RESUMEN
Contact sites between the endoplasmic reticulum (ER) and the plasma membrane (PM) play a crucial role in governing calcium regulation and lipid homeostasis. Despite their significance, the factors regulating their spatial distribution on the PM remain elusive. Inspired by observations in cardiomyocytes, where ER-PM contact sites concentrate on tubular PM invaginations known as transverse tubules (T-tubules), we hypothesize that the PM curvature plays a role in ER-PM contact formation. Through precise control of PM invaginations, we show that PM curvatures locally induce the formation of ER-PM contacts in cardiomyocytes. Intriguingly, the junctophilin family of ER-PM tethering proteins, specifically expressed in excitable cells, is the key player in this process, while the ubiquitously expressed extended synaptotagmin 2 does not show a preference for PM curvature. At the mechanistic level, we find that the low complexity region (LCR) and the MORN motifs of junctophilins can independently bind to the PM, but both the LCR and MORN motifs are required for targeting PM curvatures. By examining the junctophilin interactome, we identify a family of curvature-sensing proteins, Eps15-homology domain containing proteins (EHDs), that interact with the MORN_LCR motifs and facilitate junctophilins' preferential tethering to curved PM. These findings highlight the pivotal role of PM curvature in the formation of ER-PM contacts in cardiomyocytes and unveil a novel mechanism for the spatial regulation of ER-PM contacts through PM curvature modulation.
RESUMEN
The development of biomaterials capable of regulating cellular processes and guiding cell fate decisions has broad implications in tissue engineering, regenerative medicine, and cell-based assays for drug development and disease modeling. Recent studies have shown that three-dimensional (3D) nanoscale physical cues such as nanotopography can modulate various cellular processes like adhesion and endocytosis by inducing nanoscale curvature on the plasma and nuclear membranes. Two-dimensional (2D) biochemical cues such as protein micropatterns can also regulate cell function and fate by controlling cellular geometries. Development of biomaterials with precise control over nanoscale physical and biochemical cues can significantly influence programming cell function and fate. In this study, we utilized a laser-assisted micropatterning technique to manipulate the 2D architectures of cells on 3D nanopillar platforms. We performed a comprehensive analysis of cellular and nuclear morphology and deformation on both nanopillar and flat substrates. Our findings demonstrate the precise engineering of single cell architectures through 2D micropatterning on nanopillar platforms. We show that the coupling between the nuclear and cell shape is disrupted on nanopillar surfaces compared to flat surfaces. Furthermore, our results suggest that cell elongation on nanopillars enhances nanopillar-induced endocytosis. We believe our platform serves as a versatile tool for further explorations into programming cell function and fate through combined physical cues that create nanoscale curvature on cell membranes and biochemical cues that control the geometry of the cell.
Asunto(s)
Microambiente Celular , Endocitosis , Ingeniería de Tejidos/métodos , Materiales Biocompatibles/química , Humanos , Propiedades de Superficie , Nanoestructuras/química , Animales , Forma de la Célula , Adhesión CelularRESUMEN
Realizing the full potential of organoids and assembloids to model neural development and disease will require improved methods for long-term, minimally invasive recording of electrical activity. Current technologies, such as patch clamp, penetrating microelectrodes, planar electrode arrays and substrate-attached flexible electrodes, do not allow chronic recording of organoids in suspension, which is necessary to preserve architecture. Inspired by kirigami art, we developed flexible electronics that transition from a two-dimensional to a three-dimensional basket-like configuration with either spiral or honeycomb patterns to accommodate the long-term culture of organoids in suspension. Here we show that this platform, named kirigami electronics (KiriE), integrates with and enables chronic recording of cortical organoids for up to 120 days while preserving their morphology, cytoarchitecture and cell composition. We demonstrate integration of KiriE with optogenetic and pharmacological manipulation and modeling phenotypes related to a genetic disease. Moreover, KiriE can capture corticostriatal connectivity in assembloids following optogenetic stimulation. Thus, KiriE will enable investigation of disease and activity patterns underlying nervous system assembly.
RESUMEN
Organoids and assembloids have emerged as a promising platform to model aspects of nervous system development. Longterm, minimally-invasive recordings in these multi-cellular systems are essential for developing disease models. Current technologies, such as patch-clamp, penetrating microelectrodes, planar electrode arrays and substrate-attached flexible electrodes, do not, however, allow chronic recording of organoids in suspension, which is necessary to preserve their architecture. Inspired by the art of kirigami, we developed flexible electronics that transition from a 2D pattern to a 3D basketlike configuration to accommodate the long-term culture of organoids in suspension. This platform, named kirigami electronics (KiriE), integrates with and enables chronic recording of cortical organoids while preserving morphology, cytoarchitecture, and cell composition. KiriE can be integrated with optogenetic and pharmacological stimulation and model disease. Moreover, KiriE can capture activity in cortico-striatal assembloids. Moving forward, KiriE could reveal disease phenotypes and activity patterns underlying the assembly of the nervous system.
RESUMEN
Integrin-mediated focal adhesions are the primary architectures that transmit forces between the extracellular matrix (ECM) and the actin cytoskeleton. Although focal adhesions are abundant on rigid and flat substrates that support high mechanical tensions, they are sparse in soft three-dimensional (3D) environments. Here we report curvature-dependent integrin-mediated adhesions called curved adhesions. Their formation is regulated by the membrane curvatures imposed by the topography of ECM protein fibres. Curved adhesions are mediated by integrin Évß5 and are molecularly distinct from focal adhesions and clathrin lattices. The molecular mechanism involves a previously unknown interaction between integrin ß5 and a curvature-sensing protein, FCHo2. We find that curved adhesions are prevalent in physiological conditions, and disruption of curved adhesions inhibits the migration of some cancer cell lines in 3D fibre matrices. These findings provide a mechanism for cell anchorage to natural protein fibres and suggest that curved adhesions may serve as a potential therapeutic target.
Asunto(s)
Uniones Célula-Matriz , Adhesiones Focales , Adhesión Celular/fisiología , Uniones Célula-Matriz/metabolismo , Adhesiones Focales/metabolismo , Integrinas/genética , Integrinas/metabolismo , Matriz Extracelular/metabolismoRESUMEN
The cell membrane is characterized by a rich variety of topographical features such as local protrusions or invaginations. Curvature-sensing proteins, including the Bin/Amphiphysin/Rvs (BAR) or epsin N-terminal homology (ENTH) family proteins, sense the bending sharpness and the positive/negative sign of these topographical features to induce subsequent intracellular signaling. A number of assays have been developed to study curvature-sensing properties of proteins in vitro, but it is still challenging to probe low curvature regime with the diameter of curvature from hundreds of nanometers to micrometers. It is particularly difficult to generate negative membrane curvatures with well-defined curvature values in the low curvature regime. In this work, we develop a nanostructure-based curvature sensing (NanoCurvS) platform that enables quantitative and multiplex analysis of curvature-sensitive proteins in the low curvature regime, in both negative and positive directions. We use NanoCurvS to quantitatively measure the sensing range of a negative curvature-sensing protein IRSp53 (an I-BAR protein) and a positive curvature-sensing protein FBP17 (an F-BAR protein). We find that, in cell lysates, the I-BAR domain of IRSp53 is able to sense shallow negative curvatures with the diameter-of-curvature up to 1500 nm, a range much wider than previously expected. NanoCurvS is also used to probe the autoinhibition effect of IRSp53 and the phosphorylation effect of FBP17. Therefore, the NanoCurvS platform provides a robust, multiplex, and easy-to-use tool for quantitative analysis of both positive and negative curvature-sensing proteins.
Asunto(s)
Transducción de Señal , Membrana Celular/metabolismoRESUMEN
Mammalian cells adhere to the extracellular matrix (ECM) and sense mechanical cues through integrin-mediated adhesions 1, 2 . Focal adhesions and related structures are the primary architectures that transmit forces between the ECM and the actin cytoskeleton. Although focal adhesions are abundant when cells are cultured on rigid substrates, they are sparse in soft environments that cannot support high mechanical tensions 3 . Here, we report a new class of integrin-mediated adhesions, curved adhesions, whose formation is regulated by membrane curvature instead of mechanical tension. In soft matrices made of protein fibres, curved adhesions are induced by membrane curvatures imposed by the fibre geometry. Curved adhesions are mediated by integrin ÉVß5 and are molecularly distinct from focal adhesions and clathrin lattices. The molecular mechanism involves a previously unknown interaction between integrin ß5 and a curvature-sensing protein FCHo2. We find that curved adhesions are prevalent in physiologically relevant environments. Disruption of curved adhesions by knocking down integrin ß5 or FCHo2 abolishes the migration of multiple cancer cell lines in 3D matrices. These findings provide a mechanism of cell anchorage to natural protein fibres that are too soft to support the formation of focal adhesions. Given their functional importance for 3D cell migration, curved adhesions may serve as a therapeutic target for future development.
RESUMEN
Phase contrast microscopy has played a central role in the development of modern biology, geology, and nanotechnology. It can visualize the structure of translucent objects that remains hidden in regular optical microscopes. The optical layout of a phase contrast microscope is based on a 4 f image processing setup and has essentially remained unchanged since its invention by Zernike in the early 1930s. Here, we propose a conceptually new approach to phase contrast imaging that harnesses the non-local optical response of a guided-mode-resonator metasurface. We highlight its benefits and demonstrate the imaging of various phase objects, including biological cells, polymeric nanostructures, and transparent metasurfaces. Our results showcase that the addition of this non-local metasurface to a conventional microscope enables quantitative phase contrast imaging with a 0.02π phase accuracy. At a high level, this work adds to the growing body of research aimed at the use of metasurfaces for analog optical computing.
Asunto(s)
Microscopía , Nanoestructuras , Microscopía de Contraste de Fase , Procesamiento de Imagen Asistido por Computador , GeologíaRESUMEN
Unintended binding of small-molecule drugs to ion channels affects electrophysiological properties of cardiomyocytes and potentially leads to arrhythmia and heart failure. The waveforms of intracellular action potentials reflect the coordinated activities of cardiac ion channels and serve as a reliable means for assessing drug toxicity, but the implementation is limited by the low throughput of patch clamp for intracellular recording measurements. In the last decade, several new technologies are being developed to address this challenge. We recently developed the nanocrown electrode array (NcEA) technology that allows robust, parallel, and long-duration recording of intracellular action potentials (iAPs). Here, we demonstrate that NcEAs allow comparison of iAP waveforms before and after drug treatment from the same cell. This self-referencing comparison not only shows distinct drug effects of sodium, potassium, and calcium blockers, but also reveals subtle differences among three subclasses of sodium channel blockers with sub-millisecond accuracy. Furthermore, self-referencing comparison unveils heterogeneous drug responses among different cells. In our study, whole-panel simultaneous intracellular recording can be reliably achieved with â¼94% success rate. The average duration of intracellular recording is â¼30 min and some last longer than 2 h. With its high reliability, long recording duration, and easy-to-use nature, NcEA would be useful for iAP-based preclinical drug screening.
Asunto(s)
Técnicas Biosensibles , Cardiotoxicidad , Potenciales de Acción/fisiología , Calcio/metabolismo , Evaluación Preclínica de Medicamentos , Humanos , Canales Iónicos/metabolismo , Miocitos Cardíacos/metabolismo , Potasio/metabolismo , Reproducibilidad de los Resultados , Sodio/metabolismo , Bloqueadores de los Canales de Sodio/farmacologíaRESUMEN
The glycocalyx is a shell of heavily glycosylated proteins and lipids distributed on the cell surface of nearly all cell types. Recently, it has been found that bulky transmembrane glycoproteins such as MUC1 can modulate membrane shape by inducing membrane protrusions. In this work, we examine the reciprocal relationship of how membrane shape affects MUC1's spatial distribution on the cell membrane and its biological significance. By employing nanopatterned surfaces and membrane-sculpting proteins to manipulate membrane curvature, we show that MUC1 avoids positively-curved membranes (membrane invaginations) and accumulates on negatively-curved membranes (membrane protrusions). MUC1's curvature sensitivity is dependent on the length and the extent of glycosylation of its ectodomain, with large and highly glycosylated forms preferentially staying out of positive curvature. Interestingly, MUC1's avoidance of positive membrane curvature enables it to escape from endocytosis and being removed from the cell membrane. These findings also suggest that the truncation of MUC1's ectodomain, often observed in breast and ovarian cancers, may enhance its endocytosis and potentiate its intracellular accumulation and signaling.
Asunto(s)
Endocitosis , Glicoproteínas , Membrana Celular , Fibras de la Dieta , Glicosilación , Proteínas de la MembranaRESUMEN
Surface topography on the scale of tens of nanometers to several micrometers substantially affects cell adhesion, migration, and differentiation. Recent studies using electron microscopy and super-resolution microscopy provide insight into how cells interact with surface nanotopography; however, the complex sample preparation and expensive imaging equipment required for these methods makes them not easily accessible. Expansion microscopy (ExM) is an affordable approach to image beyond the diffraction limit, but ExM cannot be readily applied to image the cell-material interface as most materials do not expand. Here, we develop a protocol that allows the use of ExM to resolve the cell-material interface with high resolution. We apply the technique to image the interface between U2OS cells and nanostructured substrates as well as the interface between primary osteoblasts with titanium dental implants. The high spatial resolution enabled by ExM reveals that although AP2 and F-actin both accumulate at curved membranes induced by vertical nanostructures, they are spatially segregated. Using ExM, we also reliably image how osteoblasts interact with roughened titanium implant surfaces below the diffraction limit; this is of great interest to understand osseointegration of the implants but has up to now been a significant technical challenge due to the irregular shape, the large volume, and the opacity of the titanium implants that have rendered them incompatible with other super-resolution techniques. We believe that our protocol will enable the use of ExM as a powerful tool for cell-material interface studies.
Asunto(s)
Microscopía , Titanio , Titanio/química , Propiedades de Superficie , Oseointegración , OsteoblastosRESUMEN
Drug-induced cardiotoxicity arises primarily when a compound alters the electrophysiological properties of cardiomyocytes. Features of intracellular action potentials (iAPs) are powerful biomarkers that predict proarrhythmic risks. In the last decade, a number of vertical nanoelectrodes have been demonstrated to achieve parallel and minimally-invasive iAP recordings. However, the large variability in success rate and signal strength have hindered nanoelectrodes from being broadly adopted for proarrhythmia drug assessment. In this work, we develop vertically-aligned nanocrown electrodes that are mechanically robust and achieve > 99% success rates in obtaining intracellular access through electroporation. We validate the accuracy of nanocrown electrode recordings by simultaneous patch clamp recording from the same cell. Finally, we demonstrate that nanocrown electrodes enable prolonged iAP recording for continual monitoring of the same cells upon the sequential addition of four incremental drug doses. Our technology development provides an advancement towards establishing an iAP screening assay for preclinical evaluation of drug-induced arrhythmogenicity.
Asunto(s)
Fenómenos Electrofisiológicos , Miocitos Cardíacos , Potenciales de Acción/fisiología , Electrodos , Electroporación , Miocitos Cardíacos/fisiologíaRESUMEN
Plasma membrane topography has been shown to strongly influence the behavior of many cellular processes such as clathrin-mediated endocytosis, actin rearrangements, and others. Recent studies have used three-dimensional (3D) nanostructures such as nanopillars to imprint well-defined membrane curvatures (the "nano-bio interface"). In these studies, proteins and their interactions were probed by two-dimensional fluorescence microscopy. However, the low resolution and limited axial detail of such methods are not optimal to determine the relative spatial position and distribution of proteins along a 100 nm-diameter object, which is below the optical diffraction limit. Here, we introduce a general method to explore the nanoscale distribution of proteins at the nano-bio interface with 10-20 nm precision using 3D single-molecule super-resolution (SR) localization microscopy. This is achieved by combining a silicone-oil immersion objective and 3D double-helix point spread function microscopy. We carefully adjust the objective to minimize spherical aberrations between quartz nanopillars and the cell. To validate the 3D SR method, we imaged the 3D shape of surface-labeled nanopillars and compared the results with electron microscopy measurements. Turning to transmembrane-anchored labels in cells, the high quality 3D SR reconstructions reveal the membrane tightly wrapping around the nanopillars. Interestingly, the cytoplasmic protein AP-2 involved in clathrin-mediated endocytosis accumulates along the nanopillar above a specific threshold of 1/R (the reciprocal of the radius) membrane curvature. Finally, we observe that AP-2 and actin preferentially accumulate at positive Gaussian curvature near the pillar caps. Our results establish a general method to investigate the nanoscale distribution of proteins at the nano-bio interface using 3D SR microscopy.
Asunto(s)
Actinas , Imagen Individual de Molécula , Actinas/metabolismo , Imagen Individual de Molécula/métodos , Microscopía Fluorescente/métodos , Membrana Celular/metabolismo , Clatrina/metabolismoRESUMEN
Both substrate stiffness and surface topography regulate cell behavior through mechanotransduction signaling pathways. Such intertwined effects suggest that engineered surface topographies might substitute or cancel the effects of substrate stiffness in biomedical applications. However, the mechanisms by which cells recognize topographical features are not fully understood. Here we demonstrate that the presence of nanotopography drastically alters cell behavior such that neurons and stem cells cultured on rigid glass substrates behave as if they were on soft hydrogels. With atomic force microscopy, we show that rigid nanotopography resembles the effects of soft hydrogels in reducing cell stiffness and membrane tension. Further, we reveal that nanotopography reduces focal adhesions and cell stiffness by enhancing the endocytosis and the subsequent removal of integrin receptors. This mechanistic understanding will support the rational design of nanotopography that directs cells on rigid materials to behave as if they were on soft ones.
Asunto(s)
Adhesiones Focales , Mecanotransducción Celular , Endocitosis , Integrinas , Células MadreRESUMEN
Heparin and heparan sulfate are glycosaminoglycans that modulate numerous biological processes. The desire to capture the structural diversity responsible for their functions provides notable issues during synthesis, including site-selective sulfonation, stereoselective glycosylation and the sheer number of probable targets at hand. With current advances in synthetic approaches, carbohydrate chemists generate these complex targets by chemical and enzymatic methods. Fondaparinux and a number of polysaccharides have been synthesized to probe anticoagulation and other biological functions. Moreover, a trove of structural information could be obtained by many analytical methods, which provide hints to the potential protein-binding sequences within the sugar chain. Further structure-activity relationship studies help unveil the secrets of the heparin/heparan sulfate code, providing potential candidates for drug development.