a-nanobar-supported-lipid-bilayer-system-for-study-membrane-curvature

A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins <em>in vitro</em>

Membrane curvature plays important roles&#160;in various essential processes of cells, such as cell migration, cell division, and vesicle trafficking. It ...

Nanyang Technological University

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.

The Role of Membrane Curvature in Nanoscale Topography-Induced Intracellular Signaling.

This paper introduces a facile and scalable method to generate a layer of antibacterial coating on microneedles. The antibacterial coating (i.e., zinc oxide nanobushes) is generated on the surface of gold-coated polystyrene microneedles using the hydrothermal growth method. The antimicrobial property is examined using the agar diffusion test with both gram-positive and gram-negative bacteria.

In Situ Generation of Zinc Oxide Nanobushes on Microneedles as Antibacterial Coating.

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.

A nanostructure platform for live-cell manipulation of membrane curvature.

Plasma membrane (PM) curvature defines cell shape and intracellular organelle morphologies and is a fundamental cell property. Growth/proliferation is more stimulated in flatter cells than the same cells in elongated shapes. PM-anchored K-Ras small GTPase regulates cell growth/proliferation and plays key roles in cancer. The lipid-anchored K-Ras form nanoclusters selectively enriched with specific phospholipids, such as phosphatidylserine (PS), for efficient effector recruitment and activation. K-Ras function may, thus, be sensitive to changing lipid distribution at membranes with different curvatures. Here, we used complementary methods to manipulate membrane curvature of intact/live cells, native PM blebs, and synthetic liposomes. We show that the spatiotemporal organization and signaling of an oncogenic mutant K-Ras  favor flatter membranes with low curvature. Our findings are consistent with the more stimulated growth/proliferation in flatter cells. Depletion of endogenous PS abolishes K-Ras  PM curvature sensing. In cells and synthetic bilayers, only mixed-chain PS species, but not other PS species tested, mediate K-Ras  membrane curvature sensing. Thus, K-Ras nanoclusters act as relay stations to convert mechanical perturbations to mitogenic signaling.

Membrane curvature sensing of the lipid-anchored K-Ras small GTPase.

The RAS oncogenes are frequently mutated in human cancers and among the three isoforms (,  and ),  is the most frequently mutated oncogene. Here, we demonstrate that a subset of flavaglines, a class of natural anti-tumour drugs and chemical ligands of prohibitins, inhibit RAS GTP loading and oncogene activation in cells at nanomolar concentrations. Treatment with rocaglamide, the first discovered flavagline, inhibited the nanoclustering of KRAS, but not HRAS and NRAS, at specific phospholipid-enriched plasma membrane domains. We further demonstrate that plasma membrane-associated prohibitins directly interact with KRAS, phosphatidylserine and phosphatidic acid, and these interactions are disrupted by rocaglamide but not by the structurally related flavagline FL1. Depletion of prohibitin-1 phenocopied the rocaglamide-mediated effects on KRAS activation and stability. We also demonstrate that flavaglines inhibit the oncogenic growth of KRAS-mutated cells and that treatment with rocaglamide reduces non-small-cell lung carcinoma (NSCLC) tumour nodules in autochthonous KRAS-driven mouse models without severe side effects. Our data suggest that it will be promising to further develop flavagline derivatives as specific KRAS inhibitors for clinical applications

A subset of flavaglines inhibits KRAS nanoclustering and activation.

Membrane curvature has emerged as an intriguing physical principle underlying biological signaling and membrane trafficking. The CIP4/FBP17/Toca-1 F-BAR subfamily is unique in the BAR family because its structurally folded F-BAR domain does not contain any hydrophobic motifs that insert into membrane. Although widely assumed so, whether the banana-shaped F-BAR domain alone can sense curvature has never been experimentally demonstrated. Using a nanobar-supported lipid bilayer system, we found that the F-BAR domain of FBP17 displayed minimal curvature sensing . In comparison, an alternatively spliced intrinsically disordered region (IDR) adjacent to the F-BAR domain has the membrane curvature-sensing ability greatly exceeding that of F-BAR domain alone. In living cells, the presence of the IDR delayed the recruitment of FBP17 in curvature-coupled cortical waves. Collectively, we propose that contrary to the common belief, FBP17's curvature-sensing capability largely originates from IDR, and not the F-BAR domain alone.

Comparative Study of Curvature Sensing Mediated by F-BAR and an Intrinsically Disordered Region of FBP17.

Batteries for contact lenses fabricated by conventional methods could cause severe damage to the eyes if broken. Herein, we present flexible aqueous batteries that operate in tears and provide a safe power supply to smart contact lenses. Nanocomposite flexible electrodes of carbon nanotubes and Prussian blue analogue nanoparticles for cathode and anode were embedded in UV-polymerized hydrogel as not only a soft contact lens but also an ion-permeable separator. The battery exhibited a discharging capacity of 155 μAh in an aqueous electrolyte of 0.15 M Na-ions and 0.02 M K-ions, equivalent to the ionic concentration of tears. The power supply was enough to operate a low-power static random-access memory. In addition, we verified the mechanical stability, biocompatibility and compatibility with a contact lens cleaning solution. It could ultimately enable a safe power supply for smart contact lenses without risk of injury due to the leakage or breakage of the battery.

Tear-Based Aqueous Batteries for Smart Contact Lenses Enabled by Prussian Blue Analogue Nanocomposites.

Intracellular delivery of advanced therapeutics, including biologicals and supramolecular agents, is complex because of the natural biological barriers that have evolved to protect the cell. Efficient delivery of therapeutic nucleic acids, proteins, peptides and nanoparticles is crucial for clinical adoption of emerging technologies that can benefit disease treatment through gene and cell therapy. Nanoneedles are arrays of vertical high-aspect-ratio nanostructures that can precisely manipulate complex processes at the cell interface, enabling effective intracellular delivery. This emerging technology has already enabled the development of efficient and non-destructive routes for direct access to intracellular environments and delivery of cell-impermeant payloads. However, successful implementation of this technology requires knowledge of several scientific fields, making it complex to access and adopt by researchers who are not directly involved in developing nanoneedle platforms. This presents an obstacle to the widespread adoption of nanoneedle technologies for drug delivery. This tutorial aims to equip researchers with the knowledge required to develop a nanoinjection workflow. It discusses the selection of nanoneedle devices, approaches for cargo loading and strategies for interfacing to biological systems and summarises an array of bioassays that can be used to evaluate the efficacy of intracellular delivery.

Tutorial: using nanoneedles for intracellular delivery.

Despite the increasing prevalence of obesity, the current medications, which act indirectly on the central nervous system to suppress appetite or on the gastrointestinal tract to inhibit fat absorption, suffer from poor effectiveness and side effects. Here, we developed a transdermal mild photothermal therapy directly acting on the root of evil (subcutaneous white adipose depot) to induce its ameliorating remodeling (browning, lipolysis, and apoptosis), based on the injectable thermoresponsive hydrogel encapsulated with copper sulfide nanodots. Further, combining pharmaceutical therapy with codelivery of mirabegron leads to a strong therapeutic synergy. This method not only ensures high effectiveness and low side effects due to localized and targeted application but also remotely creates significant improvements in systemic metabolism. Specifically, as compared to the untreated group, it totally inhibits obesity development in high-fat-diet fed mice (15% less in body weight) with decreased masses of both subcutaneous (40%) and visceral fats (54%), reduced serum levels of cholesterol (54%)/triglyceride (18%)/insulin (74%)/glucose (45%), and improved insulin sensitivity (65% less in insulin resistance index). This self-administrable method is amenable for long-term home-based treatment. Finally, multiple interconnected signaling pathways are revealed, providing mechanistic insights to develop effective strategies to combat obesity and associated metabolic disorders.

Transdermal Photothermal-Pharmacotherapy to Remodel Adipose Tissue for Obesity and Metabolic Disorders.

As a dominant oncogenic protein, Ras is well-known to segregate into clusters on the plasma membrane for activating downstream signaling. However, current technologies for direct measurements of Ras clustering are limited to sophisticated high-resolution techniques like electron microscopy and fluorescence lifetime imaging. To further promote fundamental investigations and the related drug development, we hereby introduce a nanobar-based platform which effectively guides Ras clusters into quantifiable patterns in live cells that is resolvable under conventional microscopy. Major Ras isoforms, K-Ras, H-Ras, and N-Ras, were differentiated, as well as their highly prevalent oncogenic mutants G12V and G13D. Moreover, the isoform specificity and the sensitivity of a Ras inhibitor were successfully characterized on nanobars. We envision that this nanobar-based platform will serve as an effective tool to read Ras clustering on the plasma membrane, enabling a novel avenue both to decipher Ras regulations and to facilitate anti-Ras drug development.

Patterning of Oncogenic Ras Clustering in Live Cells Using Vertically Aligned Nanostructure Arrays.

Neuroblastoma is a hard-to-treat childhood cancer that is well known for the heterogeneity of its clinical phenotypes. Although the risk levels of neuroblastoma have been defined from a complex matrix of clinical and tumor biological factors to guide treatment, the accuracy in predicting cancer relapse and related fatality is still poor in many cases, where heterogeneity with subpopulations in highly malignant or drug-resistant tumors is believed to be underestimated by the current analysis methods. Therefore, new technologies to probe neuroblastoma heterogeneity are needed for the improvement of risk stratification. In this study, we introduce the nanopillar-guided subnuclear morphology as an effective indicator for heterogeneity evaluation among individual neuroblastoma cells. Nuclear polymorphisms, especially the generation of subnuclear irregularities, are well-known markers of high cancer metastasis risk and poor prognosis. By quantitatively evaluating the orientation of nanopillar-guided nuclear envelope features in neuroblastoma cells, we identified two subpopulations with differential motilities and EMT marker levels. Moreover, with endogenous expression, cells with high levels of the nuclear structure protein lamin A exhibit anisotropic deformation on nanopillars and migrate faster than low-lamin A cells, indicating a greater potential for metastasis. Overexpression of lamin A, however, reduces both the coherency and migration speed, suggesting that subpopulations with similar lamin A levels may have different metastatic potentials. We further verified that nanopillar-generated nuclear deformation patterns can quantitatively reveal individual cells' responses to anti-cancer drug treatment. Overall, we envision that the nanopillar-based assessment of subnuclear irregularities brings new additions to our toolkits for both precise risk stratification in neuroblastoma and the evaluation of related anti-cancer therapeutics.

Revealing the heterogeneity in neuroblastoma cells  nanopillar-guided subnuclear deformation.

For more than a century, abnormal nuclei in tumor cells, presenting subnuclear invaginations and folds on the nuclear envelope, have been known to be associated with high malignancy and poor prognosis. However, current nuclear morphology analysis focuses on the features of the entire nucleus, overlooking the malignancy-related subnuclear features in nanometer scale. The main technical challenge is to probe such tiny and randomly distributed features inside cells. We here employ nanopillar arrays to guide subnuclear features into ordered patterns, enabling their quantification as a strong indicator of cell malignancy. Both breast and liver cancer cells were validated as well as the quantification of nuclear abnormality heterogeneity. The alterations of subnuclear patterns were also explored as effective readouts for drug treatment. We envision that this nanopillar-enabled quantification of subnuclear abnormal features in tumor cells opens a new angle in characterizing malignant cells and studying the unique nuclear biology in cancer.

Article	Year
The Role of Membrane Curvature in Nanoscale Topography-Induced Intracellular Signaling. Accounts of chemical research\| PubMed ID: 29648779	2018
In Situ Generation of Zinc Oxide Nanobushes on Microneedles as Antibacterial Coating. SLAS technology\| PubMed ID: 30500311	2019
A nanostructure platform for live-cell manipulation of membrane curvature. Nature protocols\| PubMed ID: 31101905	2019
Membrane curvature sensing of the lipid-anchored K-Ras small GTPase. Life science alliance\| PubMed ID: 31296567	2019
A subset of flavaglines inhibits KRAS nanoclustering and activation. Journal of cell science\| PubMed ID: 32501281	2020
Comparative Study of Curvature Sensing Mediated by F-BAR and an Intrinsically Disordered Region of FBP17. iScience\| PubMed ID: 33205024	2020
Tear-Based Aqueous Batteries for Smart Contact Lenses Enabled by Prussian Blue Analogue Nanocomposites. Nano letters\| PubMed ID: 33533624	2021
Tutorial: using nanoneedles for intracellular delivery. Nature protocols\| PubMed ID: 34426708	2021
Transdermal Photothermal-Pharmacotherapy to Remodel Adipose Tissue for Obesity and Metabolic Disorders. ACS nano\| PubMed ID: 34979079	2022
Patterning of Oncogenic Ras Clustering in Live Cells Using Vertically Aligned Nanostructure Arrays. Nano letters\| PubMed ID: 35044178	2022
Revealing the heterogeneity in neuroblastoma cells nanopillar-guided subnuclear deformation. Nanoscale\| PubMed ID: 35133394	2022
Guiding Irregular Nuclear Morphology on Nanopillar Arrays for Malignancy Differentiation in Tumor Cells. Nano letters\| PubMed ID: 35969027	2022

Wenting Zhao

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