Nanotopography modulates intracellular excitable systems through cytoskeleton actuation.

Cellular sensing of most environmental cues involves receptors that affect a signal-transduction excitable network (STEN), which is coupled to a cytoskeletal excitable network (CEN). We show that the mechanism of sensing of nanoridges is fundamentally different. CEN activity occurs preferentially on nanoridges, whereas STEN activity is constrained between nanoridges. In the absence of STEN, waves disappear, but long-lasting F-actin puncta persist along the ridges. When CEN is suppressed, wave propagation is no longer constrained by nanoridges. A computational model reproduces these experimental observations. Our findings indicate that nanotopography is sensed directly by CEN, whereas STEN is only indirectly affected due to a CEN-STEN feedback loop. These results explain why texture sensing is robust and acts cooperatively with multiple other guidance cues in complex, in vivo microenvironments.

Contractility, focal adhesion orientation, and stress fiber orientation drive cancer cell polarity and migration along wavy ECM substrates.

Contact guidance is a powerful topographical cue that induces persistent directional cell migration. Healthy tissue stroma is characterized by a meshwork of wavy extracellular matrix (ECM) fiber bundles, whereas metastasis-prone stroma exhibit less wavy, more linear fibers. The latter topography correlates with poor prognosis, whereas more wavy bundles correlate with benign tumors. We designed nanotopographic ECM-coated substrates that mimic collagen fibril waveforms seen in tumors and healthy tissues to determine how these nanotopographies may regulate cancer cell polarization and migration machineries. Cell polarization and directional migration were inhibited by fibril-like wave substrates above a threshold amplitude. Although polarity signals and actin nucleation factors were required for polarization and migration on low-amplitude wave substrates, they did not localize to cell leading edges. Instead, these factors localized to wave peaks, creating multiple "cryptic leading edges" within cells. On high-amplitude wave substrates, retrograde flow from large cryptic leading edges depolarized stress fibers and focal adhesions and inhibited cell migration. On low-amplitude wave substrates, actomyosin contractility overrode the small cryptic leading edges and drove stress fiber and focal adhesion orientation along the wave axis to mediate directional migration. Cancer cells of different intrinsic contractility depolarized at different wave amplitudes, and cell polarization response to wavy substrates could be tuned by manipulating contractility. We propose that ECM fibril waveforms with sufficiently high amplitude around tumors may serve as "cell polarization barriers," decreasing directional migration of tumor cells, which could be overcome by up-regulation of tumor cell contractility.

Excitable systems: A new perspective on the cellular impact of elongate mineral particles.

We investigate how the geometry of elongate mineral particles (EMPs) in contact with cells influences esotaxis, a recently discovered mechanism of texture sensing. Esotaxis is based on cytoskeletal waves and oscillations that are nucleated, shaped, and steered by the texture of the surroundings. We find that all EMPs studied trigger an esotactic response in macrophages, and that this response dominates cytoskeletal activity in these immune cells. In contrast, epithelial cells show little to no esotactic response to the EMPs. These results are consistent with the distinct interactions of both cell types with ridged nanotopographies of dimensions comparable to those of asbestiform EMPs. Our findings raise the question of whether narrow, asbestiform EMPs may also dominate cytoskeletal activity in other types of immune cells that exhibit similar esotactic effects. These findings, together with prior studies of esotaxis, lead us to the hypothesis that asbestiform EMPs suppress the migration of immune cells and activate immune signaling, thereby outcompeting signals that would normally stimulate the immune system in nearby tissue.

Nanotopography modulates cytoskeletal organization and dynamics during T cell activation.

Exposure to MHC-antigen complexes on the surface of antigen-presenting cells (APCs) activates T cells, inducing the formation of the immune synapse (IS). Antigen detection at the APC surface is thus a critical step in the adaptive immune response. The physical properties of antigen-presenting surfaces encountered by T cells in vivo are believed to modulate T cell activation and proliferation. Although stiffness and ligand mobility influence IS formation, the effect of the complex topography of the APC surface on this process is not well understood. Here we investigate how nanotopography modulates cytoskeletal dynamics and signaling during the early stages of T cell activation using high-resolution fluorescence microscopy on nanofabricated surfaces with parallel nanoridges of different spacings. We find that although nanoridges reduce the maximum spread area as compared with cells on flat surfaces, the ridges enhance the accumulation of actin and the signaling kinase ZAP-70 at the IS. Actin polymerization is more dynamic in the presence of ridges, which influence the directionality of both actin flows and microtubule (MT) growth. Our results demonstrate that the topography of the activating surface exerts both global effects on T cell morphology and local changes in actin and MT dynamics, collectively influencing T cell signaling.

Actin Dynamics as a Multiscale Integrator of Cellular Guidance Cues.

Migrating cells must integrate multiple, competing external guidance cues. However, it is not well understood how cells prioritize among these cues. We investigate external cue integration by monitoring the response of wave-like, actin-polymerization dynamics, the driver of cell motility, to combinations of nanotopographies and electric fields in neutrophil-like cells. The electric fields provide a global guidance cue, and approximate conditions at wound sites in vivo. The nanotopographies have dimensions similar to those of collagen fibers, and act as a local esotactic guidance cue. We find that cells prioritize guidance cues, with electric fields dominating long-term motility by introducing a unidirectional bias in the locations at which actin waves nucleate. That bias competes successfully with the wave guidance provided by the bidirectional nanotopographies.

Cortical waves mediate the cellular response to electric fields.

Electrotaxis, the directional migration of cells in a constant electric field, is important in regeneration, development, and wound healing. Electrotaxis has a slower response and a smaller dynamic range than guidance by other cues, suggesting that the mechanism of electrotaxis shares both similarities and differences with chemical-gradient-sensing pathways. We examine a mechanism centered on the excitable system consisting of cortical waves of biochemical signals coupled to cytoskeletal reorganization, which has been implicated in random cell motility. We use electro-fused giant Dictyostelium discoideum cells to decouple waves from cell motion and employ nanotopographic surfaces to limit wave dimensions and lifetimes. We demonstrate that wave propagation in these cells is guided by electric fields. The wave area and lifetime gradually increase in the first 10 min after an electric field is turned on, leading to more abundant and wider protrusions in the cell region nearest the cathode. The wave directions display 'U-turn' behavior upon field reversal, and this switch occurs more quickly on nanotopography. Our results suggest that electric fields guide cells by controlling waves of signal transduction and cytoskeletal activity, which underlie cellular protrusions. Whereas surface receptor occupancy triggers both rapid activation and slower polarization of signaling pathways, electric fields appear to act primarily on polarization, explaining why cells respond to electric fields more slowly than to other guidance cues.

Fluorinated interphase enables reversible aqueous zinc battery chemistries.

Metallic zinc is an ideal anode due to its high theoretical capacity (820 mAh g-1), low redox potential (-0.762 V versus the standard hydrogen electrode), high abundance and low toxicity. When used in aqueous electrolyte, it also brings intrinsic safety, but suffers from severe irreversibility. This is best exemplified by low coulombic efficiency, dendrite growth and water consumption. This is thought to be due to severe hydrogen evolution during zinc plating and stripping, hitherto making the in-situ formation of a solid-electrolyte interphase (SEI) impossible. Here, we report an aqueous zinc battery in which a dilute and acidic aqueous electrolyte with an alkylammonium salt additive assists the formation of a robust, Zn2+-conducting and waterproof SEI. The presence of this SEI enables excellent performance: dendrite-free zinc plating/stripping at 99.9% coulombic efficiency in a Ti||Zn asymmetric cell for 1,000 cycles; steady charge-discharge in a Zn||Zn symmetric cell for 6,000 cycles (6,000 h); and high energy densities (136 Wh kg-1 in a Zn||VOPO4 full battery with 88.7% retention for >6,000 cycles, 325 Wh kg-1 in a Zn||O2 full battery for >300 cycles and 218 Wh kg-1 in a Zn||MnO2 full battery with 88.5% retention for 1,000 cycles) using limited zinc. The SEI-forming electrolyte also allows the reversible operation of an anode-free pouch cell of Ti||ZnxVOPO4 at 100% depth of discharge for 100 cycles, thus establishing aqueous zinc batteries as viable cell systems for practical applications.

Quantifying topography-guided actin dynamics across scales using optical flow.

The dynamic rearrangement of the actin cytoskeleton is an essential component of many mechanotransduction and cellular force generation pathways. Here we use periodic surface topographies with feature sizes comparable to those of in vivo collagen fibers to measure and compare actin dynamics for two representative cell types that have markedly different migratory modes and physiological purposes: slowly migrating epithelial MCF10A cells and polarizing, fast-migrating, neutrophil-like HL60 cells. Both cell types exhibit reproducible guidance of actin waves (esotaxis) on these topographies, enabling quantitative comparisons of actin dynamics. We adapt a computer-vision algorithm, optical flow, to measure the directions of actin waves at the submicron scale. Clustering the optical flow into regions that move in similar directions enables micron-scale measurements of actin-wave speed and direction. Although the speed and morphology of actin waves differ between MCF10A and HL60 cells, the underlying actin guidance by nanotopography is similar in both cell types at the micron and submicron scales.

Actin Cytoskeleton and Focal Adhesions Regulate the Biased Migration of Breast Cancer Cells on Nanoscale Asymmetric Sawteeth.

Physical guidance from the underlying matrix is a key regulator of cancer invasion and metastasis. We explore the effects of surface topography on the migration phenotype of multiple breast cancer cell lines using aligned nanoscale ridges and asymmetric sawtooth structures. Both benign and metastatic breast cancer cells preferentially move parallel to nanoridges, with enhanced speeds compared to flat surfaces. In contrast, asymmetric sawtooth structures unidirectionally bias the movement of breast cancer cells in a cell-type-dependent manner. Quantitative analysis shows that the level of bias in cell migration increases when cells move with higher speeds or with higher directional persistence. Live-cell imaging studies further reveal that actin polymerization waves are unidirectionally guided by the sawteeth in the same direction as the cell motion. High-resolution fluorescence imaging and scanning electron microscopy studies reveal that two breast cancer cell lines with opposite migrational profiles exhibit profoundly different cell cortical plasticity and focal adhesion patterns. These results suggest that the overall migration response of cancer cells to surface topography is directly related to the underlying cytoskeletal architectures and dynamics, which are regulated by both intrinsic and extrinsic factors.

Replication of biocompatible, nanotopographic surfaces.

The ability of cells to sense and respond to nanotopography is being implicated as a key element in many physiological processes such as cell differentiation, immune response, and wound healing, as well as in pathologies such as cancer metastasis. To understand how nanotopography affects cellular behaviors, new techniques are required for the mass production of biocompatible, rigid nanotopographic surfaces. Here we introduce a method for the rapid and reproducible production of biocompatible, rigid, acrylic nanotopographic surfaces, and for the functionalization of the surfaces with adhesion-promoting molecules for cell experiments. The replica surfaces exhibit high optical transparency, which is advantageous for high-resolution, live-cell imaging. As a representative application, we demonstrate that epithelial cells form focal adhesions on surfaces composed of nanoscale ridges and grooves, and that the focal adhesions prefer to localize on the nanoridges. We further demonstrate that both F-actin and microtubules align along the nanoridges, but only F-actin aligns along the nanogrooves. The mass production of nanotopographic surfaces opens the door to the investigation of the effect of physical cues on the spatial distribution and the dynamics of intracellular proteins, and to the study of the mechanism of mechanosensing in processes such as cell migration, phagocytosis, division, and differentiation.

One-pot facile synthesis of Janus particles with tailored shape and functionality.

This communication describes a novel strategy for the synthesis of silica Janus particles with controlled shape and functionality using a facile wet-chemical approach.