Biology subjects

Conteduca, D.

Publications and source records attributed to Conteduca, D..

3 recordsLinked to original sources

bioRxiv2023

Multiple Intermediates in the Detergent-Induced Fusion of Lipid Vesicles

Detergent-induced vesicle interactions, critical for applications including virus inactivation, varies according to the detergent type and membrane composition, but the underlying mechanistic details remain underexplored. Here, we use a lipid mixing assay based on Forster resonance energy transfer (FRET), and single-vesicle characterization approaches to identify that sub-micron sized vesicles are induced to fuse by the non-ionic detergent Triton-X 100. We demonstrate that the process is a multi-step mechanism, characterized by discrete values of FRET efficiency between membrane-embedded fluorophores, and involves permeabilization, vesicle docking, hemi-fusion and full lipid mixing at sub-solubilizing detergent concentrations. We also dissect the kinetics of vesicle fusion to surface-tethered vesicles using a label-free quartz-crystal microbalance with dissipation monitoring approach, opening a platform for biotechnology applications. The presented strategies provide mechanistic insight into the dynamics of vesicle fusion and have implications for applications including drug delivery and sensor development where transport and manipulation of encapsulated cargo is essential.

biophysics↗

bioRxiv2022

Crowding induced morphological changes in synthetic lipid vesicles determined using smFRET

Lipid vesicles are valuable mesoscale molecular confinement vessels for studying membrane mechanics and lipid-protein interactions, and they have found utility among bio-inspired technologies including drug delivery vehicles. While vesicle morphology can be modified by changing the lipid composition and introducing fusion or pore-forming proteins and detergents, the influence of extramembrane crowding on vesicle morphology has remained under explored owing to a lack of experimental tools capable of capturing morphological changes on the nanoscale. Here, we use biocompatible polymers to simulate molecular crowding in vitro, and through combinations of FRET spectroscopy, lifetime analysis, dynamic light scattering and single-vesicle imaging, we characterize how crowding regulates vesicle morphology. We show that both freely-diffusing and surface-tethered vesicles fluorescently tagged with the DiI and DiD FRET pair undergo compaction in response to modest concentrations of sorbitol, polyethylene glycol and Ficoll. A striking observation is that sorbitol results in irreversible compaction, whereas the influence of high molecular weight PEG-based crowders was found to be reversible. Regulation of molecular crowding allows for precise control of vesicle architecture in vitro, with vast implications for drug delivery and vesicle trafficking systems. Furthermore, our observations of vesicle compaction may also serve to act as a mechanosensitive readout of extramembrane crowding.

biophysics↗

bioRxiv2022

Tween-20 induces the structural remodelling of single lipid vesicles

The interaction of Tween-20 with lipid membranes is crucial for a number of biotechnological applications including viral inactivation and membrane protein extraction, but the underlying mechanistic details have remained elusive. Evidence from ensemble assays supports a global model of Tween-20 induced membrane disruption that broadly encompasses association of the surfactant with the membrane surface, membrane fragmentation and the release of mixed micelles to solution, but whether this process involves intermediate and dynamic transitions between regimes is an open question. In search of the mechanistic origins of membrane disruption, increasing focus is put on identifying Tween-20 interactions with highly controllable model membranes. In light of this, and to unveil quantitative mechanistic details, we employed highly interdisciplinary biophysical approaches, including quartz-crystal microbalance with dissipation monitoring, steady-state and time-resolved fluorescence and FRET spectroscopy, dynamic light scattering, fluorescence correlation spectroscopy, wide-field single-vesicle imaging and scanning electron microscopy, to interrogate the interactions between Tween-20 and both freely-diffusing and surface-immobilized model-membrane vesicles. Using ultrasensitive sensing approaches, we discovered that Tween-20 leads to a stepwise and phase-dependent structural remodelling of sub-micron sized vesicles that includes permeabilization and swelling, even at detergent concentrations below the critical micellar concentration. These insights into the structural perturbation of lipid vesicles upon Tween-20 interaction highlight the impact on vesicle conformation prior to complete solubilization, and the tools presented may have general relevance for probing the interaction between lipid vesicles and a wide variety of disruptive agents.

biophysics↗