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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.

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.

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.

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.

Over the past decade, there has been growing interest in developing biosensors and devices with nanoscale and vertical topography. Vertical nanostructures induce spontaneous cell engulfment, which enhances the cell–probe coupling efficiency and the sensitivity of biosensors. Although local membranes in contact with the nanostructures are found to be fully fluidic for lipid and membrane protein diffusions, cells appear to actively sense and respond to the surface topography presented by vertical nanostructures. For future development of biodevices, it is important to understand how cells interact with these nanostructures and how their presence modulates cellular function and activities. How cells recognize nanoscale surface topography has been an area of active research for two decades before the recent biosensor works. Extensive studies show that surface topographies in the range of tens to hundreds of nanometers can significantly affect cell functions, behaviors, and ultimately the cell fate. For example, titanium implants having rough surfaces are better for osteoblast attachment and host–implant integration than those with smooth surfaces. At the cellular level, nanoscale surface topography has been shown by a large number of studies to modulate cell attachment, activity, and differentiation. However, a mechanistic understanding of how cells interact and respond to nanoscale topographic features is still lacking. In this Account, we focus on some recent studies that support a new mechanism that local membrane curvature induced by nanoscale topography directly acts as a biochemical signal to induce intracellular signaling, which we refer to as the curvature hypothesis. The curvature hypothesis proposes that some intracellular proteins can recognize membrane curvatures of a certain range at the cell-to-material interface. These proteins then recruit and activate downstream components to modulate cell signaling and behavior. We discuss current technologies allowing the visualization of membrane deformation at the cell membrane-to-substrate interface with nanometer precision and demonstrate that vertical nanostructures induce local curvatures on the plasma membrane. These local curvatures enhance the process of clathrin-mediated endocytosis and affect actin dynamics. We also present evidence that vertical nanostructures can induce significant deformation of the nuclear membrane, which can affect chromatin distribution and gene expression. Finally, we provide a brief perspective on the curvature hypothesis and the challenges and opportunities for the design of nanotopography for manipulating cell behavior. Accepted version

Arabidopsis cryptochrome 2 (CRY2) can simultaneously undergo light-dependent CRY2–CRY2 homo-oligomerization and CRY2–CIB1 hetero-dimerization, both of which have been widely used to optically control intracellular processes. Applications using CRY2–CIB1 interaction desire minimal CRY2 homo-oligomerization to avoid unintended complications, while those utilizing CRY2–CRY2 interaction prefer robust homo-oligomerization. However, selecting the type of CRY2 interaction has not been possible as the molecular mechanisms underlying CRY2 interactions are unknown. Here we report CRY2–CIB1 and CRY2–CRY2 interactions are governed by well-separated protein interfaces at the two termini of CRY2. N-terminal charges are critical for CRY2–CIB1 interaction. Moreover, two C-terminal charges impact CRY2 homo-oligomerization, with positive charges facilitating oligomerization and negative charges inhibiting it. By engineering C-terminal charges, we develop CRY2high and CRY2low with elevated or suppressed oligomerization respectively, which we use to tune the levels of Raf/MEK/ERK signaling. These results contribute to our understanding of the mechanisms underlying light-induced CRY2 interactions and enhance the controllability of CRY2-based optogenetic systems., Cryptochrome 2 (CRY2) can form light-regulated CRY2-CRY2 homo-oligomers or CRY2-CIB1 hetero-dimers, but modulating these interactions is difficult owing to the lack of interaction mechanism. Here the authors identify the interactions facilitating homo-oligomers and introduce mutations to create low and high oligomerization versions.

Clathrin-mediated endocytosis (CME) involves nanoscale bending and inward budding of the plasma membrane, by which cells regulate both the distribution of membrane proteins and the entry of extracellular species1,2. Extensive studies have shown that CME proteins actively modulate the plasma membrane curvature1,3,4. However, the reciprocal regulation of how plasma membrane curvature affects the activities of endocytic proteins is much less explored, despite studies suggesting that membrane curvature itself can trigger biochemical reactions5-8. This gap in our understanding is largely due to technical challenges in precisely controlling the membrane curvature in live cells. In this work, we use patterned nanostructures to generate well-defined membrane curvatures ranging from +50 nm to -500 nm radius of curvature. We find that the positively curved membranes are CME hotspots, and that key CME proteins, clathrin and dynamin, show a strong preference toward positive membrane curvatures with a radius < 200 nm. Of ten CME related proteins we examined, all show preferences to positively curved membrane. By contrast, other membrane-associated proteins and non-CME endocytic protein, caveolin1, show no such curvature preference. Therefore, nanostructured substrates constitute a novel tool for investigating curvature-dependent processes in live cells.

The interface between biological cells and non-biological surfaces profoundly influences cellular activities, chronic tissue responses, and ultimately the success of medical implants. Materials in contact with cells can be plastics, metal, ceramics or other synthetic materials, and their surfaces vary widely in chemical compositions, stiffness, topography and levels of roughness. To understand the molecular mechanism of how cells and tissues respond to different materials, it is of critical importance to directly visualize the cell-material interface at the relevant length scale of nanometers. Conventional ultrastructural analysis by transmission electron microscopy (TEM) often requires substrate removal before microtome sectioning, which is not only challenging for most substrates but also can cause structural distortions of the interface. Here, we present a new method for in situ examination of the cell-to-material interface at any desired cellular location, based on focused-ion beam milling and scanning electron microscopy imaging (FIB-SEM). This method involves a thin-layer plastification procedure that preserves adherent cells as well as enhances the contrast of biological specimen. We demonstrate that this unique procedure allows the visualization of cell-to-material interface and intracellular structures with 10nm resolution, compatible with a variety of materials and surface topographies, and capable of volume and multi-directional imaging. We expect that this method will be very useful for studies of cell-to-material interactions and also suitable for in vivo studies such as examining osteoblast adhesion and new bone formation in response to titanium implants.

The measurement of the electrophysiology of human pluripotent stem cell-derived cardiomyocytes is critical for their biomedical applications, from disease modeling to drug screening. Yet, a method that enables the high-throughput intracellular electrophysiology measurement of single cardiomyocytes in adherent culture is not available. To address this area, we have fabricated vertical nanopillar electrodes that can record intracellular action potentials from up to 60 single beating cardiomyocytes. Intracellular access is achieved by highly localized electroporation, which allows for low impedance electrical access to the intracellular voltage. Herein, we demonstrate that this method provides the accurate measurement of the shape and duration of intracellular action potentials, validated by patch clamp, and can facilitate cellular drug screening and disease modeling using human pluripotent stem cells. This study validates the use of nanopillar electrodes for myriad further applications of human pluripotent stem cell-derived cardiomyocytes such as cardiomyocyte maturation monitoring and electrophysiology-contractile force correlation.

The stability and deformability of the cell nucleus are important to many biological processes like migration, proliferation, polarization. When cells are exposed to mechanical force, the force will be transmitted via cytoskeleton to the nucleus, induce shape deformation of the nuclear envelopes, and even change the configurations of nucleoskeletons. However, current techniques for studying nuclear mechanics are limited for studying inducing the effects of subcellular force perturbation in live cells. Here we developed a novel assay of using vertical nanopillar arrays to study the mechanical coupling between cell nucleus and cytoskeleton in live cells. Our results showed that nanopillars can induce deformation of nuclear envelope. By changing the geometry of the nanopillars or the stiffness of the nucleus, we can control the degree of nuclear deformation. Also, cytoskeletons such as actin and intermediate filaments were showed to play important roles in inducing and preventing nuclear deformation, respectively. Furthermore, we showed that mechanical perturbation of the nuclear envelope can cause the reorganization of nuclear lamina, which give the clue that cell nucleus itself may be able to sense and respond to mechanical signals. Overall, vertical nanopillars provide a long-term and non-invasive force to create a subcellular nuclear perturbation, and can be used as a tool for studying nuclear mechanotransduction in live cells.