No abstract

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 the 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.

Vertically aligned nanopillars can serve as excellent electrical, optical and mechanical platforms for biological studies. However, revealing the nature of the interface between the cell and the nanopillar is very challenging. In particular, a matter of debate is whether the cell membrane remains intact around the nanopillar. Here we present a detailed characterization of the cell-nanopillar interface by transmission electron microscopy. We examined cortical neurons growing on nanopillars with diameter 50−500 nm and heights 0.5−2 μm. We found that on nanopillars less than 300 nm in diameter, the cell membrane wraps around the entirety of the nanopillar without the nanopillar penetrating into the interior of the cell. On the other hand, the cell sits on top of arrays of larger, closely spaced nanopillars. We also observed that the membrane-surface gap of both cell bodies and neurites is smaller for nanopillars than for a flat substrate. These results support a tight interaction between the cell membrane and the nanopillars and previous findings of excellent sealing in electrophysiology recordings using nanopillar electrodes.

Cell migration in a cultured neuronal network presents an obstacle to selectively measuring the activity of the same neuron over a long period of time. Here we report the use of nanopillar arrays to pin the position of neurons in a noninvasive manner. Vertical nanopillars protruding from the surface serve as geometrically better focal adhesion points for cell attachment than a flat surface. The cell body mobility is significantly reduced from 57.8 µm on flat surface to 3.9 µm on nanopillars over five day period. Yet, neurons growing on nanopillar arrays show a growth pattern that does not differ in any significant way from that seen on a flat substrate. Notably, while the cell bodies of neurons are efficiently anchored by the nanopillars, the axons and dendrites are free to grow and elongate into the surrounding area to develop a neuronal network, which opens up opportunities for long-term study of the same neurons in connected networks. KeywordsNanopillar; neuron; cell growth; cell migration; extracellular recording A fundamental understanding of neural network formation, transmission and remodeling requires measurements of individual neuron activities including firing threshold, firing rate and temporal sequence1. Extracellular approaches such as patterned multi-electrode arrays (MEA)2 -4 and planar field effect transistors4 have been successful in simultaneously measuring multi-cell activities over an extended period of time and thus have provided valuable information regarding the development and formation of neuronal networks. For example, the emergence of synchronous electrical firing pattern and developmental changes of the network activity have been observed in networks of cultured cortical neurons5 -7. In such studies, dissociated neurons obtained from fetal or neonatal brains are cultured atop the embedded electrodes or transistors. Electric signals generated by a neuron can be detected extracellularly if there is an electrode in close contact. However, it has been difficult to consistently measure the activity of the same neuron over a long term period. This difficulty is partly due to neuron mobility and partly due to lack of neuron-to-electrode specificity8. Neurons cultured on a flat substrate tend to migrate over time, especially in the first few weeks9 , 10. The migration range can be as long as hundreds of micrometers and well beyond the detection range of a single electrode or transistor. As a result, patterned electrodes or transistors are not always monitoring the activity of the same neuron as neurons move around. This presents a challenge to monitoring individual neuron activities in a neuronal network for an extended time (up to months), which demands stable and specific neuron-electrode correspondence.* To whom correspondence should be addressed. BC, bcui@stanford.edu; YC, yicui@stanford.edu. Considerable efforts have been put forth to control the migration of neurons and thus to improve the neuron-electrode interface11 -16. The first approach is to promote neuron-toelectrode atta...

The mechanical stability and deformability of the cell nucleus are crucial to many biological processes, including migration, proliferation and polarization. In vivo, the cell nucleus is frequently subjected to deformation on a variety of length and time scales, but current techniques for studying nuclear mechanics do not provide access to subnuclear deformation in live functioning cells. Here we introduce arrays of vertical nanopillars as a new method for the in situ study of nuclear deformability and the mechanical coupling between the cell membrane and the nucleus in live cells. Our measurements show that nanopillar-induced nuclear deformation is determined by nuclear stiffness, as well as opposing effects from actin and intermediate filaments. Furthermore, the depth, width and curvature of nuclear deformation can be controlled by varying the geometry of the nanopillar array. Overall, vertical nanopillar arrays constitute a novel approach for non-invasive, subcellular perturbation of nuclear mechanics and mechanotransduction in live cells.

16(Keywords: clathrin, dynamin, endocytosis, membrane curvature, curvature sensing, 17 nanopillar, nanowire, nanostructure.)18 not peer-reviewed)

Observing individual molecules in a complex environment by fluorescence microscopy is becoming increasingly important in biological and medical research, for which critical reduction of observation volume is required. Here, we demonstrate the use of vertically aligned silicon dioxide nanopillars to achieve below-the-diffraction-limit observation volume in vitro and inside live cells. With a diameter much smaller than the wavelength of visible light, a transparent silicon dioxide nanopillar embedded in a nontransparent substrate restricts the propagation of light and affords evanescence wave excitation along its vertical surface. This effect creates highly confined illumination volume that selectively excites fluorescence molecules in the vicinity of the nanopillar. We show that this nanopillar illumination can be used for in vitro single-molecule detection at high fluorophore concentrations. In addition, we demonstrate that vertical nanopillars interface tightly with live cells and function as highly localized light sources inside the cell. Furthermore, specific chemical modification of the nanopillar surface makes it possible to locally recruit proteins of interest and simultaneously observe their behavior within the complex, crowded environment of the cell.

The photochromic molecule diarylethene works as a "toggle switch" for biocompatible fluorescence polymer dots and enables fluorescence switching in biological samples.