which affects membrane organization, facilitating signal transduction for rapid proliferation of normal and cancer epithelial cells.

The cellular plasma membrane is the barrier over which cells exchange materials and communicate with their surroundings, and thus plays the central role in cellular sensing and metabolism. Therefore, the investigation of plasma membrane organization and dynamics is required for understanding of cellular functions. The plasma membrane is a heterogeneous matrix. The presence of structures such as lipid and protein domains and the cytoskeleton meshwork poses a hindrance to the free diffusion of membrane associated biomolecules. However, these domains and the cytoskeleton meshwork barriers are below the optical diffraction limit with potentially short lifetimes and are not easily detected even in super-resolution microscopy. Therefore, dynamic measurements are often used to indirectly prove the existence of domains and barriers by analyzing the mode of diffusion of probe molecules. One of these tools is the Fluorescence Correlation Spectroscopy (FCS) diffusion law. The FCS diffusion law is a plot of diffusion time (τd) versus observation area. For at least three different diffusive modes - free, domain confined, and meshwork hindered hop diffusion - the expected plots have been characterized, typically by its y-intercept (τ0) when fit with a linear model, and have been verified in many cases. However, a description of τ0 has only been given for pure diffusive modes. But in many experimental cases it is not evident that a protein will undergo only one kind of diffusion, and thus the interpretation of the τ0 value is problematic. Here, we therefore address the question about the absolute value of τ0 in the case of complex diffusive modes, i.e. when either one molecule is domain confined and cytoskeleton hindered or when two molecules exhibit different diffusive behavior at the same position in a sample. In addition, we investigate how τ0 changes when the diffusive mode of a probe alters upon disruption of domains or the cytoskeleton by drug treatments. By a combination of experimental studies and simulations, we show that τ0 is not influenced equally by the different diffusive modes as typically found in cellular environments, and that it is the relative change of τ0 rather than its absolute value that provides information on the mode of diffusion.

In this research we investigate the connection between the cytoplasmic machinery of apoptosis and the plasma membrane organization by studying the coupling of caspase-3 activation and inhibition with PS exposure and the change of lipid order in plasma membrane sensed by a fluorescent membrane probe NR12S. First, we performed in silico molecular dynamics simulations, which suggest that the mechanism of response of NR12S to lipid order may combine both sensitivity to membrane polarity/hydration and change in the fluorophore orientation. Second, cellular studies revealed that upon triggering apoptosis with IPA-3 and camptothecin the NR12S response is similar to that observed after decrease of lipid order induced by cholesterol depletion, 7-ketocholesterol enrichment or sphingomyelin hydrolysis. NR12S response can be influenced by a caspase-3 inhibitor Z-DEVD-FMK. Flow cytometry data further indicate that the NR12S response correlates with the response of FITC-labeled DEVD-FMK peptide and GFP-labeled Annexin V on the whole time scale (0–24 h) of apoptosis induction by camptothecin. We conclude that fine changes in lipid order observed by NR12S are coupled with early steps of cellular events in apoptosis.

Killer T cells (cytotoxic T lymphocytes, CTLs) maintain immune homoeostasis by eliminating virus-infected and cancerous cells. CTLs achieve this by forming an immunological synapse with their targets and secreting a pore-forming protein (perforin) and pro-apoptotic serine proteases (granzymes) into the synaptic cleft. Although the CTL and the target cell are both exposed to perforin within the synapse, only the target cell membrane is disrupted, while the CTL is invariably spared. How CTLs escape unscathed remains a mystery. Here, we report that CTLs achieve this via two protective properties of their plasma membrane within the synapse: high lipid order repels perforin and, in addition, exposed phosphatidylserine sequesters and inactivates perforin. The resulting resistance of CTLs to perforin explains their ability to kill target cells in rapid succession and to survive these encounters. Furthermore, these mechanisms imply an unsuspected role for plasma membrane organization in protecting cells from immune attack., Cytotoxic T lymphocytes (CTLs) eliminate virus-infected and cancerous cells by secreting the pore-forming protein (perforin) and pro-apoptotic serine proteases (granzymes). Here authors show that two mechanisms protect the membranes of CTLs from disruption by perforin and granzymes.

Background: Aspirin-exacerbated respiratory disease (AERD) is a syndrome characterized by rhinosinusitis nasal polyps asthma and aspirin intolerance. An imbalance of eicosanoid metabolism with overproduction of cysteinyl leukotrienes (CysLTs) has been associated with AERD however, the precise mechanisms underlying AERD are not well understood Aim: To establish the transcriptome of the nasal airway epithelial cells derived from AERD patients in order to uncover the genes patterns during this disease. Methods: Nasal airway epithelial cells were isolated from 12 AERD patients and 6 healthy controls. In the AERD group cells were grown from both the affected- (nasal polyps) and non-affected nasal mucosa. RNA was poly (A) selected and then sequenced with 25 million reads per sample on the Illumina HiSeq 2000 platform. Results: Comparative transcriptome profiling of nasal epithelial cells was successfully achieved in AERD (affected and non-affected nasal mucosa) and normal healthy controls: Twenty genes had twofold mean regulation expression differences or greater. Seven genes were upregulated, and thirteen genes were down-regulated. Differentially regulated genes included cell adhesion, inflammation, defense and immunity, protein binding and metabolic process. Significantly enriched pathways were plasma membrane organization, macroautophagy, leukotriene metabolic process, and embryonic development. Conclusion: the present study reveals novel genes transcripts involved in the disease pathogenesis which may become potential molecular targets for therapeutic intervention in AERD.

There are 2 populations of T lymphocytes, αβ T and γδ T cells, that can be distinguished by the expression of either an αβ TCR or a γδ TCR, respectively. Pairing of the Ag binding heterodimer, which consists of TCR-α/TCR-β (TCRαβ) or TCR-γ/TCR-δ (TCRγδ), with proteins of the CD3 complex forms the complete αβ or γδ TCR. Despite some similarities in the structure of TCRαβ and TCRγδ and the shared subunits of the CD3 complex, the 2 receptors differ in important aspects. These include the assembly geometry of the complex, the glycosylation pattern, the plasma membrane organization, as well as the accessibility of signaling motifs in the CD3 intracellular tails. These differences are reflected in the different demands and outcomes of ligand-induced signaling. It was shown that exposure of the proline-rich sequence (PRS) in CD3ε occurs with all activating αβ TCR ligands and is required to induce αβ TCR signaling. In sharp contrast, CD3ε PRS exposure was not induced by binding of those ligands to the γδ TCR that have been studied. Further, signaling by the γδ TCR occurs independently of CD3ε PRS exposure. Interestingly, it can be enhanced by anti-CD3ε Ab-induced enforcement of CD3ε PRS exposure. This review contrasts these two similar, but different immune receptors.

Epithelial cells have a polarised distribution of protein complexes on the lateral membrane and are present as a polygonal array dominated by hexagons. Metazoan embryogenesis enables the study of temporal formation of the polygonal array and mechanisms that regulate its distribution. The plasma membrane of the syncytialDrosophilablastoderm embryo is organized as a polygonal array during cortical division cycles with an apical membrane and lateral furrow in between adjacent nuclei. We find that polygonal plasma membrane organization arises in syncytial division cycle 11 and hexagon dominance occurs with increase in furrow length in cycle 12. This is coincident with DE-cadherin and Bazooka enrichment at edges and the septin, Peanut enrichment at vertices of the base of the furrow. DE-cadherin depletion leads to loss of hexagon dominance. Bazooka and Peanut depletion leads to a delay in occurrence of hexagon dominance from nuclear cycle 12 to 13. Hexagon dominance in Bazooka and Peanut mutants occurs with furrow extension and correlates with increase in DE-cadherin in syncytial cycle 13. We conclude that a change in polarity complex distribution leads to loss of furrow stability thereby changing the polygonal organization of the blastoderm embryo.Highlight Summary for TOCMetazoan embryogenesis starts with the formation of polygonal epithelial-like cells. We show that hexagon dominance in polygonal epithelial-like plasma membrane organization occurs in nuclear cycle 12 in the syncytial blastodermDrosophilaembryo. DE-cadherin and Bazooka distribution along the lateral furrow regulates this hexagon dominance.

Human endometrial epithelium (EE) is composed of a multitude of proteins, amongst which those localized on the plasma membrane [plasma membrane proteins (PMPs)] are of critical relevance in the early stages of implantation. Evidence supports the key role of few PMPs in implantation. However, many remain unidentified, as efforts have not been made till date to generate the plasma membrane proteome of human EE cells, using a gel-free approach. This study presents a protein catalog of the PMP enriched fraction of Ishikawa cell line; often used as an in vitro model for embryo-adhesive EE. Liquid chromatography with tandem mass spectrometry identified 3,598 proteins. Of these, 1,963 proteins were annotated for their membrane localization. Of 1,963 proteins, 1,321 were found to have a transmembrane domain and 43 proteins had glycophosphatidylinositol (GPI) anchor. Extensive data mining revealed endometrial expression of 943 proteins reported in humans and/or rodents. Further, quantitative alterations were observed in the plasma membrane proteome on the perturbation of intracellular trafficking. Silencing of Rab11a (known for its role in plasma membrane organization) expression caused alteration in the abundance of 74 proteins. Caveolin-1 and EpCAM levels were reduced whereas Rab4a abundance increased in the PMP extracts of Rab11a deficient cells, compared with control cells. Briefly, the study reports the identity of several novel plasma membrane-localized proteins. A major spin-off of the study is the identification of novel proteins trafficked by Rab11a to the plasma membrane. Targeted analysis of novel PMPs may reveal their specific roles in endometrial receptivity and implantation.

Inorganic polyphosphate (polyP) is crucial for adaptive reactions and stress response in microorganisms. A convenient model to study the role of polyP in yeast is the Saccharomyces cerevisiae strain CRN/PPN1 that overexpresses polyphosphatase Ppn1 with stably decreased polyphosphate level. In this study, we combined the whole-transcriptome sequencing, fluorescence microscopy, and polyP quantification to characterize the CRN/PPN1 response to manganese and oxidative stresses. CRN/PPN1 exhibits enhanced resistance to manganese and peroxide due to its pre-adaptive state observed in normal conditions. The pre-adaptive state is characterized by up-regulated genes involved in response to an external stimulus, plasma membrane organization, and oxidation/reduction. The transcriptome-wide data allowed the identification of particular genes crucial for overcoming the manganese excess. The key gene responsible for manganese resistance is PHO84 encoding a low-affinity manganese transporter: Strong PHO84 down-regulation in CRN/PPN1 increases manganese resistance by reduced manganese uptake. On the contrary, PHM7, the top up-regulated gene in CRN/PPN1, is also strongly up-regulated in the manganese-adapted parent strain. Phm7 is an unannotated protein, but manganese adaptation is significantly impaired in &Delta, phm7, thus suggesting its essential function in manganese or phosphate transport.

Differential regulation of the μ-opioid receptor (MOP) has been linked to the development of opioid tolerance and dependence which both limit the clinical use of opioid analgesics. At a cellular level, MOP regulation occurs via receptor phosphorylation, desensitization, plasma membrane redistribution, and internalization. Here, we used fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) to detect and quantify ligand-dependent changes in the plasma membrane organization of MOP expressed in human embryonic kidney (HEK293) cells. The low internalizing agonist morphine and the antagonist naloxone did not alter constitutive MOP plasma membrane organization. In contrast, the internalizing agonist DAMGO changed MOP plasma membrane organization in a pertussis toxin-insensitive manner and by two mechanisms. Firstly, it slowed MOP diffusion in a manner that was independent of internalization but dependent on GRK2/3. Secondly, DAMGO reduced the surface receptor number and the proportion of mobile receptors, and increased receptor clustering in a manner that was dependent on clathrin-mediated endocytosis. Overall, these results suggest the existence of distinct sequential MOP reorganization events at the plasma membrane and provide insights into the specific protein interactions that control MOP plasma membrane organization.

Rho GTPases play critical roles in cell proliferation and cell death in many species. As in animal cells, cells of the budding yeast Saccharomyces cerevisiae undergo regulated cell death under various physiological conditions and upon exposure to external stress. The Rho5 GTPase is necessary for oxidant-induced cell death, and cells expressing a constitutively active GTP-locked Rho5 are hypersensitive to oxidants. Yet how Rho5 regulates yeast cell death has been poorly understood. To identify genes that are involved in the Rho5-mediated cell death program, we performed two complementary genome-wide screens: one screen for oxidant-resistant deletion mutants and another screen for Rho5-associated proteins. Functional enrichment and interaction network analysis revealed enrichment for genes in pathways related to metabolism, transport, and plasma membrane organization. In particular, we find that ATG21, which is known to be involved in the CVT (Cytoplasm-to-Vacuole Targeting) pathway and mitophagy, is necessary for cell death induced by oxidants. Cells lacking Atg21 exhibit little cell death upon exposure to oxidants even when the GTP-locked Rho5 is expressed. Moreover, Atg21 interacts with Rho5 preferentially in its GTP-bound state, suggesting that Atg21 is a downstream target of Rho5 in oxidant-induced cell death. Given the high degree of conservation of Rho GTPases and autophagy from yeast to human, this study may provide insight into regulated cell death in eukaryotes in general.

The ability to resist copper toxicity is important for microbial pathogens to survive attack by innate immune cells. A sur7Δ mutant of the fungal pathogen Candida albicans exhibits decreased virulence that correlates with increased sensitivity to copper, as well as defects in other stress responses and morphogenesis. Previous studies indicated that copper kills sur7Δ cells by a mechanism distinct from the known resistance pathways involving the Crp1 copper exporter or the Cup1 metallothionein. Since Sur7 resides in punctate plasma membrane domains known as MCC/eisosomes, we examined overexpression of SUR7 and found that it rescued the copper sensitivity of a mutant that fails to form MCC/eisosomes (pil1Δ lsp1Δ), indicating that these domains act to facilitate Sur7 function. Genetic screening identified new copper-sensitive mutants, the strongest of which were similar to sur7Δ in having altered plasma membranes due to defects in membrane trafficking, cortical actin, and morphogenesis (rvs161Δ, rvs167Δ, and arp2Δ arp3Δ). Consistent with the mutants having altered plasma membrane organization, they were all more readily permeabilized by copper, which is known to bind phosphatidylserine and phosphatidylethanolamine and cause membrane damage. Although these phospholipids are normally localized to the intracellular leaflet of the plasma membrane, their exposure on the surface of the copper-sensitive mutants was indicated by increased susceptibility to membrane damaging agents that bind to these phospholipids. Increased copper sensitivity was also detected for a drs2Δ mutant, which lacks a phospholipid flippase that is involved in maintaining phospholipid asymmetry. Copper binds phosphatidylserine with very high affinity, and deleting CHO1 to prevent phosphatidylserine synthesis rescued the copper sensitivity of sur7Δ cells, confirming a major role for phosphatidylserine in copper sensitivity. These results highlight how proper plasma membrane architecture protects fungal pathogens from copper and attack by the immune system, thereby opening up new avenues for therapeutic intervention., Author summary The transition metal copper is used by the innate immune system to attack microbial pathogens. To better understand how the human fungal pathogen Candida albicans resists this type of stress, we screened for mutants that were more susceptible to killing by copper. Interestingly, we identified a new class of copper-sensitive mutants whose plasma membranes are more readily permeabilized by copper. The common characteristic of these new copper-sensitive mutants is that they have an altered cell surface, which weakened their resistance to copper. These results help to explain the toxic effects of copper and suggest novel therapeutic strategies for fungal infections.

Epidermal growth factor receptor (EGFR) signaling drives the formation of many types of cancer, including colon cancer. Docosahexaenoic acid (DHA, 22∶6Δ4,7,10,13,16,19), a chemoprotective long-chain n-3 polyunsaturated fatty acid suppresses EGFR signaling. However, the mechanism underlying this phenotype remains unclear. Therefore, we used super-resolution microscopy techniques to investigate the mechanistic link between EGFR function and DHA-induced alterations to plasma membrane nanodomains. Using isogenic in vitro (YAMC and IMCE mouse colonic cell lines) and in vivo (Drosophila, wild type and Fat-1 mice) models, cellular DHA enrichment via therapeutic nanoparticle delivery, endogenous synthesis, or dietary supplementation reduced EGFR-mediated cell proliferation and downstream Ras/ERK signaling. Phospholipid incorporation of DHA reduced membrane rigidity and the size of EGFR nanoclusters. Similarly, pharmacological reduction of plasma membrane phosphatidic acid (PA), phosphatidylinositol-4,5-bisphosphate (PIP2) or cholesterol was associated with a decrease in EGFR nanocluster size. Furthermore, in DHA-treated cells only the addition of cholesterol, unlike PA or PIP2, restored EGFR nanoscale clustering. These findings reveal that DHA reduces EGFR signaling in part by reshaping EGFR proteolipid nanodomains, supporting the feasibility of using membrane therapy, i.e., dietary/drug-related strategies to target plasma membrane organization, to reduce EGFR signaling and cancer risk., Summary Cellular membrane phospholipid enrichment of DHA suppresses EGFR-mediated phenotypes by reducing EGFR nanocluster formation across a variety of in vitro and in vivo models.

Whereas important discoveries made by single-particle tracking have changed our view of the plasma membrane organization and motor protein dynamics in the past three decades, experimental studies of intracellular processes using single-particle tracking are rather scarce because of the lack of three-dimensional (3D) tracking capacity. In this study we use a newly developed 3D single-particle tracking method termed TSUNAMI (Tracking of Single particles Using Nonlinear And Multiplexed Illumination) to investigate epidermal growth factor receptor (EGFR) trafficking dynamics in live cells at 16/43 nm ( xy / z ) spatial resolution, with track duration ranging from 2 to 10 min and vertical tracking depth up to tens of microns. To analyze the long 3D trajectories generated by the TSUNAMI microscope, we developed a trajectory analysis algorithm, which reaches 81% segment classification accuracy in control experiments (termed simulated movement experiments). When analyzing 95 EGF-stimulated EGFR trajectories acquired in live skin cancer cells, we find that these trajectories can be separated into three groups—immobilization (24.2%), membrane diffusion only (51.6%), and transport from membrane to cytoplasm (24.2%). When EGFRs are membrane-bound, they show an interchange of Brownian diffusion and confined diffusion. When EGFRs are internalized, transitions from confined diffusion to directed diffusion and from directed diffusion back to confined diffusion are clearly seen. This observation agrees well with the model of clathrin-mediated endocytosis.

Background Plasma membrane organization is a mechanistic target of n-3 (ω-3) polyunsaturated fatty acids. Previous studies show that eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) differentially disrupt plasma membrane molecular order to enhance the frequency and function of B lymphocytes. However, it is not known whether EPA and DHA affect the plasma membrane organization of B lymphomas differently to influence their function. Objective We tested whether EPA and DHA had different effects on membrane order in B lymphomas and liposomes and studied their effects on B-lymphoma growth. Methods B lymphomas were treated with 25 μmol EPA, DHA, or serum albumin control/L for 24 h. Membrane order was measured with fluorescence polarization, and cellular fatty acids (FAs) were analyzed with GC. Growth was quantified with a viability assay. (2)H nuclear magnetic resonance (NMR) studies were conducted on deuterated phospholipid bilayers. Results Treating Raji, Ramos, and RPMI lymphomas for 24 h with 25 μmol EPA or DHA/L lowered plasma membrane order by 10-40% relative to the control. There were no differences between EPA and DHA on membrane order for the 3 cell lines. FA analyses revealed complex changes in response to EPA or DHA treatment and a large fraction of EPA was converted to docosapentaenoic acid (DPA; 22:5n-3). NMR studies, which were used to understand why EPA and DHA had similiar membrane effects, showed that phospholipids containing DPA, similar to DHA, were more ordered than those containing EPA. Finally, treating B lymphomas with 25 μmol EPA or DHA/L did not increase the frequency of B lymphomas compared with controls. Conclusions The results establish that 25 μmol EPA and DHA/L equally disrupt membrane order and do not promote B lymphoma growth. The data open a new area of investigation, which is how EPA's conversion to DPA substantially moderates its influence on membrane properties.

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are a class of membrane proteins containing a soluble protein attached by a conserved glycolipid anchor to the external leaflet of the plasma membrane. In polarized epithelial cells, GPI-APs are predominantly sorted to the apical surface in the trans-Golgi network (TGN) by clustering in sphingolipid- and cholesterol-dependent microdomains (or rafts), which have been proposed to act as apical sorting platforms. Recent data indicate that the mechanisms of GPI-AP sorting, occurring in the Golgi, control both the membrane transport of GPI-APs and their specific activity at the apical surface of fully polarized epithelial cells. Here, we discuss the most recent findings and the factors regulating apical sorting of GPI-APs at the Golgi in polarized epithelial cells. We also underline the differences in the plasma membrane organization of GPI-APs between polarized and non-polarized cells supporting the existence of various mechanisms that control GPI-AP organization in different cell types.

Candida albicans is a human fungal pathogen capable of causing lethal systemic infections. The plasma membrane plays key roles in virulence because it not only functions as a protective barrier, it also mediates dynamic functions including secretion of virulence factors, cell wall synthesis, invasive hyphal morphogenesis, endocytosis, and nutrient uptake. Consistent with this functional complexity, the plasma membrane is composed of a wide array of lipids and proteins. These components are organized into distinct domains that will be the topic of this review. Some of the plasma membrane domains that will be described are known to act as scaffolds or barriers to diffusion, such as MCC/eisosomes, septins, and sites of contact with the endoplasmic reticulum. Other zones mediate dynamic processes, including secretion, endocytosis, and a special region at hyphal tips that facilitates rapid growth. The highly organized architecture of the plasma membrane facilitates the coordination of diverse functions and promotes the pathogenesis of C. albicans.

The surface of eukaryotic cells is a multi-component fluid bilayer in which glycosylphosphatidylinositol (GPI)-anchored proteins are an abundant constituent. In this review, we discuss the complex nature of the organization and dynamics of GPI-anchored proteins at multiple spatial and temporal scales. Different biophysical techniques have been utilized for understanding this organization, including fluorescence correlation spectroscopy, fluorescence recovery after photobleaching, single particle tracking, and a number of super resolution methods. Major insights into the organization and dynamics have also come from exploring the short-range interactions of GPI-anchored proteins by fluorescence (or Förster) resonance energy transfer microscopy. Based on the nanometer to micron scale organization, at the microsecond to the second time scale dynamics, a picture of the membrane bilayer emerges where the lipid bilayer appears inextricably intertwined with the underlying dynamic cytoskeleton. These observations have prompted a revision of the current models of plasma membrane organization, and suggest an active actin-membrane composite.

CD37 is a tetraspanin expressed on mature B cells where it orchestrates plasma membrane organization, receptor signaling, cell migration and adhesion. As CD37 is abundantly expressed on many mature B cell-derived malignancies, it represents an attractive target for new antibody therapies. DuoHexaBody®-CD37 is a novel biparatopic bispecific CD37 antibody with an Fc domain engineered to enhance antibody hexamerization upon binding to CD37 on the plasma membrane. DuoHexaBody-CD37 was shown to induce potent complement-dependent cytotoxicity (CDC) of malignant B-cell lines and primary chronic lymphocytic leukemia and non-Hodgkin lymphoma patient samples (Oostindie et al., Blood 2018 132:4170; van der Horst et al., Blood 2018 132:4179). Here, we demonstrate that the DuoHexaBody-CD37 mechanism of action also encompasses FcγR-mediated effector functions, including antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Daudi and Raji cells opsonized with DuoHexaBody-CD37 induced FcγRIIa and FcγRIIIa signaling in luciferase reporter assays. In agreement with efficient FcγR engagement, opsonization with DuoHexaBody-CD37 resulted in dose-dependent ADCC and ADCP by human healthy donor peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDMs), respectively. In a 4-hour Chromium-51 release assay, DuoHexaBody-CD37 induced ADCC in Daudi cells with PMBCs from 11 out of 12 tested donors (average EC50 of 9.87 [±9.98] ng/mL; maximum kill 20.4 [±9.2]%), and in Raji cells with 1 out of 3 donors. Image- and flow cytometry-based assays with MDMs illustrated that in presence of DuoHexaBody-CD37, Daudi cells were efficiently engulfed by effector cells from 3 different donors (average percentage phagocytic macrophages 29.7 ± 7.6%) resulting in target cell depletion (maximum depletion 75.9 [±18.0]%). Potent anti-tumor activity of DuoHexaBody-CD37 in vivo was reported in a screening approach using B-cell lymphoma patient-derived xenograft (PDX) models with single-mouse treatment groups. Two weekly doses of 5 mg/kg DuoHexaBody-CD37 resulted in strong tumor growth inhibition (tumor stasis or tumor regression) in 3/9 models compared to untreated tumors. Follow-up cohort PDX studies with eight mice per group confirmed potent, dose-dependent anti-tumor activity of DuoHexaBody-CD37 at doses as low as 1 mg/kg. In summary, DuoHexaBody-CD37 induces efficient tumor cell kill through CDC, ADCC and ADCP in vitro and shows potent anti-tumor activity in B-cell lymphoma PDX models in vivo. These data further strengthen the rationale for exploring the safety and efficacy of DuoHexaBody-CD37 in B-cell malignancy patients. Citation Format: Laurens P. Kil, Simone C. Oostindie, Kristin Strumane, Hilma J. van der Horst, Berris van Kessel, Marije B. Overdijk, Andreas Lingnau, Marcel Brandhorst, Jeroen van den Brakel, Margaret A. Lindorfer, Ronald P. Taylor, Martine E. Chamuleau, Tuna Mutis, A. Kate Sasser, Janine Schuurman, Paul W. Parren, Frank J. Beurskens, Esther C. Breij. Multifaceted mechanism of action of DuoHexaBody-CD37 involves both complement- and Fc gamma receptor-mediated cytotoxicity in pre-clinical B-cell lymphoma models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4544.

Ras proteins are small GTPases that regulate cellular growth and proliferation. The three RAS genes KRAS, HRAS and NRAS are ubiquitously expressed and share almost complete sequence homology; despite this they exhibit isoform-specific signaling in vitro and in vivo. Differences in localization, including nanoclustering on the cell surface, have been suggested to determine isoform-specific signaling. Currently, over 20 Ras effectors have been identified, although most work has focused on the Raf-MAP kinase and PtdIns 3-kinase (PI3K) pathways downstream of Ras. It has been shown that Ras isoform-specific signaling is highly context-dependent with significant cell type-dependent differences in coupling to canonical effector pathways. While the main Ras signaling network is understood, proteins regulating localization and interactions within the Ras network are still being identified. Here, we have performed a comprehensive analysis of the Ras isoform-specific interactome with particular interest in the role of cell surface nanoclustering and plasma membrane organization. We have screened the proteome microenvironment in living mammalian cells via APEX2, an engineered promiscuous peroxidase, which biotinylates vicinal proteins to the protein of interest. Here, APEX2-tagged Ras was used to identify proximal proteins to KRAS, HRAS and NRAS using mass spectrometry. Our preliminary data have shown that APEX2 is a suitable screening tool for the Ras proteome microenvironment. This method has allowed us to observe differences between the Ras isoforms, which are undergoing validation. Citation Format: Stephanie P. Mo, Judy M. Coulson, Ian A. Prior. Screening the Ras proteome microenvironment using APEX2 [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A11.

The trafficking of G protein coupled-receptors (GPCRs) is one of the most exciting areas in cell biology because of recent advances demonstrating that GPCR signaling is spatially encoded. GPCRs, acting in a diverse array of physiological systems, can have differential signaling consequences depending on their subcellular localization. At the plasma membrane, GPCR organization could fine-tune the initial stages of receptor signaling by determining the magnitude of signaling and the type of effectors to which receptors can couple. This organization is mediated by the lipid composition of the plasma membrane, receptor-receptor interactions, and receptor interactions with intracellular scaffolding proteins. GPCR organization is subsequently changed by ligand binding and the regulated endocytosis of these receptors. Activated GPCRs can modulate the dynamics of their own endocytosis through changing clathrin-coated pit dynamics, and through the scaffolding adaptor protein β-arrestin. This endocytic regulation has signaling consequences, predominantly through modulation of the MAPK cascade. This review explores what is known about receptor sorting at the plasma membrane, protein partners that control receptor endocytosis, and the ways in which receptor sorting at the plasma membrane regulates downstream trafficking and signaling.

Since the publication of the fluid mosaic as a relevant model for biological membranes, accumulating evidence has revealed the outstanding complexity of the composition and organization of the plant plasma membrane (PM). Powerful new methodologies have uncovered the remarkable multiscale and multicomponent heterogeneity of PM subcompartmentalization, and this is emerging as a general trait with different features and properties. It is now evident that the dynamics of such a complex organization are intrinsically related to signaling pathways that regulate key physiological processes. Listing and linking recent progress in precisely qualifying these heterogeneities will help to draw an integrated picture of the plant PM. Understanding the key principles governing such a complex dynamic organization will contribute to deciphering the crucial role of the PM in cell physiology.