Interfacing soft materials with biological systems holds considerable promise for both biosensors and recording live cells. However, the interface between cells and organic substrates is not well studied, despite its crucial role in the effectiveness of the device. Furthermore, well-known cell adhesion enhancers, such as microgrooves, have not been implemented on these surfaces. Here, we present a nanoscale characterization of the cell–substrate interface for 3D laser-patterned organic electrodes by combining electrochemical impedance spectroscopy (EIS) and scanning electron microscopy/focused ion beam (SEM/FIB). We demonstrate that introducing 3D micropatterned grooves on organic surfaces enhances the cell adhesion of electrogenic cells.Keywords: biointerface; electrochemical impedance spectroscopy; femtosecond laser; organic bioelectronics; PEDOT:PSS; scanning electron microscopy/focused ion beam;

The interface between cells and nonbiological surfaces regulates cell attachment, chronic tissue responses, and ultimately the success of medical implants or biosensors. Clinical and laboratory studies show that topological features of the surface profoundly influence cellular responses; for example, titanium surfaces with nano- and microtopographical structures enhance osteoblast attachment and host–implant integration as compared to a smooth surface. To understand how cells and tissues respond to different topographical features, it is of critical importance to directly visualize the cell–material interface at the relevant nanometer length scale. Here, we present a method for in situ examination of the cell-to-material interface at any desired location, based on focused ion beam milling and scanning electron microscopy imaging to resolve the cell membrane-to-material interface with 10 nm resolution. By examining how cell membranes interact with topographical features such as nanoscale protrusions or invaginations, we discovered that the cell membrane readily deforms inward and wraps around protruding structures, but hardly deforms outward to contour invaginating structures. This asymmetric membrane response (inward vs outward deformation) causes the cleft width between the cell membrane and the nanostructure surface to vary by more than an order of magnitude. Our results suggest that surface topology is a crucial consideration for the development of medical implants or biosensors whose performances are strongly influenced by the cell-to-material interface. We anticipate that the method can be used to explore the direct interaction of cells/tissue with medical devices such as metal implants in the future.Keywords: cell−material interface; focused ion beam; nanostructures; scanning electron microscopy; ultrathin resin plasticization;

Clinical studies of circulating tumor cells (CTC) have stringent demands for high capture purity and high capture efficiency. Nanostructured surfaces have been shown to significantly increase the capture efficiency yet suffer from low capture purity. Here we introduce a dual-functional lipid coating on nanostructured surfaces. The lipid coating serves both as an effective passivation layer that helps prevent nonspecific cell adhesion and as a functionalized layer for antibody-based specific cell capture. In addition, the fluidity of lipid bilayers enables antibody clustering that enhances the cell–surface interaction for efficient cell capture. As a result, the lipid-coating method helps promote both the capture efficiency and capture purity of nanostructure-based CTC capture.

•We developed an optogenetic tool to control organelle distributions in cells•Motor proteins can be recruited to organelle membrane by dimerization of CRY2-CIB1•Organelles can be driven toward cell nucleus or toward cell periphery by light•Light-induced organelle transport can be spatially controlled at subcellular regionIntracellular transport and distribution of organelles play important roles in diverse cellular functions, including cell polarization, intracellular signaling, cell survival, and apoptosis. Here, we report an optogenetic strategy to control the transport and distribution of organelles by light. This is achieved by optically recruiting molecular motors onto organelles through the heterodimerization of Arabidopsis thaliana cryptochrome 2 (CRY2) and its interacting partner CIB1. CRY2 and CIB1 dimerize within subseconds upon exposure to blue light, which requires no exogenous ligands and low intensity of light. We demonstrate that mitochondria, peroxisomes, and lysosomes can be driven toward the cell periphery upon light-induced recruitment of kinesin, or toward the cell nucleus upon recruitment of dynein. Light-induced motor recruitment and organelle movements are repeatable, reversible, and can be achieved at subcellular regions. This light-controlled organelle redistribution provides a new strategy for studying the causal roles of organelle transport and distribution in cellular functions in living cells.Figure optionsDownload full-size imageDownload high-quality image (248 K)Download as PowerPoint slide

The photoreceptor cryptochrome 2 (CRY2) has become a powerful optogenetic tool that allows light-inducible manipulation of various signaling pathways and cellular processes in mammalian cells with high spatiotemporal precision and ease of application. However, it has also been shown that the behavior of CRY2 under blue light is complex, as the photoexcited CRY2 can both undergo homo-oligomerization and heterodimerization by binding to its dimerization partner CIB1. To better understand the light-induced CRY2 activities in mammalian cells, this article systematically characterizes CRY2 homo-oligomerization in different cellular compartments, as well as how CRY2 homo-oligomerization and heterodimerization activities affect each other. Quantitative analysis reveals that membrane-bound CRY2 has drastically enhanced oligomerization activity compared to that of its cytoplasmic form. While CRY2 homo-oligomerization and CRY2-CIB1 heterodimerization could happen concomitantly, the presence of certain CIB1 fusion proteins can suppress CRY2 homo-oligomerization. However, the homo-oligomerization of cytoplasmic CRY2 can be significantly intensified by its recruitment to the membrane via interaction with the membrane-bound CIB1. These results contribute to the understanding of the light-inducible CRY2-CRY2 and CRY2-CIB1 interaction systems and can be used as a guide to establish new strategies utilizing the dual optogenetic characteristics of CRY2 to probe cellular processes.Keywords: CRY2-CIB1 dimerization; cryptochrome 2; light control; oligomerization; optogenetics

U0126 is a potent and selective inhibitor of MEK1 and MEK2 kinases. It has been widely used as an inhibitor for the Ras/Raf/MEK/ERK signaling pathway with over 5000 references on the NCBI PubMed database. In particular, U0126 has been used in a number of studies to show that inhibition of the Raf/MEK/ERK pathway protects neuronal cells against oxidative stress. Here, we report that U0126 can function as an antioxidant that protects PC12 cells against a number of different oxidative-stress inducers. This protective effect of U0126 is independent of its function as a MEK inhibitor, as several other MEK inhibitors failed to show similar protective effects. U0126 reduces reactive oxygen species (ROS) in cells. We further demonstrate that U0126 is a direct ROS scavenger in vitro, and the oxidation products of U0126 exhibit fluorescence. Our finding that U0126 is a strong antioxidant signals caution for its future usage as a MEK inhibitor and for interpreting some previous results.Keywords: antioxidant; cell death; MEK inhibitor; oxidative stress; U0126

In this issue of Chemistry & Biology, Kim and colleagues describe their work on optogenetic control of fibroblast growth factor receptor (FGFR) signaling. By engineering a chimeric receptor, the authors demonstrate that FGFR intracellular signaling can be controlled in space and time by blue light.

In this study, cyclometalated iridium(III) complex-doped polymer dots were synthesized and shown to emit luminescence upon X-ray irradiation, potentially serving as a new probe for molecular imaging during X-ray computed tomography.

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.

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

Brain derived neurotrophic factor (BDNF) plays a key role in the growth, development and maintenance of the central and peripheral nervous systems. Exogenous BDNF activates its membrane receptors at the axon terminal, and subsequently sends regulation signals to the cell body. To understand how a BDNF signal propagates in neurons, it is important to follow the trafficking of BDNF after it is internalized at the axon terminal. Here we labeled BDNF with bright, photostable quantum dots (QD-BDNF) and followed the axonal transport of QD-BDNF in real time in hippocampal neurons. We showed that QD-BDNF was able to bind BDNF receptors and activate downstream signaling pathways. When QD-BDNF was applied to the distal axons of hippocampal neurons, it was observed to be actively transported toward the cell body at an average speed of 1.11 ± 0.05 μm s−1. A closer examination revealed that QD-BDNF was transported by both discrete endosomes and multivesicular body-like structures. Our results showed that QD-BDNF could be used to track the movement of exogenous BDNF in neurons over long distances and to study the signaling organelles that contain BDNF.

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 a flat surface to 3.9 μm on nanopillars over a 5 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.

We present a detailed motion analysis of retrograde nerve growth factor (NGF) endosomes in axons to show that mechanical tugs-of-war and intracellular motor regulation are complimentary features of the near-unidirectional endosome directionality. We used quantum dots to fluorescently label NGF and acquired trajectories of retrograde quantum-dot-NGF-endosomes with <20-nm accuracy at 32 Hz in microfluidic neuron cultures. Using a combination of transient motion analysis and Bayesian parsing, we partitioned the trajectories into sustained periods of retrograde (dynein-driven) motion, constrained pauses, and brief anterograde (kinesin-driven) reversals. The data shows many aspects of mechanical tugs-of-war and multiple-motor mechanics in NGF-endosome transport. However, we found that stochastic mechanical models based on in vitro parameters cannot simulate the experimental data, unless the microtubule-binding affinity of kinesins on the endosome is tuned down by 10 times. Specifically, the simulations suggest that the NGF-endosomes are driven on average by 5–6 active dyneins and 1–2 downregulated kinesins. This is also supported by the dynamics of endosomes detaching under load in axons, showcasing the cooperativity of multiple dyneins and the subdued activity of kinesins. We discuss the possible motor coordination mechanism consistent with motor regulation and tugs-of-war for future investigations.

Microtubules are essential cytoskeletal tracks for cargo transportation in axons and also serve as the primary structural scaffold of neurons. Structural assembly, stability, and dynamics of axonal microtubules are of great interest for understanding neuronal functions and pathologies. However, microtubules are so densely packed in axons that their separations are well below the diffraction limit of light, which precludes using optical microscopy for live-cell studies. Here, we present a single-molecule imaging method capable of resolving individual microtubules in live axons. In our method, unlabeled microtubules are revealed by following individual axonal cargos that travel along them. We resolved more than six microtubules in a 1 μm diameter axon by real-time tracking of endosomes containing quantum dots. Our live-cell study also provided direct evidence that endosomes switch between microtubules while traveling along axons, which has been proposed to be the primary means for axonal cargos to effectively navigate through the crowded axoplasmic environment.

In this study, cyclometalated iridium(III) complex-doped polymer dots were synthesized and shown to emit luminescence upon X-ray irradiation, potentially serving as a new probe for molecular imaging during X-ray computed tomography.

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