The population context is a driver of the heterogeneous response of epithelial cells to interferons.

Isogenic cells respond in a heterogeneous manner to interferon. Using a micropatterning approach combined with high-content imaging and spatial analyses, we characterized how the population context (position of a cell with respect to neighboring cells) of epithelial cells affects their response to interferons. We identified that cells at the edge of cellular colonies are more responsive than cells embedded within colonies. We determined that this spatial heterogeneity in interferon response resulted from the polarized basolateral interferon receptor distribution, making cells located in the center of cellular colonies less responsive to ectopic interferon stimulation. This was conserved across cell lines and primary cells originating from epithelial tissues. Importantly, cells embedded within cellular colonies were not protected from viral infection by apical interferon treatment, demonstrating that the population context-driven heterogeneous response to interferon influences the outcome of viral infection. Our data highlights that the behavior of isolated cells does not directly translate to their behavior in a population, placing the population context as one important factor influencing heterogeneity during interferon response in epithelial cells.

Poly(lipoic acid)-Based Nanoparticles as Self-Organized, Biocompatible, and Corona-Free Nanovectors.

Herein we present an innovative approach to produce biocompatible, degradable, and stealth polymeric nanoparticles based on poly(lipoic acid), stabilized by a PEG-ended surfactant. Taking advantage of the well-known thiol-induced polymerization of lipoic acid, a universal and nontoxic nanovector consisted of a solid cross-linked polymeric matrix of lipoic acid monomers was prepared and loaded with active species with a one-step protocol. The biological studies demonstrated a high stability in biological media, the virtual absence of "protein" corona in biological fluids, the absence of acute toxicity in vitro and in vivo, complete clearance from the organism, and a relevant preference for short-term accumulation in the heart. All these features make these nanoparticles candidates as a promising tool for nanomedicine.

Role of Clathrin Light Chains in Regulating Invadopodia Formation.

One of the most fundamental processes of the cell is the uptake of molecules from the surrounding environment. Clathrin-mediated endocytosis (CME) is the best-described uptake pathway and regulates nutrient uptake, protein and lipid turnover at the plasma membrane (PM), cell signaling, cell motility and cell polarity. The main protein in CME is clathrin, which assembles as a triskelion-looking building block made of three clathrin heavy chains and three clathrin light chains. Compared to clathrin heavy chains (CHCs), the role of the two isoforms of clathrin light chains (CLCA and CLCB) is poorly understood. Here, we confirm that the simultaneous deletion of both CLCA/B causes abnormal actin structures at the ventral PM and we describe them, for the first time, as functional invadopodia rather than disorganized actin-cytoskeleton assembly sites. Their identification is based on the occurrence of common invadopodia markers as well as functional invadopodia activity characterized by an increased local proteolytic activity of the extracellular matrix proteins. We demonstrate that CLCA/B deletion impacts the intracellular trafficking and recovery of the matrix metalloproteinase 14 (MMP14) leading to its accumulation at the plasma membrane and induction of invadopodia formation. Importantly, we show that invadopodia formation can be prevented by depletion of MMP14. As such, we propose that CLCA/B regulate invadopodia formation by regulating MMP14 delivery to the plasma membrane.

Ultrasmall Gold Nanoparticle Cellular Uptake: Influence of Transient Bionano Interactions.

Ultrasmall nanoparticles are attracting an increasing interest for a variety of biomedical applications, from therapeutic targeting to imaging, in virtue of the peculiar behavior shown in vivo (i.e., efficient renal clearance, low liver accumulation, etc.). In evaluating their potential to overcome some of the challenges that larger particles have faced, it is important to understand their mechanisms of interaction with the cell membrane in relation to the biological environment and their tendency to transiently interact with biomolecules. In this work, the mechanism of cellular uptake across a range of serum concentrations is investigated using 2 nm gold nanoparticles with different surface chemistries as a model. The data suggest that despite their ultrasmall size, for these nanoparticles, internalization occurs via energy-dependent processes, and the surface chemistry could play a key role in determining the modality of the transient protein interaction, especially in conditions close to the in vivo scenario (large excess of the protein content). These aspects might be exploited to define novel targeting strategies.

A Three-Dimensional Cell Culture Platform for Long Time-Scale Observations of Bio-Nano Interactions.

We know surprisingly little about the long-term outcomes for nanomaterials interacting with organisms. To date, most of what we know is derived from in vivo studies that limit the range of materials studied and the scope of advanced molecular biology tools applied. Long-term in vitro nanoparticle studies are hampered by a lack of suitable models, as standard cell culture techniques present several drawbacks, while technical limitations render current three-dimensional (3D) cellular spheroid models less suited. Now, by controlling the kinetic processes of cell assembly and division in a non-Newtonian culture medium, we engineer reproducible cell clusters of controlled size and phenotype, leading to a convenient and flexible long-term 3D culture that allows nanoparticle studies over many weeks in an in vitro setting. We present applications of this model for the assessment of intracellular polymeric and silica nanoparticle persistence and found that hydrocarbon-based polymeric nanoparticles undergo no apparent degradation over long time periods with no obvious biological impact, while amorphous silica nanoparticles degrade at different rates over several weeks, depending on their synthesis method.

Magnetic One-Step Purification of His-Tagged Protein by Bare Iron Oxide Nanoparticles.

Magnetic separation is a promising alternative to conventional methods in downstream processing. This can facilitate easier handling, fewer processing steps, and more sustainable processes. Target materials can be extracted directly from crude cell lysates in a single step by magnetic nanoadsorbents with high-gradient magnetic fishing (HGMF). Additionally, the use of hazardous consumables for reducing downstream processing steps can be avoided. Here, we present proof of principle of one-step magnetic fishing from crude Escherichia coli cell lysate of a green fluorescent protein (GFP) with an attached hexahistidine (His6)-tag, which is used as the model target molecule. The focus of this investigation is the upscale to a liter scale magnetic fishing process in which a purity of 91% GFP can be achieved in a single purification step from cleared cell lysate. The binding through the His6-tag can be demonstrated, since no significant binding of nontagged GFP toward bare iron oxide nanoparticles (BIONs) can be observed. Nonfunctionalized BIONs with primary particle diameters of around 12 nm, as used in the process, can be produced with a simple and low-cost coprecipitation synthesis. Thus, HGMF with BIONs might pave the way for a new and greener era of downstream processing.