Membrane Domains Research Articles

Apical PAR protein caps orient the mitotic spindle in C. elegans early embryos

During embryonic development, oriented cell divisions are important for patterned tissue growth and cell fate specification. Cell division orientation is controlled in part by asymmetrically localized polarity proteins, which establish functional domains of the cell membrane and interact with microtubule regulators to position the mitotic spindle. For example, in the 8-cell mouse embryo, apical polarity proteins form caps on the outside, contact-free surface of the embryo that position the mitotic spindle to execute asymmetric cell division. A similar radial or "inside-outside" polarity is established at an early stage in many other animal embryos, but in most cases, it remains unclear how inside-outside polarity is established and how it influences downstream cell behaviors. Here, we explore inside-outside polarity in C.elegans somatic blastomeres using spatiotemporally controlled protein degradation and live embryo imaging. We show that PAR polarity proteins, which form apical caps at the center of the contact-free membrane, localize dynamically during the cell cycle and contribute to spindle orientation and proper cell positioning. Surprisingly, isolated single blastomeres lacking cell contacts are able to break symmetry and form PAR-3/atypical protein kinase C (aPKC) caps. Polarity caps form independently of actomyosin flows and microtubules and can regulate spindle orientation in cooperation with the key polarity kinase aPKC. Together, our results reveal a role for apical polarity caps in regulating spindle orientation in symmetrically dividing cells and provide novel insights into how these structures are formed.

Cell surface plasticity in response to shape change in the whole organism

Plasma membrane rupture can result in catastrophic cell death. The skeletal muscle fiber plasma membrane, the sarcolemma, provides an extreme example of a membrane subject to mechanical stress since these cells specifically evolved to generate contraction and movement. A quantitative model correlating ultrastructural remodeling of surface architecture with tissue changes invivo is required to understand how membrane domains contribute to the shape changes associated with tissue deformation in whole animals. We and others have shown that loss of caveolae, small invaginations of the plasma membrane particularly prevalent in the muscle sarcolemma, renders the plasma membrane more susceptible to rupture during stretch.1,2,3 While it is thought that caveolae are able to flatten and be absorbed into the bulk membrane to buffer local membrane expansion, a direct demonstration of this model invivo has been unachievable since it would require measurement of caveolae at the nanoscale combined with detailed whole-animal morphometrics under conditions of perturbation. Here, we describe the development and application of the "active trapping model" where embryonic zebrafish are immobilized in a curved state that mimics natural body axis curvature during an escape response. The model is amenable to multiscale, multimodal imaging including high-resolution whole-animal three-dimensional quantitative electron microscopy. Using the active trapping model, we demonstrate the essential role of caveolae in maintaining sarcolemmal integrity and quantify the specific contribution of caveolar-derived membrane to surface expansion. We show that caveolae directly contribute to an increase in plasma membrane surface area under physiologically relevant membrane deformation conditions.

Rafting on the Evidence for Lipid Raft-like Domains as Hubs Triggering Environmental Toxicants' Cellular Effects.

The plasma membrane lipid rafts are cholesterol- and sphingolipid-enriched domains that allow regularly distributed, sub-micro-sized structures englobing proteins to compartmentalize cellular processes. These membrane domains can be highly heterogeneous and dynamic, functioning as signal transduction platforms that amplify the local concentrations and signaling of individual components. Moreover, they participate in cell signaling routes that are known to be important targets of environmental toxicants affecting cell redox status and calcium homeostasis, immune regulation, and hormonal functions. In this work, the evidence that plasma membrane raft-like domains operate as hubs for toxicants' cellular actions is discussed, and suggestions for future research are provided. Several studies address the insertion of pesticides and other organic pollutants into membranes, their accumulation in lipid rafts, or lipid rafts' disruption by polychlorinated biphenyls (PCBs), benzo[a]pyrene (B[a]P), and even metals/metalloids. In hepatocytes, macrophages, or neurons, B[a]P, airborne particulate matter, and other toxicants caused rafts' protein and lipid remodeling, oxidative changes, or amyloidogenesis. Different studies investigated the role of the invaginated lipid rafts present in endothelial cells in mediating the vascular inflammatory effects of PCBs. Furthermore, in vitro and in vivo data strongly implicate raft-localized NADPH oxidases, the aryl hydrocarbon receptor, caveolin-1, and protein kinases in the toxic mechanisms of occupational and environmental chemicals.

The role of lipid-modified proteins in cell wall synthesis and signaling.

The plant cell wall is a complex and dynamic extracellular matrix. Plant primary cell walls are the first line of defense against pathogens and regulate cell expansion. Specialized cells deposit a secondary cell wall that provides support and permits water transport. The composition and organization of the cell wall varies between cell types and species, contributing to the extensibility, stiffness, and hydrophobicity required for its proper function. Recently, many of the proteins involved in the biosynthesis, maintenance, and remodeling of the cell wall have been identified as being post-translationally modified with lipids. These modifications exhibit diverse structures and attach to proteins at different sites, which defines the specific role played by each lipid modification. The introduction of relatively hydrophobic lipid moieties promotes the interaction of proteins with membranes and can act as sorting signals, allowing targeted delivery to the plasma membrane regions and secretion into the apoplast. Disruption of lipid modification results in aberrant deposition of cell wall components and defective cell wall remodeling in response to stresses, demonstrating the essential nature of these modifications. Although much is known about which proteins bear lipid modifications, many questions remain regarding the contribution of lipid-driven membrane domain localization and lipid heterogeneity to protein function in cell wall metabolism. In this update, we highlight the contribution of lipid modifications to proteins involved in the formation and maintenance of plant cell walls, with a focus on the addition of glycosylphosphatidylinositol anchors, N-myristoylation, prenylation, and S-acylation.

Dynamic association of the intramembrane proteases SPPL2a/b and their substrates with tetraspanin-enriched microdomains

Signal peptide peptidase-like 2a and b (SPPL2a/b) are aspartyl intramembrane proteases and cleave tail-anchored proteins as well as N-terminal fragments (NTFs) derived from type II-oriented transmembrane proteins. How these proteases recruit substrates and cleavage is regulated, is still incompletely understood. We found that SPPL2a/b localize to detergent-resistant membrane (DRM) domains with the characteristics of tetraspanin-enriched microdomains (TEMs). Based on this, association with several tetraspanins was evaluated. We demonstrate that not only SPPL2a/b but also their substrates tumor necrosis factor (TNF) and CD74 associate with tetraspanins like CD9, CD81, and CD82 and/or TEMs and analyze the stability of these complexes in different detergents. CD9 and CD81 deficiency has protease- and substrate-selective effects on SPPL2a/b function. Our findings suggest that reciprocal interactions with tetraspanins may assist protease-substrate encounters of SPPL2a/b within the membrane. Beyond SPP/SPPL proteases, this supports previous concepts that tetraspanins facilitate membrane-embedded proteolytic processes.

Characterizing the transmembrane domains of ADAM10 and BACE1 and the impact of membrane composition

The β-secretase, BACE1, and the α-secretase, ADAM10, are known to competitively cleave amyloid precursor protein (APP) in the amyloid cascades of Alzheimer’s disease. Cleavage of APP by BACE1 produces a 99-residue C-terminal peptide (APP-C99) that is subsequently cleaved by γ-secretase to form amyloid-β (Aβ) protein, whereas cleavage of APP by ADAM10 is nonamyloidogenic. It has been speculated that ADAM10/APP and BACE1/APP interactions are regulated by colocalization within and outside of liquid-ordered membrane domains; however, the mechanism of this regulation and the character of the proteins’ transmembrane domains are not well understood. In this work, we have developed and characterized minimal congener sequences for the transmembrane domains of ADAM10 and BACE1 using a multiscale modeling approach combining both temperature replica exchange and conventional molecular dynamics simulations based on the coarse-grained Martini2.2 and all-atom CHARMM36 force fields. Our results show that membrane composition impacts the character of the transmembrane domains of BACE1 and ADAM10, adding credence to the speculation that membrane domains are involved in the etiology of Alzheimer’s disease.

A new family of proteins is required for tethering of Casparian strip membrane domain and nutrient homoeostasis in rice.

Cell-cell junctions are essential for multicellular organisms to maintain nutrient homoeostasis. A plant-type tight junction, the Casparian strip (CS)-Casparian strip membrane domain (CSD) that seals the paracellular space between adjacent endodermal cells, has been known for more than one hundred years. However, the molecular basis of this structure remains unknown. Here we report that a new family of proteins containing a glycine/alanine/proline-rich domain, a lectin domain and a secretory signal peptide (GAPLESS) mediates tethering of the plasma membrane to the CS in rice. The GAPLESS proteins are specifically localized in the CS of root endodermal cells, and loss of their functions results in a disabled cell-cell junction and disrupted nutrient homoeostasis. The GAPLESS protein forms a tight complex with OsCASP1 in the plasma membrane, thereby mediating the CS-CSD junction. This study provides valuable insights into the junctional complex of plant endodermal cells, shedding light on our understanding of nutrient homoeostasis in crops and the cell junctions of eukaryotes.

Muscarinic Receptor Stimulation Does Not Inhibit Voltage-dependent Ca2+ Channels in Rat Adrenal Medullary Chromaffin Cells.

Adrenal medullary chromaffin (AMC) and sympathetic ganglion cells are derived from the neural crest and show a similar developmental path. Thus, these two cell types have many common properties in membrane excitability and signaling. However, AMC cells function as endocrine cells while sympathetic ganglion cells are neurons. In rat sympathetic ganglion cells, muscarinic M1 and M4 receptors mediate excitation and inhibition via suppression of M-type K+ channels and suppression of voltage-dependent Ca2+ channels, respectively. On the other hand, M1 receptor stimulation in rat AMC cells also produces excitation by suppressing TWIK-related acid sensitive K+ (TASK) channels. However, whether M4 receptors are coupled with voltage-dependent Ca2+ channel suppression is unclear. We explore this issue electrophysiologically and biochemically. Electrical stimulation of nerve fibers in rat adrenal glands trans-synaptically increased the Ca2+ signal in AMC cells. This electrically evoked increased Ca2+ signal was not altered during muscarine-induced increase in Ca2+ signal, whereas it decreased significantly during a GABA-induced increase, due to a shunt effect of increased Cl- conductance. The whole-cell current recordings revealed that voltage-dependent Ca2+ currents in AMC cells were suppressed by adenosine triphosphate, but not by muscarinic agonists. The fractionation analysis and immunocytochemistry indicated that CaV1.2 Ca2+ channels and M4 receptors are located in the raft and non-raft membrane domains, respectively. We concluded that muscarinic stimulation in rat AMC cells does not produce voltage-dependent Ca2+ channel inhibition. This lack of muscarinic inhibition is at least partly due to physical separation of voltage-dependent Ca2+ channels and M4 receptors in the plasma membrane.

Promoting the activity of a receptor tyrosine phosphatase with a novel pH-responsive transmembrane agonist inhibits cancer-associated phenotypes.

Cell signaling by receptor protein tyrosine kinases (RTKs) is tightly controlled by the counterbalancing actions of receptor protein tyrosine phosphatases (RPTPs). Due to their role in attenuating the signal-initiating potency of RTKs, RPTPs have long been viewed as therapeutic targets. However, the development of activators of RPTPs has remained limited. We previously reported that the homodimerization of a representative member of the RPTP family (protein tyrosine phosphatase receptor J or PTPRJ) is regulated by specific transmembrane (TM) residues. Disrupting this interaction by single point mutations promotes PTPRJ access to its RTK substrates (e.g., EGFR and FLT3), reduces RTK's phosphorylation and downstream signaling, and ultimately antagonizes RTK-driven cell phenotypes. Here, we designed and tested a series of first-in-class pH-responsive TM peptide agonists of PTPRJ that are soluble in aqueous solution but insert as a helical TM domain in lipid membranes when the pH is lowered to match that of the acidic microenvironment of tumors. The most promising peptide reduced EGFR's phosphorylation and inhibited cancer cell EGFR-driven migration and proliferation, similar to the PTPRJ's TM point mutations. Developing tumor-selective and TM-targeting peptide binders of critical RPTPs could afford a potentially transformative approach to studying RPTP's selectivity mechanism without requiring less specific inhibitors and represent a novel class of therapeutics against RTK-driven cancers.

Modelling BK Polyomavirus dissemination and cytopathology using polarized human renal tubule epithelial cells.

Most humans have a lifelong imperceptible BK Polyomavirus (BKPyV) infection in epithelial cells lining the reno-urinary tract. In kidney transplant recipients, unrestricted high-level replication of donor-derived BKPyV in the allograft underlies polyomavirus-associated nephropathy, a condition with massive epithelial cell loss and inflammation causing premature allograft failure. There is limited understanding on how BKPyV disseminates throughout the reno-urinary tract and sometimes causes kidney damage. Tubule epithelial cells are tightly connected and have unique apical and basolateral membrane domains with highly specialized functions but all in vitro BKPyV studies have been performed in non-polarized cells. We therefore generated a polarized cell model of primary renal proximal tubule epithelial cells (RPTECs) and characterized BKPyV entry and release. After 8 days on permeable inserts, RPTECs demonstrated apico-basal polarity. BKPyV entry was most efficient via the apical membrane, that in vivo faces the tubular lumen, and depended on sialic acids. Progeny release started between 48 and 58 hours post-infection (hpi), and was exclusively detected in the apical compartment. From 72 hpi, cell lysis and detachment gradually increased but cells were mainly shed by extrusion and the barrier function was therefore maintained. The decoy-like cells were BKPyV infected and could transmit BKPyV to uninfected cells. By 120 hpi, the epithelial barrier was disrupted by severe cytopathic effects, and BKPyV entered the basolateral compartment mimicking the interstitial space. Addition of BKPyV-specific neutralizing antibodies to this compartment inhibited new infections. Taken together, we propose that during in vivo low-level BKPyV replication, BKPyV disseminates inside the tubular system, thereby causing minimal damage and delaying immune detection. However, in kidney transplant recipients lacking a well-functioning immune system, replication in the allograft will progress and eventually cause denudation of the basement membrane, leading to an increased number of decoy cells, high-level BKPyV-DNAuria and DNAemia, the latter a marker of allograft damage.

Golgi retention and oncogenic KIT signaling via PLCγ2-PKD2-PI4KIIIβ activation in gastrointestinal stromal tumor cells

Most gastrointestinal stromal tumors (GISTs) develop due to gain-of-function mutations in the tyrosine kinase gene, KIT. We recently showed that mutant KIT mislocalizes to the Golgi area and initiates uncontrolled signaling. However, the molecular mechanisms underlying its Golgi retention remain unknown. Here, we show that protein kinase D2 (PKD2) is activated by the mutant, which causes Golgi retention of KIT. In PKD2-inhibited cells, KIT migrates from the Golgi region to lysosomes and subsequently undergoes degradation. Importantly, delocalized KIT cannot trigger downstream activation. In the Golgi/trans-Golgi network (TGN), KIT activates the PKD2-phosphatidylinositol 4-kinase IIIβ (PKD2-PI4KIIIβ) pathway through phospholipase Cγ2 (PLCγ2) to generate a PI4P-rich membrane domain, where the AP1-GGA1 complex is aberrantly recruited. Disruption of any factors in this cascade results in the release of KIT from the Golgi/TGN. Our findings show the molecular mechanisms underlying KIT mislocalization and provide evidence for a strategy for inhibition of oncogenic signaling.

Cooperative regulation of C1-domain membrane recruitment polarizes atypical protein kinase C.

Recruitment of the Par complex protein atypical protein kinase C (aPKC) to a specific membrane domain is a key step in the polarization of animal cells. While numerous proteins and phospholipids interact with aPKC, how these interactions cooperate to control its membrane recruitment has been unknown. Here, we identify aPKC's C1 domain as a phospholipid interaction module that targets aPKC to the membrane of Drosophila neural stem cells (NSCs). The isolated C1 binds the NSC membrane in an unpolarized manner during interphase and mitosis and is uniquely sufficient among aPKC domains for targeting. Other domains, including the catalytic module and those that bind the upstream regulators Par-6 and Bazooka, restrict C1's membrane targeting activity-spatially and temporally-to the apical NSC membrane during mitosis. Our results suggest that aPKC polarity results from cooperative activation of autoinhibited C1-mediated membrane binding activity.

Membrane Domain Anti-Registration Induces an Intrinsic Transmembrane Potential.

Plasma membrane segregation into various nanoscale membrane domains is driven by distinct interactions between diverse lipids and proteins. Among them, liquid-ordered (Lo) membrane domains are defined as "lipid rafts" and liquid-disordered (Ld) ones as "lipid non-rafts". Using model membrane systems, both intra-leaflet and inter-leaflet dynamics of these membrane domains are widely studied. Nevertheless, the biological impact of the latter, which is accompanied by membrane domain registration/anti-registration, is far from clear. Hence, in this work, we studied the biological relevance of the membrane domain anti-registration using both all-atom molecular dynamics (MD) simulations and confocal fluorescence microscopy. All-atom MD simulations suggested an intrinsic transmembrane potential for the case of the membrane anti-registration (Lo/Ld). Meanwhile, confocal fluorescence microscopy experiments of HeLa and 293T cell lines indicated that membrane cholesterol depletion could significantly alter the transmembrane potential of cells. Considering differences in the cholesterol content between Lo and Ld membrane domains, our confocal fluorescence microscopy experiments are consistent with our all-atom MD simulations. In short, membrane domain anti-registration induces local membrane asymmetry and, thus, an intrinsic transmembrane potential.

The LDL receptor is regulated by membrane cholesterol as revealed by fluorescence fluctuation analysis

Membrane cholesterol-rich domains have been shown to be important for regulating a range of membrane protein activities. Low-density lipoprotein receptor (LDLR)-mediated internalization of cholesterol-rich LDL particles is tightly regulated by feedback mechanisms involving intracellular sterol sensors. Since LDLR plays a role in maintaining cellular cholesterol homeostasis, we explore the role that membrane domains may have in regulating LDLR activity. We expressed a fluorescent LDLR-mEGFP construct in HEK293T cells and imaged the unligated receptor or bound to an LDL/DiI fluorescent ligand using total internal reflection fluorescence microscopy. We studied the receptor’s spatiotemporal dynamics using fluorescence fluctuation analysis methods. Image cross correlation spectroscopy reveals a lower LDL-to-LDLR binding fraction when membrane cholesterol concentrations are augmented using cholesterol esterase, and a higher binding fraction when the cells are treated with methyl-β-cyclodextrin) to lower membrane cholesterol. This suggests that LDLR’s ability to metabolize LDL particles is negatively correlated to membrane cholesterol concentrations. We then tested if a change in activity is accompanied by a change in membrane localization. Image mean-square displacement analysis reveals that unligated LDLR-mEGFP and ligated LDLR-mEGFP/LDL-DiI constructs are transiently confined on the cell membrane, and the size of their confinement domains increases with augmented cholesterol concentrations. Receptor diffusion within the domains and their domain-escape probabilities decrease upon treatment with methyl-β-cyclodextrin, consistent with a change in receptor populations to more confined domains, likely clathrin-coated pits. We propose a feedback model to account for regulation of LDLR within the cell membrane: when membrane cholesterol concentrations are high, LDLR is sequestered in cholesterol-rich domains. These LDLR populations are attenuated in their efficacy to bind and internalize LDL. However, when membrane cholesterol levels drop, LDL has a higher binding affinity to its receptor and the LDLR transits to nascent clathrin-coated domains, where it diffuses at a slower rate while awaiting internalization.

The Membrane Phase Transition Gives Rise to Responsive Plasma Membrane Structure and Function.

Several groups have recently reported evidence for the emergence of domains in cell plasma membranes when membrane proteins are organized by ligand binding or assembly of membrane proximal scaffolds. These domains recruit and retain components that favor the liquid-ordered phase, adding to a decades-old literature interrogating the contribution of membrane phase separation in plasma membrane organization and function. Here we propose that both past and present observations are consistent with a model in which membranes have a high compositional susceptibility, arising from their thermodynamic state in a single phase that is close to a miscibility phase transition. This rigorous framework naturally allows for both transient structure in the form of composition fluctuations and long-lived structure in the form of induced domains. In this way, the biological tuning of plasma membrane composition enables a responsive compositional landscape that facilitates and augments cellular biochemistry vital to plasma membrane functions.

Phospholipids can regulate complex I assembly independent of their role in maintaining mitochondrial membrane integrity

Several phospholipid (PL) molecules are intertwined with some mitochondrial complex I (CI) subunits in the membrane domain of CI, but their function is unclear. We report that when the Drosophila melanogaster ortholog of the intramitochondrial PL transporter, STARD7, is severely disrupted, assembly of the oxidative phosphorylation (OXPHOS) system is impaired, and the biogenesis of several CI subcomplexes is hampered. However, intriguingly, a restrained knockdown of STARD7 impairs the incorporation of NDUFS5 and NDUFA1 into the proximal part of the CI membrane domain without directly affecting the incorporation of subunits in the distal part of the membrane domain, OXPHOS complexes already assembled, or mitochondrial cristae integrity. Importantly, the restrained knockdown of STARD7 appears to induce a modest amount of cardiolipin remodeling, indicating that there could be some alteration in the composition of the mitochondrial phospholipidome. We conclude that PLs can regulate CI biogenesis independent of their role in maintaining mitochondrial membrane integrity.

Quinone Catalysis Modulates Proton Transfer Reactions in the Membrane Domain of Respiratory Complex I.

Complex I is a redox-driven proton pump that drives electron transport chains and powers oxidative phosphorylation across all domains of life. Yet, despite recently resolved structures from multiple organisms, it still remains unclear how the redox reactions in Complex I trigger proton pumping up to 200 Å away from the active site. Here, we show that the proton-coupled electron transfer reactions during quinone reduction drive long-range conformational changes of conserved loops and trans-membrane (TM) helices in the membrane domain of Complex I from Yarrowia lipolytica. We find that the conformational switching triggers a π → α transition in a TM helix (TM3ND6) and establishes a proton pathway between the quinone chamber and the antiporter-like subunits, responsible for proton pumping. Our large-scale (>20 μs) atomistic molecular dynamics (MD) simulations in combination with quantum/classical (QM/MM) free energy calculations show that the helix transition controls the barrier for proton transfer reactions by wetting transitions and electrostatic effects. The conformational switching is enabled by re-arrangements of ion pairs that propagate from the quinone binding site to the membrane domain via an extended network of conserved residues. We find that these redox-driven changes create a conserved coupling network within the Complex I superfamily, with point mutations leading to drastic activity changes and mitochondrial disorders. On a general level, our findings illustrate how catalysis controls large-scale protein conformational changes and enables ion transport across biological membranes.

Photo-Lipids: Light-Sensitive Nano-Switches to Control Membrane Properties.

Biological membranes are described as a complex mixture of lipids and proteins organized according to thermodynamic principles. This chemical and spatial complexity can lead to specialized functional membrane domains enriched with specific lipids and proteins. The interaction between lipids and proteins restricts their lateral diffusion and range of motion, thus altering their function. One approach to investigating these membrane properties is to use chemically accessible probes. In particular, photo-lipids, which contain a light-sensitive azobenzene moiety that changes its configuration from trans- to cis- upon light irradiation, have recently gained popularity for modifying membrane properties. These azobenzene-derived lipids serve as nanotools for manipulating lipid membranes in vitro and in vivo. Here, we will discuss the use of these compounds in artificial and biological membranes as well as their application in drug delivery. We will focus mainly on changes in the membrane's physical properties as well as lipid membrane domains in phase-separated liquid-ordered/liquid-disordered bilayers driven by light, and how these changes in membrane physical properties alter transmembrane protein function.

Fluctuations of formin binding in the generation of membrane patterns

Circular actin waves that propagate on the substrate-attached membrane of Dictyostelium cells separate two distinct membrane domains from each other: an inner territory rich in phosphatidyl-(3,4,5) trisphosphate (PIP3) and an external area decorated with the PIP3-degrading 3-phosphatase PTEN. During wave propagation, the inner territory increases at the expense of the external area. Beyond a size limit, the inner territory becomes unstable, breaking into an inner and an external domain. The sharp boundary between these domains is demarcated by the insertion of an actin wave. During the conversion of inner territory to external area, the state of the membrane fluctuates, as visualized by dynamic landscapes of formin B binding. Here we analyze the formin B fluctuations in relation to three markers of the membrane state: activated Ras, PIP3, and PTEN.

Molecular mechanism of GPCR spatial organization at the plasma membrane.

G-protein-coupled receptors (GPCRs) mediate many critical physiological processes. Their spatial organization in plasma membrane (PM) domains is believed to encode signaling specificity and efficiency. However, the existence of domains and, crucially, the mechanism of formation of such putative domains remain elusive. Here, live-cell imaging (corrected for topography-induced imaging artifacts) conclusively established the existence of PM domains for GPCRs. Paradoxically, energetic coupling to extremely shallow PM curvature (<1 µm-1) emerged as the dominant, necessary and sufficient molecular mechanism of GPCR spatiotemporal organization. Experiments with different GPCRs, H-Ras, Piezo1 and epidermal growth factor receptor, suggest that the mechanism is general, yet protein specific, and can be regulated by ligands. These findings delineate a new spatiomechanical molecular mechanism that can transduce to domain-based signaling any mechanical or chemical stimulus that affects the morphology of the PM and suggest innovative therapeutic strategies targeting cellular shape.