Sara Anselmo

Protein aggregation strongly depends on the local environment, yet investigating it in compartmentalized, high-surface-area volumes remains challenging. Here, we present an optically controlled platform for inducing and monitoring protein aggregation in microliter droplets. Local pH changes are generated in situ via photoconversion of 2-nitrobenzaldehyde, enabling noninvasive environmental control without mechanical mixing or direct perturbation of the sample. By combining fluorescein-based pH measurements with Raster Image Correlation Spectroscopy (RICS), pH variations and protein diffusion were monitored. As a model system, we investigated β-lactoglobulin, whose association state strongly depends on pH. Light-induced acidification progressively shifted the protein toward its isoelectric region, resulting in a measurable decrease in the diffusion coefficient consistent with the formation of larger supramolecular species. Furthermore, modulation of electrostatic interactions through NaCl addition revealed the sensitivity of the system to environmental factors governing the balance between electrostatic repulsion and short-range attractive interactions. Under these conditions, the platform enabled the observation of aggregation behaviors that were significantly less pronounced in corresponding bulk conditions. Overall, this droplet-based approach allows for the precise manipulation of local chemistry and quantitative analysis of biomolecular self-assembly, making it readily extendable to various noninvasive, time-resolved monitoring applications.

Ferritin, a physiological iron-storage protein, has emerged as a highly attractive platform for drug delivery owing to its biocompatibility, structural robustness, and intrinsic ability to encapsulate and protect therapeutic cargo within its hollow nanocage. Building upon previous studies that established the baseline characteristics of engineered ferritin mutants in comparison to the wild-type protein, this work specifically investigates and directly compares the thermal stability profiles of two distinct mutated variants. These variants of human H-chain ferritin, obtained through targeted site-directed mutagenesis, feature either four or six tryptophan residues per subunit, strategically positioned toward the inner cavity of the protein shell. These modifications were intended to enhance hydrophobic interactions with guest molecules while preserving the native quaternary architecture. Temperature-dependent changes in surface hydrophobicity and solvent accessibility were probed using the environment-sensitive fluorescent dye ANS, enabling a comparative assessment of the conformational behavior of the two mutants. Overall, this study highlights how targeted modulation of the internal cavity composition of ferritin can tune both its physicochemical properties and stability, providing insights relevant for the rational design of ferritin-based nanoplatforms for biomedical applications.

Lipid rafts are dynamic microdomains in the membrane, rich in cholesterol and sphingolipids, that are critical for biological processes like cell signalling, membrane trafficking, and protein organization. Their essential role is claimed in both physiological and pathological conditions, including cancer, neurodegenerative diseases, and viral infections, making them a key area of research. Fluorescence-based approaches, including super-resolution fluorescence microscopy techniques, enable precise analysis of the organization, dynamics, and interactions of these microdomains, thanks also to the innovative design of appropriate fluorescent probes. Moreover, these non-invasive approaches allow for the study of live cells, facilitating the collection of quantitative data under physiologically relevant conditions. This review synthesizes the latest insights into the role of lipid rafts in biological and pathological processes and underscores how fluorescence techniques have advanced our understanding of these critical microdomains. The findings emphasize the pivotal role of lipid rafts in health and disease, providing a foundation for future research and potential therapeutic interventions.

Continuous progress has been made in the development of new molecules for therapeutic purposes. This is driven by the need to address several challenges such as molecular instability and biocompatibility, difficulties in crossing the plasma membrane, and the development of host resistance. In this context, cell-penetrating peptides (CPPs) constitute a promising tool for the development of new therapies due to their intrinsic ability to deliver therapeutic molecules to cells and tissues. These short peptides have gained increasing attention for applications in drug delivery as well as for their antimicrobial and anticancer activity but the general rules regulating the events involved in cellular uptake and in the following processes are still unclear. Here, we use fluorescence microscopy methods to analyze the interactions between the multifunctional peptide Transportan 10 (TP10) and the giant plasma membrane vesicles (GPMVs) derived from cancer cells. This aims to highlight the molecular mechanisms underlying functional interactions which bring its translocation across the membrane or cytotoxic mechanisms leading to membrane collapse and disruption. The Fluorescence Lifetime Imaging Microscopy (FLIM) method coupled with the phasor approach analysis proved to be the winning choice for following highly dynamic spatially heterogeneous events in real-time and highlighting aspects of such complex phenomena. Thanks to the presented approach, we were able to identify and monitor TP10 translocation into the lumen, internalization, and membrane-induced modifications depending on the peptide concentration regime.