RESUMO
Actin filament assembly and the regulation of its mechanical properties are fundamental processes essential for eukaryotic cell function. Residue E167 in vertebrate actins forms an inter-subunit salt bridge with residue K61 of the adjacent subunit. Saccharomyces cerevisiae actin filaments are more flexible than vertebrate filaments and have an alanine at this position (A167). Substitution of this alanine for a glutamic acid (A167E) confers Saccharomyces cerevisiae actin filaments with salt-dependent stiffness similar to vertebrate actins. We developed an optimized cryogenic electron microscopy workflow refining sample preparation and vitrification to obtain near-atomic resolution structures of wild-type and A167E mutant Saccharomyces cerevisiae actin filaments. The difference between these structures allowed us to pinpoint the potential binding site of a filament-associated cation that controls the stiffness of the filaments in vertebrate and A167E Saccharomyces cerevisiae actins. Through an analysis of previously published high-resolution reconstructions of vertebrate actin filaments, along with a newly determined high-resolution vertebrate actin structure in the absence of potassium, we identified a unique peak near residue 167 consistent with the binding of a magnesium ion. Our findings show how magnesium can contribute to filament stiffening by directly bridging actin subunits and allosterically affecting the orientation of the DNase-I binding loop of actin, which plays a regulatory role in modulating actin filament stiffness and interactions with regulatory proteins.
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Protein micropatterning enables robust control of cell positioning on electron-microscopy substrates for cryogenic electron tomography (cryo-ET). However, the combination of regulated cell boundaries and the underlying electron-microscopy substrate (EM-grids) provides a poorly understood microenvironment for cell biology. Because substrate stiffness and morphology affect cellular behavior, we devised protocols to characterize the nanometer-scale details of the protein micropatterns on EM-grids by combining cryo-ET, atomic force microscopy, and scanning electron microscopy. Measuring force displacement characteristics of holey carbon EM-grids, we found that their effective spring constant is similar to physiological values expected from skin tissues. Despite their apparent smoothness at light-microscopy resolution, spatial boundaries of the protein micropatterns are irregular at nanometer scale. Our protein micropatterning workflow provides the means to steer both positioning and morphology of cell doublets to determine nanometer details of punctate adherens junctions. Our workflow serves as the foundation for studying the fundamental structural changes governing cell-cell signaling.
Assuntos
Processamento de Imagem Assistida por Computador , Proteínas , Processamento de Imagem Assistida por Computador/métodos , Microscopia Crioeletrônica/métodos , Carbono/química , Transdução de SinaisRESUMO
Nanodiscs have become a popular tool in structure determination of membrane proteins using cryogenic electron microscopy and single particle analysis. However, the structure determination of small membrane proteins remains challenging. When the embedded protein is in the same size range as the nanodisc, the nanodisc can significantly contribute to the alignment and classification during the structure determination process. In those cases, it is crucial to minimize the heterogeneity in the nanodisc preparations to assure maximum accuracy in the classification and alignment steps of single particle analysis. Here, we introduce a new in-silico method for the characterization of nanodisc samples that is based on analyzing the Feret diameter distribution of their particle projection as imaged in the electron microscope. We validated the method with comprehensive simulation studies and show that Feret signatures can detect subtle differences in nanodisc morphologies and composition that might otherwise go unnoticed. We used the method to identify a specific biochemical nanodisc preparation with low size variations, allowing us to obtain a structure of the 23-kDa single-span membrane protein Bcl-xL while embedded in a nanodisc. Feret signature analysis can steer experimental data collection strategies, allowing more efficient use of high-end data collection hardware, as well as image analysis investments in studies where nanodiscs significantly contribute to the total volume of the full molecular species.
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Matrix stiffness and dimensionality have been shown to be major determinants of cell behavior. However, a workflow for examining nanometer-scale responses of the associated molecular machinery is not available. Here, we describe a comprehensive, quantitative workflow that permits the analysis of cells responding to mechanical and dimensionality cues in their native state at nanometer scale by cryogenic electron tomography. Using this approach, we quantified distinct cytoskeletal nanoarchitectures and vesicle phenotypes induced in human mammary epithelial cells in response to stiffness and dimensionality of reconstituted basement membrane. Our workflow closely recapitulates the microenvironment associated with acinar morphogenesis and identified distinct differences in situ at nanometer scale. Using drug treatment, we showed that molecular events and nanometer-scale rearrangements triggered by engagement of apical cell receptors with reconstituted basement membrane correspond to changes induced by reduction of cortical tension. Our approach is fully adaptable to any kind of stiffness regime, extracellular matrix composition, and drug treatment.
Assuntos
Células Epiteliais , Matriz Extracelular , Humanos , Fluxo de Trabalho , Morfogênese , Matriz Extracelular/metabolismo , Tomografia com Microscopia EletrônicaRESUMO
Patient-derived organoids and cellular spheroids recapitulate tissue physiology with remarkable fidelity. We investigated how engagement with a reconstituted basement membrane in three dimensions (3D) supports the polarized, stress resilient tissue phenotype of mammary epithelial spheroids. Cells interacting with reconstituted basement membrane in 3D had reduced levels of total and actin-associated filamin and decreased cortical actin tension that increased plasma membrane protrusions to promote negative plasma membrane curvature and plasma membrane protein associations linked to protein secretion. By contrast, cells engaging a reconstituted basement membrane in 2D had high cortical actin tension that forced filamin unfolding and endoplasmic reticulum (ER) associations. Enhanced filamin-ER interactions increased levels of PKR-like ER kinase effectors and ER-plasma membrane contact sites that compromised calcium homeostasis and diminished cell viability. Consequently, cells with decreased cortical actin tension had reduced ER stress and survived better. Consistently, cortical actin tension in cellular spheroids regulated polarized basement membrane membrane deposition and sensitivity to exogenous stress. The findings implicate cortical actin tension-mediated filamin unfolding in ER function and underscore the importance of tissue mechanics in organoid homeostasis.
Assuntos
Actinas , Retículo Endoplasmático , Actinas/metabolismo , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Células Epiteliais/metabolismo , Filaminas/metabolismo , FenótipoRESUMO
Neurotransmitter release is mediated by proteins that drive synaptic vesicle fusion with the presynaptic plasma membrane. While soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) form the core of the fusion apparatus, additional proteins play key roles in the fusion pathway. Here, we report that the C-terminal amphipathic helix of the mammalian accessory protein, complexin (Cpx), exerts profound effects on membranes, including the formation of pores and the efficient budding and fission of vesicles. Using nanodisc-black lipid membrane electrophysiology, we demonstrate that the membrane remodeling activity of Cpx modulates the structure and stability of recombinant exocytic fusion pores. Cpx had particularly strong effects on pores formed by small numbers of SNAREs. Under these conditions, Cpx increased the current through individual pores 3.5-fold, and increased the open time fraction from roughly 0.1 to 1.0. We propose that the membrane sculpting activity of Cpx contributes to the phospholipid rearrangements that underlie fusion by stabilizing highly curved membrane fusion intermediates.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas do Tecido Nervoso/química , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Células HEK293 , Humanos , Bicamadas Lipídicas/química , Fusão de Membrana/fisiologia , Simulação de Dinâmica Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Poro Nuclear/química , Poro Nuclear/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica em alfa-Hélice , Estabilidade Proteica , Vesículas Sinápticas/química , Vesículas Sinápticas/metabolismoRESUMO
With the rapid increase and accessibility of high-resolution imaging technologies of cells, the interpretation of results relies more and more on the assumption that the three-dimensional integrity of the surrounding cellular landscape is not compromised by the experimental setup. However, the only available technology for directly probing the structural integrity of whole-cell preparations at the nanoscale is electron cryo-tomography, which is time-consuming, costly, and complex. We devised an accessible, inexpensive and reliable screening assay to quickly report on the compatibility of experimental protocols with preserving the structural integrity of whole-cell preparations at the nanoscale. Our Rapid Cell Integrity Assessment (RCIA) assay is executed at room temperature and relies solely on light microscopy imaging. Using cellular electron cryo-tomography as a benchmark, we verify that RCIA accurately unveils the adverse impact of reagents and/or protocols such as those used for virus inactivation or to arrest dynamic processes on the cellular nanoarchitecture.
Assuntos
Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Células Eucarióticas/ultraestrutura , Imageamento Tridimensional/métodos , Nanoestruturas/ultraestrutura , Citoesqueleto de Actina/química , Citoesqueleto de Actina/ultraestrutura , Animais , Células Cultivadas , Células Eucarióticas/química , Células Eucarióticas/classificação , Células HeLa , Humanos , Membranas Intracelulares/química , Membranas Intracelulares/ultraestrutura , Camundongos , Microscopia de Fluorescência/métodos , Mitocôndrias/química , Mitocôndrias/ultraestrutura , Células NIH 3T3 , Nanoestruturas/química , Reprodutibilidade dos Testes , Células THP-1RESUMO
Plasmodium falciparum, the causative agent of malaria, moves by an atypical process called gliding motility. Actomyosin interactions are central to gliding motility. However, the details of these interactions remained elusive until now. Here, we report an atomic structure of the divergent Plasmodium falciparum actomyosin system determined by electron cryomicroscopy at the end of the powerstroke (Rigor state). The structure provides insights into the detailed interactions that are required for the parasite to produce the force and motion required for infectivity. Remarkably, the footprint of the myosin motor on filamentous actin is conserved with respect to higher eukaryotes, despite important variability in the Plasmodium falciparum myosin and actin elements that make up the interface. Comparison with other actomyosin complexes reveals a conserved core interface common to all actomyosin complexes, with an ancillary interface involved in defining the spatial positioning of the motor on actin filaments.
Assuntos
Citoesqueleto de Actina/metabolismo , Actomiosina/metabolismo , Movimento Celular/fisiologia , Plasmodium falciparum/fisiologia , Plasmodium falciparum/ultraestrutura , Actinas/metabolismo , Microscopia Crioeletrônica , Malária Falciparum/parasitologia , Miosinas/metabolismo , Conformação Proteica , Proteínas de Protozoários/metabolismoRESUMO
Cell-cell and cell-matrix junctions transmit mechanical forces during tissue morphogenesis and homeostasis. α-Catenin links cell-cell adhesion complexes to the actin cytoskeleton, and mechanical load strengthens its binding to F-actin in a direction-sensitive manner. Specifically, optical trap experiments revealed that force promotes a transition between weak and strong actin-bound states. Here, we describe the cryo-electron microscopy structure of the F-actin-bound αE-catenin actin-binding domain, which in solution forms a five-helix bundle. In the actin-bound structure, the first helix of the bundle dissociates and the remaining four helices and connecting loops rearrange to form the interface with actin. Deletion of the first helix produces strong actin binding in the absence of force, suggesting that the actin-bound structure corresponds to the strong state. Our analysis explains how mechanical force applied to αE-catenin or its homolog vinculin favors the strongly bound state, and the dependence of catch bond strength on the direction of applied force.
Assuntos
Actinas/química , alfa Catenina/química , Microscopia Crioeletrônica , Ligação ProteicaRESUMO
Hemophilia A, an X-linked bleeding disorder caused by deficiency of factor VIII (FVIII), is treated by protein replacement. Unfortunately, this regimen is costly due to the expense of producing recombinant FVIII as a consequence of its low-level secretion from mammalian host cells. FVIII expression activates the endoplasmic reticulum (ER) stress response, causes oxidative stress, and induces apoptosis. Importantly, little is known about the factors that cause protein misfolding and aggregation in metazoans. Here, we identified intrinsic and extrinsic factors that cause FVIII to form aggregates. We show that FVIII forms amyloid-like fibrils within the ER lumen upon increased FVIII synthesis or inhibition of glucose metabolism. Significantly, FVIII amyloids can be dissolved upon restoration of glucose metabolism to produce functional secreted FVIII. Two ER chaperone families and their cochaperones, immunoglobulin binding protein (BiP) and calnexin/calreticulin, promote FVIII solubility in the ER, where the former is also required for disaggregation. A short aggregation motif in the FVIII A1 domain (termed Aggron) is necessary and sufficient to seed ß-sheet polymerization, and BiP binding to this Aggron prevents amyloidogenesis. Our findings provide novel insight into mechanisms that limit FVIII secretion and ER protein aggregation in general and have implication for ongoing hemophilia A gene-therapy clinical trials.
Assuntos
Amiloide/química , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Fator VIII/metabolismo , Glucose/farmacologia , Chaperonas Moleculares/metabolismo , Amiloide/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Fator VIII/genética , Hemostáticos , Células Hep G2 , Humanos , Chaperonas Moleculares/genética , Edulcorantes/farmacologiaRESUMO
B cell lymphoma-2-associated X protein (BAX) plays a pivotal role in triggering cell apoptosis by permeabilizing the mitochondrial outer membrane. Contrary to previous findings, recent electron microscopy (EM) experiments showed that BAX monomers are able to perturb phospholipid nanodiscs (NDs) by forming lipidic pores. Here, we provide structural and thermodynamic interpretation of such data using multiscale resolution molecular dynamics (MD) simulations. Our results suggest that BAX is able to disrupt the stability, lateral packing and enhance the desorption propensity of the lipids in the ND, resulting in the formation of a stable toroidal-like pore. These findings prompted to re-evaluate the previously reported cryo-EM data to generate an improved reconstruction, thereby allowing for a more accurate localization of BAX in the EM map. We conclude that the reduced stability of the BAX-embedded ND eliminates the necessity of forming active BAX oligomers for its disruption.
Assuntos
Lipídeos de Membrana/química , Simulação de Dinâmica Molecular , Nanoestruturas/química , Fosfolipídeos/química , Proteína X Associada a bcl-2/química , Fenômenos Biofísicos , Microscopia Crioeletrônica/métodos , Humanos , Lipídeos de Membrana/metabolismo , Nanoestruturas/ultraestrutura , Fosfolipídeos/metabolismo , Porosidade , Multimerização Proteica , Termodinâmica , Proteína X Associada a bcl-2/metabolismoRESUMO
High-resolution imaging of hair-cell stereocilia of the inner ear has contributed substantially to our understanding of auditory and vestibular function. To provide three-dimensional views of the structure of stereocilia cytoskeleton and membranes, we developed a method for rapidly freezing unfixed stereocilia on electron microscopy grids, which allowed subsequent 3D imaging by electron cryo-tomography. Structures of stereocilia tips, shafts, and tapers were revealed, demonstrating that the actin paracrystal was not perfectly ordered. This sample-preparation and imaging procedure will allow for examination of structural features of stereocilia in a near-native state.
Assuntos
Temperatura Baixa , Tomografia com Microscopia Eletrônica/métodos , Células Ciliadas Vestibulares/ultraestrutura , Estereocílios/ultraestrutura , Animais , CamundongosRESUMO
Rac1 activation is at the core of signaling pathways regulating polarized cell migration. So far, it has not been possible to directly explore the structural changes triggered by Rac1 activation at the molecular level. Here, through a multiscale imaging workflow that combines biosensor imaging of Rac1 dynamics with electron cryotomography, we identified, within the crowded environment of eukaryotic cells, a unique nanoscale architecture of a flexible, signal-dependent actin structure. In cell regions with high Rac1 activity, we found a structural regime that spans from the ventral membrane up to a height of â¼60 nm above that membrane, composed of directionally unaligned, densely packed actin filaments, most shorter than 150 nm. This unique Rac1-induced morphology is markedly different from the dendritic network architecture in which relatively short filaments emanate from existing, longer actin filaments. These Rac1-mediated scaffold assemblies are devoid of large macromolecules such as ribosomes or other filament types, which are abundant at the periphery and within the remainder of the imaged volumes. Cessation of Rac1 activity induces a complete and rapid structural transition, leading to the absence of detectable remnants of such structures within 150 s, providing direct structural evidence for rapid actin filament network turnover induced by GTPase signaling events. It is tempting to speculate that this highly dynamical nanoscaffold system is sensitive to local spatial cues, thus serving to support the formation of more complex actin filament architectures-such as those mandated by epithelial-mesenchymal transition, for example-or resetting the region by completely dissipating.
Assuntos
Citoesqueleto/metabolismo , Citosol/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Linhagem Celular , Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , GTP Fosfo-Hidrolases/metabolismo , Humanos , Camundongos , Transdução de Sinais/fisiologiaRESUMO
Cryogenic electron tomography is the highest resolution tool available for structural analysis of macromolecular organization inside cells. Micropatterning of extracellular matrix (ECM) proteins is an established in vitro cell culture technique used to control cell shape. Recent traction force microscopy studies have shown correlation between cell morphology and the regulation of force transmission. However, it remains unknown how cells sustain increased strain energy states and localized stresses at the supramolecular level. Here, we report a technology to enable direct observation of mesoscale organization in epithelial cells under morphological modulation, using a maskless protein photopatterning method (PRIMO) to confine cells to ECM micropatterns on electron microscopy substrates. These micropatterned cell culture substrates can be used in mechanobiology research to correlate changes in nanometer-scale organization at cell-cell and cell-ECM contacts to strain energy states and traction stress distribution in the cell.
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Arp2/3 complex nucleates dendritic actin networks and plays a pivotal role in the formation of lamellipodia at the leading edge of motile cells. Mouse fibroblasts lacking functional Arp2/3 complex have the characteristic smooth, veil-like lamellipodial leading edge of wild-type cells replaced by a massive, bifurcating filopodia-like protrusions (FLPs) with fractal geometry. The nanometer-scale actin-network organization of these FLPs can be linked to the fractal geometry of the cell boundary by a self-organized criticality through the bifurcation behavior of cross-linked actin bundles. Despite the pivotal role of the Arp2/3 complex in cell migration, the cells lacking functional Arp2/3 complex migrate at rates similar to wild-type cells. However, these cells display defects in the persistence of a directional movement. We suggest that Arp2/3 complex suppresses the formation of FLPs by locally fine-tuning actin networks and favoring dendritic geometry over bifurcating bundles, giving cells a distinct evolutionary edge by providing the means for a directed movement.
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Cellular force transmission and mechanotransduction are critical in embryogenesis, normal physiology, and many diseases. Talin plays a key role in these processes by linking integrins to force-generating actomyosin. Using the previously characterized FRET-based talin tension sensor, we observed variations of tension both between and within individual focal adhesions in the same cell. Assembling and sliding adhesions showed gradients with higher talin tension toward the cell center, whereas mature, stable adhesions had uniform talin tension. Total talin accumulation was maximal in high-tension regions; by contrast, vinculin intensity was flat or maximal at the adhesion center, and actin intensity was maximal toward the cell center. To investigate mechanism, we combined talin tension imaging with cellular cryotomography to visualize the correlated actin organization at nanometer resolution. Regions of high talin tension had highly aligned linear actin filaments, whereas regions of low tension had less-well-aligned F-actin. These results reveal an orchestrated spatiotemporal relationship between talin tension, actin/vinculin localization, local actin organization, and focal adhesion dynamics.
Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , Adesões Focais/fisiologia , Talina/fisiologia , Animais , Células Cultivadas , Mecanotransdução Celular , Camundongos , Camundongos KnockoutRESUMO
Combining fluorescence microscopy with electron cryo-tomography allows, in principle, spatial localization of tagged macromolecular assemblies and structural features within the cellular environment. To allow precise localization and scale integration between the two disparate imaging modalities, accurate alignment procedures are needed. Here, we describe a marker-free method for aligning images from light or cryo-light fluorescence microscopy and from electron cryo-microscopy that takes advantage of sample support features, namely the holes in the carbon film. We find that the accuracy of this method, as judged by prediction errors of the hole center coordinates, is better than 100â¯nm.
Assuntos
Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Substâncias Macromoleculares/ultraestrutura , Microscopia de Fluorescência/métodos , Animais , Células CHO , Carbono/química , Cricetinae , Cricetulus , Microscopia Crioeletrônica/instrumentação , Tomografia com Microscopia Eletrônica/instrumentação , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Substâncias Macromoleculares/metabolismo , Camundongos , Microscopia de Fluorescência/instrumentação , Paxilina/química , Paxilina/genética , Paxilina/metabolismo , Reprodutibilidade dos Testes , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismoRESUMO
Arp2/3 complex is thought to be the primary protrusive force generator in cell migration by controlling the assembly and turnover of the branched filament network that pushes the leading edge of moving cells forward. However, mouse fibroblasts without functional Arp2/3 complex migrate at rates similar to wild-type cells, contradicting this paradigm. We show by correlative fluorescence and large-scale cryo-tomography studies combined with automated actin-network analysis that the absence of functional Arp2/3 complex has profound effects on the nano-scale architecture of actin networks. Our quantitative analysis at the single-filament level revealed that cells lacking functional Arp2/3 complex fail to regulate location-dependent fine-tuning of actin filament growth and organization that is distinct from its role in the formation and regulation of dendritic actin networks.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Fibroblastos/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Actinas/ultraestrutura , Animais , Células Cultivadas , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Fibroblastos/ultraestrutura , CamundongosRESUMO
Integrins are bidirectional, allosteric transmembrane receptors that play a central role in hemostasis and arterial thrombosis. Using cryo-electron microscopy, multireference single-particle reconstruction methods, and statistics-based computational fitting approaches, we determined three-dimensional structures of human integrin αIIbß3 embedded in a lipid bilayer (nanodiscs) while bound to domains of the cytosolic regulator talin and to extracellular ligands. We also determined the conformations of integrin in solution by itself to localize the membrane and the talin-binding site. To our knowledge, our data provide unprecedented three-dimensional information about the conformational states of intact, full-length integrin within membrane bilayers under near-physiological conditions and in the presence of cytosolic activators and extracellular ligands. We show that αIIbß3 integrins exist in a conformational equilibrium clustered around four main states. These conformations range from a compact bent nodule to two partially extended intermediate conformers and finally to a fully upright state. In the presence of nanodiscs and the two ligands, the equilibrium is significantly shifted toward the upright conformation. In this conformation, the receptor extends â¼20 nm upward from the membrane. There are no observable contacts between the two subunits other than those in the headpiece near the ligand-binding pocket, and the α- and ß-subunits are well separated with their cytoplasmic tails â¼8 nm apart. Our results indicate that extension of the ectodomain is possible without separating the legs or extending the hybrid domain, and that the ligand-binding pocket is not occluded by the membrane in any conformations of the equilibrium. Further, they suggest that integrin activation may be influenced by equilibrium shifts.
Assuntos
Membrana Celular/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Citosol/metabolismo , Humanos , Ligantes , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Nanoestruturas/química , Oligopeptídeos/metabolismo , Domínios ProteicosRESUMO
Members of the ErbB family of receptor tyrosine kinases are capable of both homointeractions and heterointeractions. Because each receptor has a unique set of binding sites for downstream signaling partners and differential catalytic activity, subtle shifts in their combinatorial interplay may have a large effect on signaling outcomes. The overexpression and mutation of ErbB family members are common in numerous human cancers and shift the balance of activation within the signaling network. Here we report the development of a spatial stochastic model that addresses the dynamics of ErbB3 homodimerization and heterodimerization with ErbB2. The model is based on experimental measures for diffusion, dimer off-rates, kinase activity, and dephosphorylation. We also report computational analysis of ErbB3 mutations, generating the prediction that activating mutations in the intracellular and extracellular domains may be subdivided into classes with distinct underlying mechanisms. We show experimental evidence for an ErbB3 gain-of-function point mutation located in the C-lobe asymmetric dimerization interface, which shows enhanced phosphorylation at low ligand dose associated with increased kinase activity.