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Intracellular electrophysiology is essential in neuroscience, cardiology, and pharmacology for studying cells' electrical properties. Traditional methods like patch-clamp are precise but low-throughput and invasive. Nanoelectrode Arrays (NEAs) offer a promising alternative by enabling simultaneous intracellular and extracellular action potential (iAP and eAP) recordings with high throughput. However, accessing intracellular potentials with NEAs remains challenging. This study presents an AI-supported technique that leverages thousands of synchronous eAP and iAP pairs from stem-cell-derived cardiomyocytes on NEAs. Our analysis revealed strong correlations between specific eAP and iAP features, such as amplitude and spiking velocity, indicating that extracellular signals could be reliable indicators of intracellular activity. We developed a physics-informed deep learning model to reconstruct iAP waveforms from extracellular recordings recorded from NEAs and Microelectrode arrays (MEAs), demonstrating its potential for non-invasive, long-term, high-throughput drug cardiotoxicity assessments. This AI-based model paves the way for future electrophysiology research across various cell types and drug interactions., Competing Interests: Competing interests: A patent application (application number 63/717,739) has been filed by the University of California San Diego and Stanford University. The named inventors on this patent application are Zeinab Jahed, Keivan Rahmani, Bianxiao Cui, and Csaba Farro. This application covers the method for intracellular recording using an AI model trained on intra- and extracellular paired data, which is a specific aspect of the manuscript. All other authors declare no competing interest. Ethics: This study does not involve experiments involving animals, human participants, or clinical samples., (© 2025. The Author(s).)

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., Competing Interests: Competing interests: Stanford University holds the patent for the generation of cortical organoids/spheroids (US11279914B2, listing S.P.P. as an inventor). Stanford University has filed a patent application that covers the devices, systems and methods of KiriE (S22-514 63/435,939, listing B.C., S.P.P., X.Y. and C.F. as inventors). The other authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Contact sites between the endoplasmic reticulum (ER) and 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, we hypothesize that 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, whereas 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 membrane occupation and recognition nexus (MORN) motifs of junctophilins can bind independently 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-that interact with the MORN_LCR motifs and facilitate the preferential tethering of junctophilins to curved PM. These findings highlight the pivotal role of PM curvature in the formation of ER-PM contacts in cardiomyocytes and unveil a mechanism for the spatial regulation of ER-PM contacts through PM curvature modulation., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Neural circuits connecting the cerebral cortex, the basal ganglia and the thalamus are fundamental networks for sensorimotor processing and their dysfunction has been consistently implicated in neuropsychiatric disorders 1-9 . These recursive, loop circuits have been investigated in animal models and by clinical neuroimaging, however, direct functional access to developing human neurons forming these networks has been limited. Here, we use human pluripotent stem cells to reconstruct an in vitro cortico-striatal-thalamic-cortical circuit by creating a four-part loop assembloid. More specifically, we generate regionalized neural organoids that resemble the key elements of the cortico-striatal-thalamic-cortical circuit, and functionally integrate them into loop assembloids using custom 3D-printed biocompatible wells. Volumetric and mesoscale calcium imaging, as well as extracellular recordings from individual parts of these assembloids reveal the emergence of synchronized patterns of neuronal activity. In addition, a multi-step rabies retrograde tracing approach demonstrate the formation of neuronal connectivity across the network in loop assembloids. Lastly, we apply this system to study heterozygous loss of ASH1L gene associated with autism spectrum disorder and Tourette syndrome and discover aberrant synchronized activity in disease model assembloids. Taken together, this human multi-cellular platform will facilitate functional investigations of the cortico-striatal-thalamic-cortical circuit in the context of early human development and in disease conditions.

TANGO1, TANGO1-Short, and cTAGE5 form stable complexes at the endoplasmic reticulum exit sites (ERES) to preferably export bulky cargoes. Their C-terminal proline-rich domain (PRD) binds Sec23A and affects COPII assembly. The PRD in TANGO1-Short was replaced with light-responsive domains to control its binding to Sec23A in U2OS cells (human osteosarcoma). TANGO1-ShortΔPRD was dispersed in the ER membrane but relocated rapidly, reversibly, to pre-existing ERES by binding to Sec23A upon light activation. Prolonged binding between the two, concentrated ERES in the juxtanuclear region, blocked cargo export and relocated ERGIC53 into the ER, minimally impacting the Golgi complex organization. Bulky collagen VII and endogenous collagen I were collected at less than 47% of the stalled ERES, whereas small cargo molecules were retained uniformly at almost all the ERES. We suggest that ERES are segregated to handle cargoes based on their size, permitting cells to traffic them simultaneously for optimal secretion., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Morphogenesis requires building stable macromolecular structures from highly dynamic proteins. Muscles are anchored by long-lasting integrin adhesions to resist contractile force. However, the mechanisms governing integrin diffusion, immobilization, and activation within developing tissues remain elusive. Here, we show that actin polymerization-driven membrane protrusions form nanotopographies that enable strong adhesion at Drosophila muscle attachment sites (MASs). Super-resolution microscopy reveals that integrins assemble adhesive belts around Arp2/3-dependent actin protrusions, forming invadosome-like structures with membrane nanotopographies. Single protein tracking shows that, during MAS development, integrins become immobile and confined within diffusion traps formed by the membrane nanotopographies. Actin filaments also display restricted motion and confinement, indicating strong mechanical connection with integrins. Using isolated muscle cells, we show that substrate nanotopography, rather than rigidity, drives adhesion maturation by regulating actin protrusion, integrin diffusion and immobilization. These results thus demonstrate that actin-polymerization-driven membrane protrusions are essential for the formation of strong integrin adhesions sites in the developing embryo, and highlight the important contribution of geometry to morphogenesis., (© 2024. The Author(s).)

Subcellular protein localization regulates protein function and can be corrupted in cancers 1 and neurodegenerative diseases 2,3 . The rewiring of localization to address disease-driving phenotypes would be an attractive targeted therapeutic approach. Molecules that harness the trafficking of a shuttle protein to control the subcellular localization of a target protein could enforce targeted protein relocalization and rewire the interactome. Here we identify a collection of shuttle proteins with potent ligands amenable to incorporation into targeted relocalization-activating molecules (TRAMs), and use these to relocalize endogenous proteins. Using a custom imaging analysis pipeline, we show that protein steady-state localization can be modulated through molecular coupling to shuttle proteins containing sufficiently strong localization sequences and expressed in the necessary abundance. We analyse the TRAM-induced relocalization of different proteins and then use nuclear hormone receptors as shuttles to redistribute disease-driving mutant proteins such as SMARCB1 Q318X , TDP43 ΔNLS and FUS R495X . TRAM-mediated relocalization of FUS R495X to the nucleus from the cytoplasm correlated with a reduction in the number of stress granules in a model of cellular stress. With methionyl aminopeptidase 2 and poly(ADP-ribose) polymerase 1 as endogenous cytoplasmic and nuclear shuttles, respectively, we demonstrate relocalization of endogenous PRMT9, SOS1 and FKBP12. Small-molecule-mediated redistribution of nicotinamide nucleotide adenylyltransferase 1 from nuclei to axons in primary neurons was able to slow axonal degeneration and pharmacologically mimic the genetic WldS gain-of-function phenotype in mice resistant to certain types of neurodegeneration 4 . The concept of targeted protein relocalization could therefore inspire approaches for treating disease through interactome rewiring., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Cytoplasmic dynein is essential for intracellular transport. Despite extensive in vitro characterizations, how the dynein motors transport vesicles by processive steps in live cells remains unclear. To dissect the molecular mechanisms of dynein, we develop optical probes that enable long-term single-particle tracking in live cells with high spatiotemporal resolution. We find that the number of active dynein motors transporting cargo switches stochastically between one and five dynein motors during long-range transport in neuronal axons. Our very bright optical probes allow the observation of individual molecular steps. Strikingly, these measurements reveal that the dwell times between steps are controlled by two temperature-dependent rate constants in which two ATP molecules are hydrolyzed sequentially during each dynein step. Thus, our observations uncover a previously unknown chemomechanical cycle of dynein-mediated cargo transport in living cells., (© 2024. The Author(s).)

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.

Chronic pain is a prevalent problem that plagues modern society, and better understanding its mechanisms is critical for developing effective therapeutics. Nerve growth factor (NGF) and its primary receptor, Tropomyosin receptor kinase A (TrkA), are known to be potent mediators of chronic pain, but there is a lack of established methods for precisely perturbing the NGF/TrkA signaling pathway in the study of pain and nociception. Optobiological tools that leverage light-induced protein-protein interactions allow for precise spatial and temporal control of receptor signaling. Previously, our lab reported a blue light-activated version of TrkA generated using light-induced dimerization of the intracellular TrkA domain, opto-iTrkA. In this work, we show that opto-iTrkA activation is able to activate endogenous ERK and Akt signaling pathways and causes the retrograde transduction of phospho-ERK signals in dorsal root ganglion (DRG) neurons. Opto-iTrkA activation also sensitizes the transient receptor potential vanilloid 1 (TRPV1) channel in cellular models, further corroborating the physiological relevance of the optobiological stimulus. Finally, we show that opto-iTrkA enables light-inducible potentiation of mechanical sensitization in mice. Light illumination enables nontraumatic and reversible (<2 days) sensitization of mechanical pain in mice transduced with opto-iTrkA, which provides a platform for dissecting TrkA pathways for nociception in vitro and in vivo .

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&#95;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.

Electrochromic optical recording (ECORE) is a label-free method that utilizes electrochromism to optically detect electrical signals in biological cells with a high signal-to-noise ratio and is suitable for long-term recording. However, ECORE usually requires a large and intricate optical setup, making it relatively difficult to transport and to study specimens on a large scale. Here, we present a Compact ECORE (CECORE) apparatus that drastically reduces the spatial footprint and complexity of the ECORE setup whilst maintaining high sensitivity. An autobalancing differential photodetector automates common-mode noise rejection, removing the need for manually adjustable optics, and a compact laser module conserves space compared to a typical laser mount. The result is a simple, easy-to-use, and relatively low cost system that achieves a sensitivity of 16.7 μV (within a factor of 5 of the shot noise limit), and reliably detects action potentials from Human-induced pluripotent stem cell (HiPSC) derived cardiomyocytes. This setup can be further improved to within 1.5 dB of the shot noise limit by filtering out power-line interference.

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 ɑvβ5 and are molecularly distinct from focal adhesions and clathrin lattices. The molecular mechanism involves a previously unknown interaction between integrin β5 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., (© 2023. The Author(s).)

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.

Protein dysregulation has been characterized as the cause of pathogenesis in many different diseases. For proteins lacking easily druggable pockets or catalytically active sites, targeted protein degradation is an attractive therapeutic approach. While several methods for targeted protein degradation have been developed, there remains a demand for lower molecular weight molecules that promote efficient degradation of their targets. In this work, we describe the synthesis and validation of a series of heterobifunctional molecules that bind a protein of interest through a small molecule ligand while targeting them to the lysosome using a short gluten peptide that leverages the TG2/LRP-1 pathway. We demonstrate that this approach can be used to effectively endocytose and degrade representative secreted, cell surface, and transmembrane proteins, notably streptavidin, the vitamin B12 receptor, cubilin, and integrin α v β 5 . Optimization of these prototypical molecules could generate pharmacologically relevant LYTAC agents.

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.

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 ɑVβ5 and are molecularly distinct from focal adhesions and clathrin lattices. The molecular mechanism involves a previously unknown interaction between integrin β5 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 β5 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.

Optical recording based on voltage-sensitive fluorescent reporters allows for spatial flexibility of measuring from desired cells, but photobleaching and phototoxicity of the fluorescent labels often limit their sensitivity and recording duration. Voltage-dependent optical absorption, rather than fluorescence, of electrochromic materials, would overcome these limitations to achieve long-term optical recording of bioelectrical signals. Electrochromic materials such as PEDOT:PSS possess the property that an applied voltage can either increase or decrease the light absorption depending on the wavelength. In this work, we harness this anticorrelated light absorption at two different wavelengths to significantly improve the signal detection. With dual-color detection, electrical activity from cells produces signals of opposite polarity, while artifacts, mechanical motions, and technical noises are uncorrelated or positively correlated. Using this technique, we are able to optically record cardiac action potentials with a high signal-to-noise ratio, 10 kHz sampling rate, >15 min recording duration, and no time-dependent degradation of the signal. Furthermore, we can reliably perform multiple recording sessions from the same culture for over 25 days.

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., (© 2022. The Author(s).)

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 ∼94% success rate. The average duration of intracellular recording is ∼30 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Advances in tridimensional (3D) culture approaches have led to the generation of organoids that recapitulate cellular and physiological features of domains of the human nervous system. Although microelectrodes have been developed for long-term electrophysiological interfaces with neural tissue, studies of long-term interfaces between microelectrodes and free-floating organoids remain limited. In this study, we report a stretchable, soft mesh electrode system that establishes an intimate in vitro electrical interface with human neurons in 3D organoids. Our mesh is constructed with poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) based electrically conductive hydrogel electrode arrays and elastomeric poly(styrene-ethylene-butylene-styrene) (SEBS) as the substrate and encapsulation materials. This mesh electrode can maintain a stable electrochemical impedance in buffer solution under 50% compressive and 50% tensile strain. We have successfully cultured pluripotent stem cell-derived human cortical organoids (hCO) on this polymeric mesh for more than 3 months and demonstrated that organoids readily integrate with the mesh. Using simultaneous stimulation and calcium imaging, we show that electrical stimulation through the mesh can elicit intensity-dependent calcium signals comparable to stimulation from a bipolar stereotrode. This platform may serve as a tool for monitoring and modulating the electrical activity of in vitro models of neuropsychiatric diseases., Competing Interests: Declaration of competing interest The authors declare no financial interests/personal relationships which may be considered as potential competing interests. Stanford holds several patents and patent applications for organoids and assembloids (SPP is listed as an inventor)., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Self-organizing neural organoids represent a promising in vitro platform with which to model human development and disease 1-5 . However, organoids lack the connectivity that exists in vivo, which limits maturation and makes integration with other circuits that control behaviour impossible. Here we show that human stem cell-derived cortical organoids transplanted into the somatosensory cortex of newborn athymic rats develop mature cell types that integrate into sensory and motivation-related circuits. MRI reveals post-transplantation organoid growth across multiple stem cell lines and animals, whereas single-nucleus profiling shows progression of corticogenesis and the emergence of activity-dependent transcriptional programs. Indeed, transplanted cortical neurons display more complex morphological, synaptic and intrinsic membrane properties than their in vitro counterparts, which enables the discovery of defects in neurons derived from individuals with Timothy syndrome. Anatomical and functional tracings show that transplanted organoids receive thalamocortical and corticocortical inputs, and in vivo recordings of neural activity demonstrate that these inputs can produce sensory responses in human cells. Finally, cortical organoids extend axons throughout the rat brain and their optogenetic activation can drive reward-seeking behaviour. Thus, transplanted human cortical neurons mature and engage host circuits that control behaviour. We anticipate that this approach will be useful for detecting circuit-level phenotypes in patient-derived cells that cannot otherwise be uncovered., (© 2022. The Author(s).)

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., (© 2022. The Author(s).)

Neurotransmitters play essential roles in regulating neural circuit dynamics both in the central nervous system as well as at the peripheral, including the gastrointestinal tract 1-3 . Their real-time monitoring will offer critical information for understanding neural function and diagnosing disease 1-3 . However, bioelectronic tools to monitor the dynamics of neurotransmitters in vivo, especially in the enteric nervous systems, are underdeveloped. This is mainly owing to the limited availability of biosensing tools that are capable of examining soft, complex and actively moving organs. Here we introduce a tissue-mimicking, stretchable, neurochemical biological interface termed NeuroString, which is prepared by laser patterning of a metal-complexed polyimide into an interconnected graphene/nanoparticle network embedded in an elastomer. NeuroString sensors allow chronic in vivo real-time, multichannel and multiplexed monoamine sensing in the brain of behaving mouse, as well as measuring serotonin dynamics in the gut without undesired stimulations and perturbing peristaltic movements. The described elastic and conformable biosensing interface has broad potential for studying the impact of neurotransmitters on gut microbes, brain-gut communication and may ultimately be extended to biomolecular sensing in other soft organs across the body., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

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.

Endothelial cells of blood vessels of the central nervous system (CNS) constitute blood-CNS barriers. Barrier properties are not intrinsic to these cells; rather they are induced and maintained by CNS microenvironment. Notably, the abluminal surfaces of CNS capillaries are ensheathed by pericytes and astrocytes. However, extrinsic factors from these perivascular cells that regulate barrier integrity are largely unknown. Here, we establish vitronectin, an extracellular matrix protein secreted by CNS pericytes, as a regulator of blood-CNS barrier function via interactions with its integrin receptor, α5, in endothelial cells. Genetic ablation of vitronectin or mutating vitronectin to prevent integrin binding, as well as endothelial-specific deletion of integrin α5, causes barrier leakage in mice. Furthermore, vitronectin-integrin α5 signaling maintains barrier integrity by actively inhibiting transcytosis in endothelial cells. These results demonstrate that signaling from perivascular cells to endothelial cells via ligand-receptor interactions is a key mechanism to regulate barrier permeability., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© 2022. The Author(s).)

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.

Mitochondria, the powerhouse of the cell, are dynamic organelles that undergo constant morphological changes. Increasing evidence indicates that mitochondria morphologies and functions can be modulated by mechanical cues. However, the mechano-sensing and -responding properties of mitochondria and the relation between mitochondrial morphologies and functions are unclear due to the lack of methods to precisely exert mechano-stimulation on and deform mitochondria inside live cells. Here, we present an optogenetic approach that uses light to induce deformation of mitochondria by recruiting molecular motors to the outer mitochondrial membrane via light-activated protein-protein hetero-dimerization. Mechanical forces generated by motor proteins distort the outer membrane, during which the inner mitochondrial membrane can also be deformed. Moreover, this optical method can achieve subcellular spatial precision and be combined with different optical dimerizers and molecular motors. This method presents a mitochondria-specific mechano-stimulator for studying mitochondria mechanobiology and the interplay between mitochondria shapes and functions., Competing Interests: Declaration of interests The authors declare no competing interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Neuromodulation is of great importance both as a fundamental neuroscience research tool for analyzing and understanding the brain function, and as a therapeutic avenue for treating brain disorders. Here, an overview of conceptual and technical progress in developing neuromodulation strategies is provided, and it is suggested that recent advances in nanotechnology are enabling novel neuromodulation modalities with less invasiveness, improved biointerfaces, deeper penetration, and higher spatiotemporal precision. The use of nanotechnology and the employment of versatile nanomaterials and nanoscale devices with tailored physical properties have led to considerable research progress. To conclude, an outlook discussing current challenges and future directions for next-generation neuromodulation modalities is presented., (© 2021 The Authors. Advanced Materials published by Wiley-VCH GmbH.)

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.

Human pluripotent stem cells have emerged as a promising in vitro model system for studying the brain. Two-dimensional and three-dimensional cell culture paradigms have provided valuable insights into the pathogenesis of neuropsychiatric disorders, but they remain limited in their capacity to model certain features of human neural development. Specifically, current models do not efficiently incorporate extracellular matrix-derived biochemical and biophysical cues, facilitate multicellular spatio-temporal patterning, or achieve advanced functional maturation. Engineered biomaterials have the capacity to create increasingly biomimetic neural microenvironments, yet further refinement is needed before these approaches are widely implemented. This Review therefore highlights how continued progression and increased integration of engineered biomaterials may be well poised to address intractable challenges in recapitulating human neural development., (© 2021. Springer Nature Limited.)

Measuring and monitoring the electrical signals transmitted between neurons is key to understanding the communication between neurons that underlies human perception, information processing, and decision-making. While electrode-based electrophysiology has been the gold standard, optical electrophysiology has opened up a new area in the past decade. Voltage-dependent fluorescent reporters enable voltage imaging with high spatial resolution and flexibility to choose recording locations. However, they exhibit photobleaching as well as phototoxicity and may perturb the physiology of the cell. Label-free optical electrophysiology seeks to overcome these hurdles by detecting electrical activities optically, without the incorporation of exogenous fluorophores in cells. For example, electrochromic optical recording detects neuroelectrical signals via a voltage-dependent color change of extracellular materials, and interferometric optical recording monitors membrane deformations that accompany electrical activities. Label-free optical electrophysiology, however, is in an early stage, and often has limited sensitivity and temporal resolution. In this Perspective, we review the recent progress to overcome these hurdles. We hope this Perspective will inspire developments of label-free optical electrophysiology techniques with high recording sensitivity and temporal resolution in the near future.

The measurement of electrical activity across systems of excitable cells underlies current progress in neuroscience, cardiac pharmacology, and neurotechnology. However, bioelectricity spans orders of magnitude in intensity, space, and time, posing substantial technological challenges. The development of methods permitting network-scale recordings with high spatial resolution remains key to studies of electrogenic cells, emergent networks, and bioelectric computation. Here, we demonstrate single-shot and label-free imaging of extracellular potentials with high resolution across a wide field-of-view. The critically coupled waveguide-amplified graphene electric field (CAGE) sensor leverages the field-sensitive optical transitions in graphene to convert electric potentials into the optical regime. As a proof-of-concept, we use the CAGE sensor to detect native electrical activity from cardiac action potentials with tens-of-microns resolution, simultaneously map the propagation of these potentials at tissue-scale, and monitor their modification by pharmacological agents. This platform is robust, scalable, and compatible with existing microscopy techniques for multimodal correlative imaging.

The physical properties of biomaterials, such as elasticity, stiffness, and surface nanotopography, are mechanical cues that regulate a broad spectrum of cell behaviors, including migration, differentiation, proliferation, and reprogramming. Among them, nanoscale surface topography, i.e. nanotopography, defines the nanoscale shape and spatial arrangement of surface elements, which directly interact with the cell membranes and stimulate changes in the cell signaling pathways. In biological systems, the effects of nanotopography are often entangled with those of other mechanical and biochemical factors. Precise engineering of 2D nanopatterns and 3D nanostructures with well-defined features has provided a powerful means to study the cellular responses to specific topographic features. In this Review, we discuss efforts in the last three years to understand how nanotopography affects membrane receptor activation, curvature-induced cell signaling, and stem cell differentiation., Competing Interests: Conflict of interest statement Nothing declared. Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Bridging integrator-1 (BIN1) is a family of banana-shaped molecules implicated in cell membrane tubulation. To understand the curvature sensitivity and functional roles of BIN1 splicing isoforms, we engineered vertical nanobars on a cell culture substrate to create high and low curvatures. When expressed individually, BIN1 isoforms with phosphoinositide-binding motifs (pBIN1) appeared preferentially at high-curvature nanobar ends, agreeing well with their membrane tubulation in cardiomyocytes. In contrast, the ubiquitous BIN1 isoform without phosphoinositide-binding motif (uBIN1) exhibited no affinity to membranes around nanobars but accumulated along Z-lines in cardiomyocytes. Importantly, in pBIN1-uBIN1 coexpression, pBIN1 recruited uBIN1 to high-curvature membranes at nanobar ends, and uBIN1 attached the otherwise messy pBIN1 tubules to Z-lines. The complementary cooperation of BIN1 isoforms (comboBIN1) represents a novel mechanism of T-tubule formation along Z-lines in cardiomyocytes. Dysregulation of BIN1 splicing, e.g., during myocardial infarction, underlied T-tubule disorganization, and correction of uBIN1/pBIN1 stoichiometry rescued T-tubule morphology in heart disease.

Understanding how a network of interconnected neurons receives, stores, and processes information in the human brain is one of the outstanding scientific challenges of our time. The ability to reliably detect neuroelectric activities is essential to addressing this challenge. Optical recording using voltage-sensitive fluorescent probes has provided unprecedented flexibility for choosing regions of interest in recording neuronal activities. However, when recording at a high frame rate such as 500 to 1,000 Hz, fluorescence-based voltage sensors often suffer from photobleaching and phototoxicity, which limit the recording duration. Here, we report an approach called electrochromic optical recording (ECORE) that achieves label-free optical recording of spontaneous neuroelectrical activities. ECORE utilizes the electrochromism of poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) thin films, whose optical absorption can be modulated by an applied voltage. Being based on optical reflection instead of fluorescence, ECORE offers the flexibility of an optical probe without suffering from photobleaching or phototoxicity. Using ECORE, we optically recorded spontaneous action potentials in cardiomyocytes, cultured hippocampal and dorsal root ganglion neurons, and brain slices. With minimal perturbation to cells, ECORE allows long-term optical recording over multiple days., Competing Interests: The authors declare no competing interest.

Advances in protein engineering have enabled the production of self-assembled protein crystals within living cells. Our recent publication demonstrates the production of ftn-PAK4, which is a ferritin-containing crystal that can mineralize iron and become magnetic when isolated. We have developed an optimized protocol for the production and isolation of PAK4-based crystals. The crystals are first grown in low-passage HEK293T cells, released using a lysis buffer containing NP-40 and DNase, and collected under careful centrifugation conditions. Our protocol maximizes the purity and yield of crystals and is quick and straightforward., Competing Interests: Competing interestsThe authors declare that there are no competing interests or conflicts of interest., (Copyright © 2020 The Authors; exclusive licensee Bio-protocol LLC.)

Brain-derived neurotrophic factor, via activation of tropomyosin receptor kinase B (TrkB), plays a critical role in neuronal proliferation, differentiation, survival, and death. Dysregulation of TrkB signaling is implicated in neurodegenerative disorders and cancers. Precise activation of TrkB signaling with spatial and temporal resolution is greatly desired to study the dynamic nature of TrkB signaling and its role in related diseases. Here we develop different optogenetic approaches that use light to activate TrkB signaling. Utilizing the photosensitive protein Arabidopsis thaliana cryptochrome 2, the light-inducible homo-interaction of the intracellular domain of TrkB in the cytosol or on the plasma membrane is able to induce the activation of downstream MAPK/ERK and PI3K/Akt signaling as well as the neurite outgrowth of PC12 cells. Moreover, we prove that such strategies are generalizable to other optical homo-dimerizers by demonstrating the optical TrkB activation based on the light-oxygen-voltage domain of aureochrome 1 from Vaucheria frigida. The results open up new possibilities of many other optical platforms to activate TrkB signaling to fulfill customized needs. By comparing all the different strategies, we find that the cryptochrome 2-integrated approach to achieve light-induced cell membrane recruitment and homo-interaction of intracellular domain of TrkB is most efficient in activating TrkB signaling. The optogenetic strategies presented are promising tools to investigate brain-derived neurotrophic factor/TrkB signaling with tight spatial and temporal control., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Receptor tyrosine kinases (RTKs) play crucial roles in human health, and their misregulation is implicated in disorders ranging from neurodegenerative diseases to cancers. The highly conserved mechanism of activation of RTKs makes them especially appealing candidates for control via optogenetic dimerization methods. This work offers a strategy for using the improved light-induced dimer (iLID) system with a constructed tandem dimer of its binding partner nano (tdnano) to build light-activatable versions of RTKs. In the absence of light, the iLID-RTK is cytosolic, monomeric, and inactive. Under blue light, the iLID + tdnano system recruits two copies of iLID-RTK to tdnano, dimerizing, and activating the RTK. We demonstrate that iLID opto-iTrkA and opto-iTrkB are capable of reproducing downstream ERK and Akt signaling only in the presence of tdnano. We further show with our opto-iTrkA that the system is compatible with multi-day and population-level activation of TrkA in PC12 cells. By leveraging genetic targeting of tdnano, we achieve RTK activation at a specific subcellular location even with whole-cell illumination, allowing us to confidently probe the impact of context on signaling outcome., (Copyright © 2020. Published by Elsevier Ltd.)

Nanoscale membrane curvature is now understood to play an active role in essential cellular processes such as endocytosis, exocytosis, and actin dynamics. Previous studies have shown that membrane curvature can directly affect protein function and intracellular signaling. However, few methods are able to precisely manipulate membrane curvature in live cells. Here, we report the development of a new method of generating nanoscale membrane curvature in live cells that is controllable, reversible, and capable of precise spatial and temporal manipulation. For this purpose, we make use of Bin/Amphiphysin/Rvs (BAR) domain proteins, a family of well-studied membrane-remodeling and membrane-sculpting proteins. Specifically, we engineered two optogenetic systems, opto-FBAR and opto-IBAR, that allow light-inducible formation of positive and negative membrane curvature, respectively. Using opto-FBAR, blue light activation results in the formation of tubular membrane invaginations (positive curvature), controllable down to the subcellular level. Using opto-IBAR, blue light illumination results in the formation of membrane protrusions or filopodia (negative curvature). These systems present a novel approach for light-inducible manipulation of nanoscale membrane curvature in live cells.

Local curvatures on the cell membrane serve as signaling hubs that promote curvature-dependent protein interactions and modulate a variety of cellular processes including endocytosis, exocytosis, and the actin cytoskeleton. However, precisely controlling the location and the degree of membrane curvature in live cells has not been possible until recently, where studies show that nanofabricated vertical structures on a substrate can imprint their shapes on the cell membrane to induce well-defined curvatures in adherent cells. Nevertheless, the intrinsic static nature of these engineered nanostructures prevents dynamic modulation of membrane curvatures. In this work, we engineer light-responsive polymer structures whose shape can be dynamically modulated by light and thus change the induced-membrane curvatures on-demand. Specifically, we fabricate three-dimensional azobenzene-based polymer structures that change from a vertical pillar to an elongated vertical bar shape upon green light illumination. We observe that U2OS cells cultured on azopolymer nanostructures rapidly respond to the topographical change of the substrate underneath. The dynamically induced high membrane curvatures at bar ends promote local accumulation of actin fibers and actin nucleator Arp2/3 complex. The ability to dynamically manipulate the membrane curvature and analyze protein response in real-time provides a new way to study curvature-dependent processes in live cells.

Surface topography profoundly influences cell adhesion, differentiation, and stem cell fate control. Numerous studies using a variety of materials demonstrate that nanoscale topographies change the intracellular organization of actin cytoskeleton and therefore a broad range of cellular dynamics in live cells. However, the underlying molecular mechanism is not well understood, leaving why actin cytoskeleton responds to topographical features unexplained and therefore preventing researchers from predicting optimal topographic features for desired cell behavior. Here we demonstrate that topography-induced membrane curvature plays a crucial role in modulating intracellular actin organization. By inducing precisely controlled membrane curvatures using engineered vertical nanostructures as topographies, we find that actin fibers form at the sites of nanostructures in a curvature-dependent manner with an upper limit for the diameter of curvature at ∼400 nm. Nanotopography-induced actin fibers are branched actin nucleated by the Arp2/3 complex and are mediated by a curvature-sensing protein FBP17. Our study reveals that the formation of nanotopography-induced actin fibers drastically reduces the amount of stress fibers and mature focal adhesions to result in the reorganization of actin cytoskeleton in the entire cell. These findings establish the membrane curvature as a key linkage between surface topography and topography-induced cell signaling and behavior., Competing Interests: The authors declare no competing interest.

Magnetogenetics is a new field that leverages genetically encoded proteins and protein assemblies that are sensitive to magnetic fields to study and manipulate cell behavior. Theoretical studies show that many proposed magnetogenetic proteins do not contain enough iron to generate substantial magnetic forces. Here, we have engineered a genetically encoded ferritin-containing protein crystal that grows inside mammalian cells. Each of these crystals contains more than 10 million ferritin subunits and is capable of mineralizing substantial amounts of iron. When isolated from cells and loaded with iron in vitro , these crystals generate magnetic forces that are 9 orders of magnitude larger than the forces from the single ferritin cages used in previous studies. These protein crystals are attracted to an applied magnetic field and move toward magnets even when internalized into cells. While additional studies are needed to realize the full potential of magnetogenetics, these results demonstrate the feasibility of engineering protein assemblies for magnetic sensing.

Membrane curvatures are involved in essential cellular processes, such as endocytosis and exocytosis, in which they are believed to act as microdomains for protein interactions and intracellular signaling. These membrane curvatures appear and disappear dynamically, and their locations are difficult or impossible to predict. In addition, the size of these curvatures is usually below the diffraction limit of visible light, making it impossible to resolve their values using live-cell imaging. Therefore, precise manipulation of membrane curvature is important to understanding how membrane curvature is involved in intracellular processes. Recent studies show that membrane curvatures can be induced by surface topography when cells are in direct contact with engineered substrates. Here, we present detailed procedures for using nanoscale structures to manipulate membrane curvatures and probe curvature-induced phenomena in live cells. We first describe detailed procedures for the design of nanoscale structures and their fabrication using electron-beam (E-beam) lithography. The fabrication process takes 2 d, but the resultant chips can be cleaned and reused repeatedly over the course of 2 years. Then we describe how to use these nanostructures to manipulate local membrane curvatures and probe intracellular protein responses, discussing surface coating, cell plating, and fluorescence imaging in detail. Finally, we describe a procedure to characterize the nanostructure-cell membrane interface using focused ion beam and scanning electron microscopy (FIB-SEM). Nanotopography-based methods can induce stable membrane curvatures with well-defined curvature values and locations in live cells, which enables the generation of a library of curvatures for probing curvature-related intracellular processes.

Cell fate is largely determined by interactions that occur at the interface between cells and their surrounding microenvironment. For this reason, especially in the field of tissue-engineering, there is a growing interest in developing techniques that allow evaluating cell-material interaction at the nanoscale, particularly focusing on cell adhesion processes. While for 2D culturing systems a consolidated series of tools already satisfy this need, in 3D environments, more closely recapitulating complex in vivo structures, there is still a lack of procedures furthering the comprehension of cell-material interactions. Here, the use of scanning electron microscopy coupled with a focused ion beam (SEM/FIB) for the characterization of cell interactions with 3D scaffolds obtained by different fabrication techniques is reported for the first time. The results clearly show the capability of the developed approach to preserve and finely resolve scaffold-cell interfaces highlighting details such as plasma membrane arrangement, extracellular matrix architecture and composition, and cellular structures playing a role in cell adhesion to the surface. It is anticipated that the developed approach will be relevant for the design of efficient cell-instructive platforms in the study of cellular guidance strategies for tissue-engineering applications as well as for in vitro 3D models., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Multielectrode arrays (MEAs) are essential tools in neural and cardiac research as they provide a means for noninvasive, multiplexed recording of extracellular field potentials with high temporal resolution. To date, the mechanical properties of the electrode material, e.g., its Young's modulus, have not been taken into consideration in most MEA designs leaving hard materials as the default choice due to their established fabrication processes. However, the cell-electrode interface is known to significantly affect some aspects of the cell's behavior. In this paper, we describe the fabrication of a soft 3D micropillar electrode array. Using this array, we proceed to successfully record action potentials from monolayer cell cultures. Specifically, our conductive hydrogel micropillar electrode showed improved signal amplitude and signal-to-noise ratio, compared with conventional hard iridium oxide micropillar electrodes of the same diameter. Taken together, our fabricated soft micropillar electrode array will provide a tissue-like Young's modulus and thus a relevant mechanical microenvironment to fundamental cardiac and neural studies., Competing Interests: The authors declare no conflict of interest.

The dynamic interface between the cellular membrane and 3D nanostructures determines biological processes and guides the design of novel biomedical devices. Despite the fact that recent advancements in the fabrication of artificial biointerfaces have yielded an enhanced understanding of this interface, there remain open questions on how the cellular membrane reacts and behaves in the presence of sharp objects on the nanoscale. Here we provide a multifaceted characterization of the cellular membrane's mechanical stability when closely interacting with high-aspect-ratio 3D vertical nanostructures, providing strong evidence that vertical nanostructures spontaneously penetrate the cellular membrane to form a steady intracellular coupling only in rare cases and under specific conditions. The cell membrane is able to conform tightly over the majority of structures with various shapes while maintaining its integrity.

Nerve growth factor/tropomyosin receptor kinase A (NGF/TrkA) signaling plays a key role in neuronal development, function, survival, and growth. The pathway is implicated in neurodegenerative disorders including Alzheimer's disease, chronic pain, inflammation, and cancer. NGF binds the extracellular domain of TrkA, leading to the activation of the receptor's intracellular kinase domain. As TrkA signaling is highly dynamic, mechanistic studies would benefit from a tool with high spatial and temporal resolution. Here we present the design and evaluation of four strategies for light-inducible activation of TrkA in the absence of NGF. Our strategies involve the light-sensitive protein Arabidopsis cryptochrome 2 and its binding partner CIB1. We demonstrate successful recapitulation of native NGF/TrkA functions by optical induction of plasma membrane recruitment and homo-interaction of the intracellular domain of TrkA. This approach activates PI3K/AKT and Raf/ERK signaling pathways, promotes neurite growth in PC12 cells, and supports survival of dorsal root ganglion neurons in the absence of NGF. This ability to activate TrkA using light bestows high spatial and temporal resolution for investigating NGF/TrkA signaling.