Silva, P. H. J. | Search bioRxiv

bioRxiv2022

Cholesterol-containing liposomes decorated with Au nanoparticles as minimal tunable fusion machinery

Membrane fusion is essential for the basal functionality of eukaryotic cells. In physiological conditions, fusion events are regulated by a wide range of specialized proteins, as well as by a finely tuned local lipid composition and ionic environment. SNARE proteins, for example, provide the mechanical energy necessary to achieve vesicle fusion in neuromediator release, and their action is assisted by other soluble proteins, membrane cholesterol, and calcium ions. Similar cooperative effects must be explored when considering synthetic approaches to achieve controlled and selective membrane fusion. Here we show that liposomes decorated with amphiphilic Au nanoparticles (AuLips) can act as minimal tunable fusion machinery. AuLips fusion is triggered by divalent ions, while the number of fusion events dramatically depends on, and can be finely tuned by, the liposome cholesterol content. Our results, obtained via a combination of experimental (Quartz-Crystal-Microbalance with Dissipation monitoring, Fluorescence assays, Small-Angle X-ray Scattering) and computational techniques (Molecular Dynamics with coarse-grained resolution), reveal new mechanistic details on the fusogenic activity of amphiphilic Au nanoparticles in synergy with membrane cholesterol, and demonstrate the ability of these synthetic nanomaterials to induce fusion regardless of the divalent ion used (Ca2+ or Mg2+). This evidence provides a novel contribution to the development of new artificial fusogenic agents for next-generation biomedical applications that require tight control of the rate of fusion events (e.g., targeted drug delivery).

biophysics↗

bioRxiv2019

Unraveling the Complexity of Amyloid Polymorphism Using Gold Nanoparticles and Cryo-EM

The misfolding and self-assembly of proteins into {beta}-sheet-rich amyloid fibrils of various structures and morphologies is a hallmark of several neurodegenerative and systemic diseases. Increasing evidence suggests that amyloid polymorphism gives rise to different strains of amyloids with distinct toxicity and pathology-spreading properties. Validating this hypothesis is challenging due to a lack of tools and methods that allow for the direct characterization of amyloid polymorphism in hydrated and complex biological samples. Here, we report on the use of 11-mercapto-1-undecanesulfonate-coated gold nanoparticles (NPs) to label the edges of synthetic, recombinant and native amyloid fibrils to assess amyloid morphological polymorphism using cryogenic transmission electron microscopy (cryo-EM). The fibrils studied were derived from amyloid proteins involved in disorders of the central nervous system (amyloid-{beta}, tau, -synuclein) and in systemic amyloidosis (a fragment of an immunoglobulin {lambda} light chain). The labeling efficiency enabled imaging and characterization of amyloid fibrils of different morphologies under hydrated conditions using cryo-EM. These NPs allowed for the visualization of morphological features that are not directly observed using standard imaging techniques, including TEM with use of the negative stain or cryo-EM imaging. We also demonstrate the use of these NPs to label native paired helical filaments (PHFs) from the postmortem brain of an Alzheimers disease patient, as well as amyloid fibrils extracted from the heart tissue of a patient suffering from systemic amyloid light-chain (AL) amyloidosis. Analysis of the cryo-EM images of amyloids decorated with NPs shows exceptional homogeneity across the fibrils derived from human tissue in comparison to fibrils aggregated in vitro. The use of these NPs enabled us to gain novel insight into the structural features that distinguish amyloid fibrils formed in vivo from those formed in cell-free in vitro systems. Our findings demonstrate that these NPs represent a powerful tool for rapid imaging and profiling of amyloid morphological polymorphism in different types of samples, including those derived from complex biological aggregates found in human tissue and animal models of amyloid diseases. These advances should not only facilitate the profiling and characterization of amyloids for structural studies by cryo-EM but also pave the way to elucidate the structural basis of amyloid strains and toxicity and possibly the correlation between the pathological and clinical heterogeneity of amyloid diseases.

neuroscience↗