RESUMO
Cellular sorting and pattern formation are crucial for many biological processes such as development, tissue regeneration, and cancer progression. Prominent physical driving forces for cellular sorting are differential adhesion and contractility. Here, we studied the segregation of epithelial cocultures containing highly contractile, ZO1/2-depleted MDCKII cells (dKD) and their wild-type (WT) counterparts using multiple quantitative, high-throughput methods to monitor their dynamical and mechanical properties. We observe a time-dependent segregation process governed mainly by differential contractility on short (<5 h) and differential adhesion on long (>5 h) timescales. The overly contractile dKD cells exert strong lateral forces on their WT neighbors, thereby apically depleting their surface area. Concomitantly, the tight junction-depleted, contractile cells exhibit weaker cell-cell adhesion and lower traction force. Drug-induced contractility reduction and partial calcium depletion delay the initial segregation but cease to change the final demixed state, rendering differential adhesion the dominant segregation force at longer timescales. This well-controlled model system shows how cell sorting is accomplished through a complex interplay between differential adhesion and contractility and can be explained largely by generic physical driving forces.
Assuntos
Modelos Biológicos , Contração Muscular , Técnicas de Cocultura , Adesão CelularRESUMO
BACKGROUND: Systemic defects in intestinal iron absorption, circulation, and retention cause iron deficiency in 50% of patients with heart failure. Defective subcellular iron uptake mechanisms that are independent of systemic absorption are incompletely understood. The main intracellular route for iron uptake in cardiomyocytes is clathrin-mediated endocytosis. METHODS: We investigated subcellular iron uptake mechanisms in patient-derived and CRISPR/Cas-edited induced pluripotent stem cell-derived cardiomyocytes as well as patient-derived heart tissue. We used an integrated platform of DIA-MA (mass spectrometry data-independent acquisition)-based proteomics and signaling pathway interrogation. We employed a genetic induced pluripotent stem cell model of 2 inherited mutations (TnT [troponin T]-R141W and TPM1 [tropomyosin 1]-L185F) that lead to dilated cardiomyopathy (DCM), a frequent cause of heart failure, to study the underlying molecular dysfunctions of DCM mutations. RESULTS: We identified a druggable molecular pathomechanism of impaired subcellular iron deficiency that is independent of systemic iron metabolism. Clathrin-mediated endocytosis defects as well as impaired endosome distribution and cargo transfer were identified as a basis for subcellular iron deficiency in DCM-induced pluripotent stem cell-derived cardiomyocytes. The clathrin-mediated endocytosis defects were also confirmed in the hearts of patients with DCM with end-stage heart failure. Correction of the TPM1-L185F mutation in DCM patient-derived induced pluripotent stem cells, treatment with a peptide, Rho activator II, or iron supplementation rescued the molecular disease pathway and recovered contractility. Phenocopying the effects of the TPM1-L185F mutation into WT induced pluripotent stem cell-derived cardiomyocytes could be ameliorated by iron supplementation. CONCLUSIONS: Our findings suggest that impaired endocytosis and cargo transport resulting in subcellular iron deficiency could be a relevant pathomechanism for patients with DCM carrying inherited mutations. Insight into this molecular mechanism may contribute to the development of treatment strategies and risk management in heart failure.
Assuntos
Cardiomiopatia Dilatada , Insuficiência Cardíaca , Células-Tronco Pluripotentes Induzidas , Deficiências de Ferro , Humanos , Miócitos Cardíacos/metabolismo , Mutação , Cardiomiopatia Dilatada/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Ferro/metabolismo , Clatrina/genética , Clatrina/metabolismo , Clatrina/farmacologiaRESUMO
Integration of multiple data sources presents a challenge for accurate prediction of molecular patho-phenotypic features in automated analysis of data from human model systems. Here, we applied a machine learning-based data integration to distinguish patho-phenotypic features at the subcellular level for dilated cardiomyopathy (DCM). We employed a human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model of a DCM mutation in the sarcomere protein troponin T (TnT), TnT-R141W, compared to isogenic healthy (WT) control iPSC-CMs. We established a multimodal data fusion (MDF)-based analysis to integrate source datasets for Ca2+ transients, force measurements, and contractility recordings. Data were acquired for three additional layer types, single cells, cell monolayers, and 3D spheroid iPSC-CM models. For data analysis, numerical conversion as well as fusion of data from Ca2+ transients, force measurements, and contractility recordings, a non-negative blind deconvolution (NNBD)-based method was applied. Using an XGBoost algorithm, we found a high prediction accuracy for fused single cell, monolayer, and 3D spheroid iPSC-CM models (≥92 ± 0.08â¯%), as well as for fused Ca2+ transient, beating force, and contractility models (>96 ± 0.04â¯%). Integrating MDF and XGBoost provides a highly effective analysis tool for prediction of patho-phenotypic features in complex human disease models such as DCM iPSC-CMs.
Assuntos
Cardiomiopatia Dilatada , Células-Tronco Pluripotentes Induzidas , Aprendizado de Máquina , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/patologia , Cardiomiopatia Dilatada/patologia , Cardiomiopatia Dilatada/metabolismo , Humanos , Fenótipo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Troponina T/metabolismo , Cálcio/metabolismoRESUMO
The cytoskeleton, an intricate network of protein filaments, motor proteins, and cross-linkers, largely determines the mechanical properties of cells. Among the three filamentous components, F-actin, microtubules, and intermediate filaments (IFs), the IF network is by far the most extensible and resilient to stress. We present a multiscale approach to disentangle the three main contributions to vimentin IF network mechanics-single-filament mechanics, filament length, and interactions between filaments-including their temporal evolution. Combining particle tracking, quadruple optical trapping, and computational modeling, we derive quantitative information on the strength and kinetics of filament interactions. Specifically, we find that hydrophobic contributions to network mechanics enter mostly via filament-elongation kinetics, whereas electrostatics have a direct influence on filament-filament interactions.
Assuntos
Filamentos Intermediários/metabolismo , Vimentina/metabolismo , Detergentes/farmacologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Íons , Modelos Biológicos , Eletricidade Estática , Fatores de TempoRESUMO
Cell adhesion molecules are crucial for a variety of biological processes, including wound healing, barrier formation and tissue homeostasis. One of them is E-cadherin which is generally found at adherent junctions between epithelial cells. To identify this molecule on the surface of cells, E-cadherin mimetic peptides with a critical amino acid sequence of HAV (histidine-alanine-valine) were synthesized and attached to solid-supported membranes covering colloidal probes. Two different functionalization strategies were established, one based on the complexation of DOGS-NTA(Ni) with a polyhistidine-tagged HAV-peptide and the other one relying on the formation of a HAV-lipopeptide using inâ situ maleimide-thiol coupling. Binding studies were performed to verify the ability of the peptides to attach to the membrane surface. Compared to the non-covalent attachment via the His-tag, we achieved a higher yield by lipopeptide formation. Colloidal probes functionalized with HAV-peptides were employed to measure the presence of E-cadherins on living cells either using video particle tracking or force spectroscopy. Here, human HaCaT cells were examined confirming the specific interaction of the HAV-peptide with the E-cadherin of the cells. Statistical methods were also used to determine the number of single-bond ruptures and the force of a single bond. These findings may be essential for the development of novel biosynthetic materials given their potential to become increasingly relevant in medical applications.
Assuntos
Caderinas , Células Epiteliais , Humanos , Caderinas/química , Caderinas/metabolismo , Linhagem Celular , Sequência de Aminoácidos , Lipopeptídeos/metabolismoRESUMO
A mechanism for full-length synaptotagmin-1 (syt-1) to interact with anionic bilayers and to promote fusion in the presence of SNAREs is proposed. Colloidal probe force spectroscopy in conjunction with tethered particle motion monitoring showed that in the absence of Ca2+ the binding of syt-1 to membranes depends on the presence and content of PI(4,5)P2. Addition of Ca2+ switches the interaction forces from weak to strong, eventually exceeding the cohesion of the C2A domain of syt-1 leading to partial unfolding of the protein. Fusion of single unilamellar vesicles equipped with syt-1 and synaptobrevin 2 with planar pore-spanning target membranes containing PS and PI(4,5)P2 shows an almost complete suppression of stalled intermediate fusion states and an accelerated fusion kinetics in the presence of Ca2+, which is further enhanced upon addition of ATP.
Assuntos
Cálcio , Fosfatidilinositol 4,5-Difosfato , Proteínas SNARE , Sinaptotagmina I , Cálcio/química , Cálcio/metabolismo , Cinética , Fusão de Membrana , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Desdobramento de Proteína , Sinaptotagmina I/química , Sinaptotagmina I/metabolismoRESUMO
Atomic force microscopy is used to study the viscoelastic properties of epithelial cells in three different states. Force relaxation data are acquired from cells in suspension, adhered but single cells, and polarized cells in a confluent monolayer using different indenter geometries comprising flat bars, pyramidal cones, and spheres. We found that the fluidity of cells increased substantially from the suspended to the adherent state. Along this line, the prestress of suspended cells generated by cortical contractility is also greater than that of cells adhering to a surface. Polarized cells that are part of a confluent monolayer form an apical cap that is soft and fluid enough to respond rapidly to mechanical challenges from wounding, changes in the extracellular matrix, osmotic stress, and external deformation. In contrast to adherent cells, cells in the suspended state show a pronounced dependence of fluidity on the external areal strain. With increasing areal strain, the suspended cells become softer and more fluid. We interpret the results in terms of cytoskeletal remodeling that softens cells in the adherent state to facilitate adhesion and spreading by relieving internal active stress. However, once the cells spread on the surface they maintain their mechanical phenotype displaying viscoelastic homeostasis.
Assuntos
Células Epiteliais , Fenômenos Mecânicos , Adesão Celular , Matriz Extracelular , Homeostase , Microscopia de Força Atômica , Estresse MecânicoRESUMO
Near-infrared (NIR) fluorophores are emerging tools for biophotonics because of their reduced scattering, increased tissue penetration and low phototoxicity. However, the library of NIR fluorophores is still limited. Here, we report the NIR fluorescence of two benzene-fused oligo-BODIPYs in their hexameric (H) and octameric (O) forms. These dyes emit bright NIR fluorescence (H: maxima 943/1075 nm, O: maxima 976/1115 nm) that can be excited in the NIR (H = 921 nm, O = 956 nm) or non-resonantly over a broad range in the visible region. The emission bands of H show a bathochromic shift and peak sharpening with increasing dye concentration. Furthermore, the emission maxima of both H and O shift up to 20 nm in solvents of different polarity. These dyes can be used as NIR ink and imaged remotely on the macroscopic level with a stand-off distance of 20 cm. We furthermore demonstrate their versatility for biophotonics by coating microscale beads and performing microrheology via NIR video particle tracking (NIR-VPT) in biopolymer (F-actin) networks. No photodamaging of the actin filaments takes place, which is typically observed for visible fluorophores and highlights the advantages of these NIR dyes.
Assuntos
Benzeno , Corantes Fluorescentes , Benzeno/toxicidade , Compostos de Boro , Fluorescência , Corantes Fluorescentes/toxicidadeRESUMO
Epithelial cells form tight barriers that line both the outer and inner surfaces of organs and cavities and therefore face diverse environmental challenges. The response to these challenges relies on the cells' dynamic viscoelastic properties, playing a pivotal role in many biological processes such as adhesion, growth, differentiation, and motility. Therefore, the cells usually adapt their viscoelastic properties to mirror the environment that determines their fate and vitality. Albeit not a high-throughput method, atomic force microscopy is still among the dominating methods to study the mechanical properties of adherent cells since it offers a broad range of forces from Piconewtons to Micronewtons at biologically significant time scales. Here, some recent work of deformation studies on epithelial cells is reviewed with a focus on viscoelastic models suitable to describe force cycle measurements congruent with the architecture of the actin cytoskeleton. The prominent role of the cortex in the cell's response to external forces is discussed also in the context of isolated cortex extracts on porous surfaces.
Assuntos
Elasticidade , Viscosidade , Células Epiteliais/citologia , Humanos , Microscopia de Força AtômicaRESUMO
Arginine (R)-rich peptides constitute the most relevant class of cell-penetrating peptides and other membrane-active peptides that can translocate across the cell membrane or generate defects in lipid bilayers such as water-filled pores. The mode of action of R-rich peptides remains a topic of controversy, mainly because a quantitative and energetic understanding of arginine effects on membrane stability is lacking. Here, we explore the ability of several oligo-arginines R[Formula: see text] and of an arginine side chain mimic R[Formula: see text] to induce pore formation in lipid bilayers employing MD simulations, free-energy calculations, breakthrough force spectroscopy and leakage assays. Our experiments reveal that R[Formula: see text] but not R[Formula: see text] reduces the line tension of a membrane with anionic lipids. While R[Formula: see text] peptides form a layer on top of a partly negatively charged lipid bilayer, R[Formula: see text] leads to its disintegration. Complementary, our simulations show R[Formula: see text] causes membrane thinning and area per lipid increase beside lowering the pore nucleation free energy. Model polyarginine R[Formula: see text] similarly promoted pore formation in simulations, but without overall bilayer destabilization. We conclude that while the guanidine moiety is intrinsically membrane-disruptive, poly-arginines favor pore formation in negatively charged membranes via a different mechanism. Pore formation by R-rich peptides seems to be counteracted by lipids with PC headgroups. We found that long R[Formula: see text] and R[Formula: see text] but not short R[Formula: see text] reduce the free energy of nucleating a pore. In short R[Formula: see text], the substantial effect of the charged termini prevent their membrane activity, rationalizing why only longer [Formula: see text] are membrane-active.
Assuntos
Arginina/química , Membrana Celular/metabolismo , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , TermodinâmicaRESUMO
Membrane-coated colloidal probes combine the benefits of solid-supported membranes with a more complex three-dimensional geometry. This combination makes them a powerful model system that enables the visualization of dynamic biological processes with high throughput and minimal reliance on fluorescent labels. Here, we want to review recent applications of colloidal probes for the study of membrane fusion. After discussing the advantages and disadvantages of some classical vesicle-based fusion assays, we introduce an assay using optical detection of fusion between membrane-coated glass microspheres in a quasi two-dimensional assembly. Then, we discuss free energy considerations of membrane fusion between supported bilayers, and show how colloidal probes can be combined with atomic force microscopy or optical tweezers to access the fusion process with even greater detail.
Assuntos
Fusão de Membrana , Bicamadas Lipídicas , Microscopia de Força Atômica , Pinças ÓpticasRESUMO
The isotype-specific composition of the keratin cytoskeleton is important for strong adhesion, force resilience, and barrier function of the epidermis. However, the mechanisms by which keratins regulate these functions are still incompletely understood. In this study, the role and significance of the keratin network for mechanical integrity, force transmission, and barrier formation were analyzed in murine keratinocytes. Following the time-course of single-cell wound closure, wild-type (WT) cells slowly closed the gap in a collective fashion involving tightly connected neighboring cells. In contrast, the mechanical response of neighboring cells was compromised in keratin-deficient cells, causing an increased wound area initially and an inefficient overall wound closure. Furthermore, the loss of the keratin network led to impaired, fragmented cell-cell junctions, and triggered a profound change in the overall cellular actomyosin architecture. Electric cell-substrate impedance sensing of cell junctions revealed a dysfunctional barrier in knockout (Kty-/-) cells compared to WT cells. These findings demonstrate that Kty-/- cells display a novel phenotype characterized by loss of mechanocoupling and failure to form a functional barrier. Re-expression of K5/K14 rescued the barrier defect to a significant extent and reestablished the mechanocoupling with remaining discrepancies likely due to the low abundance of keratins in that setting. Our study reveals the major role of the keratin network for mechanical homeostasis and barrier functionality in keratinocyte layers.
Assuntos
Queratinócitos/citologia , Queratinas/metabolismo , Animais , Fenômenos Biomecânicos , Linhagem Celular , Epiderme/metabolismo , Epiderme/ultraestrutura , Deleção de Genes , Junções Intercelulares/genética , Junções Intercelulares/metabolismo , Junções Intercelulares/ultraestrutura , Queratinócitos/metabolismo , Queratinas/genética , Queratinas/ultraestrutura , Camundongos , CicatrizaçãoRESUMO
Cell cortices are responsible for the resilience and morphological dynamics of cells. Measuring their mechanical properties is impeded by contributions from other filament types, organelles, and the crowded cytoplasm. We established a versatile concept for the precise assessment of cortical viscoelasticity based on force cycle experiments paired with continuum mechanics. Apical cell membranes of confluent MDCK II cells were deposited on porous substrates and locally deformed. Force cycles could be described with a time-dependent area compressibility modulus obeying the same power law as employed for whole cells. The reduced fluidity of apical cell membranes compared to living cells could partially be restored by reactivating myosin motors. A comparison with artificial minimal actin cortices (MACs) reveals lower stiffness and higher fluidity attributed to missing cross-links in MACs.
Assuntos
Actinas , Miosinas , Citoesqueleto , Porosidade , ViscosidadeRESUMO
Shape, dynamics, and viscoelastic properties of eukaryotic cells are primarily governed by a thin, reversibly cross-linked actomyosin cortex located directly beneath the plasma membrane. We obtain time-dependent rheological responses of fibroblasts and MDCK II cells from deformation-relaxation curves using an atomic force microscope to access the dependence of cortex fluidity on prestress. We introduce a viscoelastic model that treats the cell as a composite shell and assumes that relaxation of the cortex follows a power law giving access to cortical prestress, area-compressibility modulus, and the power law exponent (fluidity). Cortex fluidity is modulated by interfering with myosin activity. We find that the power law exponent of the cell cortex decreases with increasing intrinsic prestress and area-compressibility modulus, in accordance with previous finding for isolated actin networks subject to external stress. Extrapolation to zero tension returns the theoretically predicted power law exponent for transiently cross-linked polymer networks. In contrast to the widely used Hertzian mechanics, our model provides viscoelastic parameters independent of indenter geometry and compression velocity.
Assuntos
Actinas/química , Fibroblastos/química , Fibroblastos/citologia , Modelos Biológicos , Actinas/fisiologia , Animais , Fenômenos Biomecânicos , Linhagem Celular , Membrana Celular/química , Membrana Celular/fisiologia , Força Compressiva , Cães , Elasticidade , Microscopia de Força Atômica , Miosinas/química , Miosinas/fisiologia , Reologia/métodos , ViscosidadeRESUMO
Cellular adhesion is an intricate physical process controlled by ligand-receptor affinity, density, mobility, and external forces transmitted through the elastic properties of the cell. As a model for cellular adhesion we study the detachment of cell-sized liposomes and membrane-coated silica beads from supported bilayers using atomic force microscopy. Adhesion between the two surfaces is mediated by the interaction between the adhesive lipid anchored saccharides lactosylceramide and the ganglioside GM3. We found that force-distance curves of liposome detachment have a very peculiar, partially concave shape, reminiscent of the nonlinear extension of polymers. By contrast, detachment of membrane coated beads led to force-distance curves similar to the detachment of living cells. Theoretical modelling of the enforced detachment suggests that the non-convex force curve shape arises from the mobility of ligands provoking a switch of shapes from spherical to unduloidal during detachment.
Assuntos
Lipossomos , Adesão Celular , Ligantes , Membranas , Microscopia de Força AtômicaRESUMO
In the plasma membrane of eukaryotic cells, proteins and lipids are organized in clusters, the latter ones often called lipid domains or "lipid rafts." Recent findings highlight the dynamic nature of such domains and the key role of membrane geometry and spatial boundaries. In this study, we used porous substrates with different pore radii to address precisely the extent of the geometric constraint, permitting us to modulate and investigate the size and mobility of lipid domains in phase-separated continuous pore-spanning membranes (PSMs). Fluorescence video microscopy revealed two types of liquid-ordered (lo) domains in the freestanding parts of the PSMs: (i) immobile domains that were attached to the pore rims and (ii) mobile, round-shaped lo domains within the center of the PSMs. Analysis of the diffusion of the mobile lo domains by video microscopy and particle tracking showed that the domains' mobility is slowed down by orders of magnitude compared with the unrestricted case. We attribute the reduced mobility to the geometric confinement of the PSM, because the drag force is increased substantially due to hydrodynamic effects generated by the presence of these boundaries. Our system can serve as an experimental test bed for diffusion of 2D objects in confined geometry. The impact of hydrodynamics on the mobility of enclosed lipid domains can have great implications for the formation and lateral transport of signaling platforms.
Assuntos
Microdomínios da Membrana/fisiologia , Difusão , HidrodinâmicaRESUMO
Intermediate filaments (IFs) are part of the cytoskeleton of eukaryotic cells and, therefore, are largely responsible for the cell's mechanical properties. IFs are characterized by a pronounced extensibility and remarkable resilience that enable them to support cells in extreme situations. Previous experiments showed that, under strain, α-helices in vimentin IFs might unfold to ß-sheets. Upon repeated stretching, the filaments soften; however, the remaining plastic strain is negligible. Here, we observe that vimentin IFs do not recover their original stiffness on reasonable time scales, and we explain these seemingly contradicting results by introducing a third, less well-defined conformational state. Reversibility on the nanoscale can be fully rescued by introducing cross-linkers that prevent transition to the ß-sheet. Our results classify IFs as a nanomaterial with intriguing mechanical properties, which is likely to play a major role for the cell's local adaption to external stimuli.
Assuntos
Filamentos Intermediários/química , Vimentina/química , Fenômenos Biomecânicos , Humanos , Nanoestruturas/química , Pinças Ópticas , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Estresse MecânicoRESUMO
Mechanical phenotyping of adherent cells has become a serious tool in cell biology to understand how cells respond to their environment and eventually to identify disease patterns such as the malignancy of cancer cells. In the steady state, homeostasis is of pivotal importance, and cells strive to maintain their internal stresses even in challenging environments and in response to external chemical and mechanical stimuli. However, a major problem exists in determining mechanical properties because many techniques, such as atomic force microscopy, that assess these properties of adherent cells locally can only address a limited number of cells and provide elastic moduli that vary substantially from cell to cell. The origin of this spread in stiffness values is largely unknown and might limit the significance of measurements. Possible reasons for the disparity are variations in cell shape and size, as well as biological reasons such as the cell cycle or polarization state of the cell. Here, we show that stiffness of adherent epithelial cells rises with increasing projected apical cell area in a nonlinear fashion. This size stiffening not only occurs as a consequence of varying cell-seeding densities, it can also be observed within a small area of a particular cell culture. Experiments with single adherent cells attached to defined areas via microcontact printing show that size stiffening is limited to cells of a confluent monolayer. This leads to the conclusion that cells possibly regulate their size distribution through cortical stress, which is enhanced in larger cells and reduced in smaller cells.
Assuntos
Tamanho Celular , Fenômenos Mecânicos , Animais , Fenômenos Biomecânicos , Adesão Celular , Cães , Células Madin Darby de Rim Canino , Fenótipo , Análise de Célula ÚnicaRESUMO
Fusion of lipid bilayers is usually prevented by large energy barriers arising from removal of the hydration shell, formation of highly curved structures, and, eventually, fusion pore widening. Here, we measured the force-dependent lifetime of fusion intermediates using membrane-coated silica spheres attached to cantilevers of an atomic-force microscope. Analysis of time traces obtained from force-clamp experiments allowed us to unequivocally assign steps in deflection of the cantilever to membrane states during the SNARE-mediated fusion with solid-supported lipid bilayers. Force-dependent lifetime distributions of the various intermediate fusion states allowed us to propose the likelihood of different fusion pathways and to assess the main free energy barrier, which was found to be related to passing of the hydration barrier and splaying of lipids to eventually enter either the fully fused state or a long-lived hemifusion intermediate. The results were compared with SNARE mutants that arrest adjacent bilayers in the docked state and membranes in the absence of SNAREs but presence of PEG or calcium. Only with the WT SNARE construct was appreciable merging of both bilayers observed.
Assuntos
Fusão de Membrana , Proteínas SNARE/química , Bicamadas Lipídicas/química , Lipossomos/química , Microscopia/métodos , Dióxido de Silício/químicaRESUMO
The thylakoid membrane of algae and land plants is characterized by its intricate architecture, comprising tightly appressed membrane stacks termed grana. The contributions of individual components to grana stack formation are not yet fully elucidated. As an in vitro model, we use supported lipid bilayers made of thylakoid lipid mixtures to study the effect of major light-harvesting complex (LHCII), different lipids, and ions on membrane stacking, seen as elevated structures forming on top of the planar membrane surface in the presence of LHCII protein. These structures were examined by confocal laser scanning microscopy, atomic force microscopy, and fluorescence recovery after photobleaching, revealing multilamellar LHCII-membrane stacks composed of connected lipid bilayers. Both native-like and non-native interactions between the LHCII complexes may contribute to membrane appression in the supported bilayers. However, applying in vivo-like salt conditions to uncharged glycolipid membranes drastically increased the level of stack formation due to enforced LHCII-LHCII interactions, which is in line with recent crystallographic and cryo-electron microscopic data [Wan, T., et al. (2014) Mol. Plant 7, 916-919; Albanese, P., et al. (2017) Sci. Rep. 7, 10067-10083]. Furthermore, we observed the nonbilayer lipid MGDG to strongly promote membrane stacking, pointing to the long-term proposed function of MGDG in stabilizing the inner membrane leaflet of highly curved margins in the periphery of each grana disc because of its negative intrinsic curvature [Murphy, D. J. (1982) FEBS Lett. 150, 19-26].