by Keyword: Cell membrane

Woythe L, Madhikar P, Feiner-Gracia N, Storm C, Albertazzi L, (2022). A Single-Molecule View at Nanoparticle Targeting Selectivity: Correlating Ligand Functionality and Cell Receptor Density Acs Nano 16, 3785-3796

Antibody-functionalized nanoparticles (NPs) are commonly used to increase the targeting selectivity toward cells of interest. At a molecular level, the number of functional antibodies on the NP surface and the density of receptors on the target cell determine the targeting interaction. To rationally develop selective NPs, the single-molecule quantitation of both parameters is highly desirable. However, techniques able to count molecules with a nanometric resolution are scarce. Here, we developed a labeling approach to quantify the number of functional cetuximabs conjugated to NPs and the expression of epidermal growth factor receptors (EGFRs) in breast cancer cells using direct stochastic optical reconstruction microscopy (dSTORM). The single-molecule resolution of dSTORM allows quantifying molecules at the nanoscale, giving a detailed insight into the distributions of individual NP ligands and cell receptors. Additionally, we predicted the fraction of accessible antibody-conjugated NPs using a geometrical model, showing that the total number exceeds the accessible number of antibodies. Finally, we correlated the NP functionality, cell receptor density, and NP uptake to identify the highest cell uptake selectivity regimes. We conclude that single-molecule functionality mapping using dSTORM provides a molecular understanding of NP targeting, aiding the rational design of selective nanomedicines.

JTD Keywords: active targeting, active targeting dstorm, antibodies, dstorm, heterogeneity, multivalency, nanomedicine, nanoparticle functionality, size, super-resolution microscopy, surface, Active targeting, Antibodies, Cell membranes, Cell receptors, Cytology, Direct stochastic optical reconstruction microscopy, Dstorm, Heterogeneity, Ligands, Medical nanotechnology, Molecules, Nanomedicine, Nanoparticle functionality, Nanoparticle targeting, Nanoparticles, Optical reconstruction, Single molecule, Stochastic systems, Stochastics, Super-resolution microscopy, Superresolution microscopy

Riera, Roger, Hogervorst, Tim P., Doelman, Ward, Ni, Yan, Pujals, Silvia, Bolli, Evangelia, Codée, Jeroen DC., van Kasteren, Sander I., Albertazzi, Lorenzo, (2021). Single-molecule imaging of glycan–lectin interactions on cells with Glyco-PAINT Nature Chemical Biology 17, 1281-1288

Most lectins bind carbohydrate ligands with relatively low affinity, making the identification of optimal ligands challenging. Here we introduce a point accumulation in nanoscale topography (PAINT) super-resolution microscopy method to capture weak glycan-lectin interactions at the single-molecule level in living cells (Glyco-PAINT). Glyco-PAINT exploits weak and reversible sugar binding to directly achieve single-molecule detection and quantification in cells and is used to establish the relative kon and koff rates of a synthesized library of carbohydrate-based probes, as well as the diffusion coefficient of the receptor-sugar complex. Uptake of ligands correlates with their binding affinity and residence time to establish structure-function relations for various synthetic glycans. We reveal how sugar multivalency and presentation geometry can be optimized for binding and internalization. Overall, Glyco-PAINT represents a powerful approach to study weak glycan-lectin interactions on the surface of living cells, one that can be potentially extended to a variety of lectin-sugar interactions.© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.

JTD Keywords: dc-sign, density, dimerization, endocytosis, lateral mobility, ligand-binding, mannose receptor, proteins, recognition, Animal, Animals, Cell membrane, Cell membrane permeability, Chemistry, Cho cell line, Cho cells, Cricetulus, Cysteine-rich domain, Kinetics, Lectin, Lectins, Ligand, Ligands, Molecular library, Multivariate analysis, Polysaccharide, Polysaccharides, Procedures, Protein binding, Single molecule imaging, Small molecule libraries, Structure activity relation, Structure-activity relationship

Nyga, Agata, Muñoz, Jose J., Dercksen, Suze, Fornabaio, Giulia, Uroz, Marina, Trepat, Xavier, Baum, Buzz, Matthews, Helen K., Conte, Vito, (2021). Oncogenic RAS instructs morphological transformation of human epithelia via differential tissue mechanics Science Advances 7,

Garcia-Parajo, M. F., (2012). The role of nanophotonics in regenerative medicine Nanotechnology in Regenerative Medicine - Methods and Protocols (Methods in Molecular Biology) (ed. Navarro, M., Planell, J. A.), Springer (New York, USA) 811, 267-284

Cells respond to biochemical and mechanical stimuli through a series of steps that begin at the molecular, nanometre level, and translate finally in global cell response. Defects in biochemical- and/or mechanical-sensing, transduction or cellular response are the cause of multiple diseases, including cancer and immune disorders among others. Within the booming field of regenerative medicine, there is an increasing need for developing and applying nanotechnology tools to bring understanding on the cellular machinery and molecular interactions at the nanoscale. Nanotechnology, nanophotonics and in particular, high-resolution-based fluorescence approaches are already delivering crucial information on the way that cells respond to their environment and how they organize their receptors to perform specialized functions. This chapter focuses on emerging super-resolution optical techniques, summarizing their principles, technical implementation, and reviewing some of the achievements reached so far.

JTD Keywords: Cell membrane organization, Nanophotonics, Near-field optical microscopy, Super-resolution optical microscopy

van Zanten, T. S., Garcia-Parajo, M. F., (2012). Super-resolution near-field optical microscopy Comprehensive Biophysics (ed. Egelman, E. H.), Elsevier (Desdren, Germany) Volume 2: Biophysical Techniques for Characterization of Cells, 144-164

Near-field optical microscopy is a technique not limited by the laws of diffraction that enables simultaneous high-resolution fluorescence and topographic measurements at the nanometer scale. This chapter highlights the intrinsic advantages of near-field optics in the study of cellular structures. The first part of the chapter lays the foundations of the near-field concept and technical implementation of near-field scanning optical microscopy (NSOM), whereas the second part of the chapter focuses on applications of NSOM to the study of model membranes and cellular structures on the plasma membrane. The last part of the chapter discusses further directions of near-field optics, including optical antennas and fluorescence correlation spectroscopy approaches in the near-field regime.

JTD Keywords: Biological membranes, Cell membrane nanoscale compartmentalization, Cellular nanodomains, Fluorescence correlation spectroscopy in reduced volumes, Immunoreceptor imaging, Lipid rafts, Near-field scanning optical microscopy, Optical nano-antennas, Shear force imaging, Single molecule detection, Super-resolution microscopy

van Zanten, Thomas S., Lopez-Bosque, M. J . , Garcia-Parajo, M. F., (2010). Imaging individual proteins and nanodomains on intact cell membranes with a probe-based optical antenna Small 6, (2), 270-275

Optical antennas that confine and enhance electromagnetic fields in a nanometric region hold great potential for nanobioimaging and biosensing. Probe-based monopole optical antennas are fabricated to enhance fields localized to <30 nm near the antenna apex in aqueous conditions. These probes are used under appropriate excitation antenna conditions to image individual antibodies with an unprecedented resolution of 26 ± 4 nm and virtually no surrounding background. On intact cell membranes in physiological conditions, the obtained resolution is 30 ± 6 nm. Importantly, the method allows individual proteins to be distinguished from nanodomains and the degree of clustering to be quantified by directly measuring physical size and intensity of individual fluorescent spots. Improved antenna geometries should lead to true live cell imaging below 10-nm resolution with position accuracy in the subnanometric range.

JTD Keywords: Cell membranes, Cell receptors, Focused ion beam milling, Nanodomains, Optical antennas