Deciphering the Structure, Growth and Assembly of Amyloid-Like Fibrils Using High-Speed Atomic Force Microscopy

Formation of fibrillar structures of proteins that deposit into aggregates has been suggested to play a key role in various neurodegenerative diseases. However mechanisms and dynamics of fibrillization remains to be elucidated. We have previously established that lithostathine, a protein overexpressed in the pre-clinical stages of Alzheimer's disease and present in the pathognomonic lesions associated with this disease, form fibrillar aggregates after its N-terminal truncation. In this paper we visualized, using high-speed atomic force microscopy (HS-AFM), growth and assembly of lithostathine protofibrils under physiological conditions with a time resolution of one image/s. Real-time imaging highlighted a very high velocity of elongation. Formation of fibrils via protofibril lateral association and stacking was also monitored revealing a zipper-like mechanism of association. We also demonstrate that, like other amyloid ß peptides, two lithostathine protofibrils can associate to form helical fibrils. Another striking finding is the propensity of the end of a growing protofibril or fibril to associate with the edge of a second fibril, forming false branching point. Taken together this study provides new clues about fibrillization mechanism of amyloid proteins

Toolbox for Non-Intrusive Structural and Functional Analysis of Recombinant VLP Based Vaccines: A Case Study with Hepatitis B Vaccine

Background: Fundamental to vaccine development, manufacturing consistency, and product stability is an understanding of the vaccine structure-activity relationship. With the virus-like particle (VLP) approach for recombinant vaccines gaining popularity, there is growing demand for tools that define their key characteristics. We assessed a suite of non-intrusive VLP epitope structure and function characterization tools by application to the Hepatitis B surface antigen (rHBsAg) VLP-based vaccine. Methodology: The epitope-specific immune reactivity of rHBsAg epitopes to a given monoclonal antibody was monitored by surface plasmon resonance (SPR) and quantitatively analyzed on rHBsAg VLPs in-solution or bound to adjuvant with a competitive enzyme-linked immunosorbent assay (ELISA). The structure of recombinant rHBsAg particles was examined by cryo transmission electron microscopy (cryoTEM) and in-solution atomic force microscopy (AFM). Principal Findings: SPR and competitive ELISA determined relative antigenicity in solution, in real time, with rapid turnaround, and without the need of dissolving the particulate aluminum based adjuvant. These methods demonstrated the nature of the clinically relevant epitopes of HBsAg as being responsive to heat and/or redox treatment. In-solution AFM and cryoTEM determined vaccine particle size distribution, shape, and morphology. Redox-treated rHBsAg enabled 3D reconstruction from CryoTEM images – confirming the previously proposed octahedral structure and the established lipidto-protei

Compression, Rupture, and Puncture of Model Membranes at the Molecular Scale

International audienceElastic properties of biological membranes are involved in a large number of membrane functionalities and activities. Conventionally characterized in terms of Young’s modulus, bending stiffness and stretching modulus, membrane mechanics can be assessed at high lateral resolution by means of atomic force microscopy (AFM). Here we show that the mechanical response of biomimetic model systems such as supported lipid bilayers (SLBs) is highly affected by the size of the AFM tip employed as a membrane indenter. Our study is focused on phase-separated fluid-gel lipid membranes at room temperature. In a small tip radius regime (≈ 2 nm) and in the case of fluid phase membranes, we show that the tip can penetrate through the membrane minimizing molecular vertical compression and in absence of molecular membrane rupture. In this case, AFM indentation experiments cannot assess the vertical membrane Young’s modulus. In agreement with the data reported in the literature, in the case of larger indenters (>2 nm) SLBs can be compressed leading to an evaluation of Young’s modulus and membrane maximal withstanding force before rupture. We show that such force increases with the indenter in agreement with the existing theoretical frame. Finally, we demonstrate that the latter has no influence on the number of molecules involved in the rupture process that is observed to be constant and rather dependent on the indenter chemical composition

Prise en charge du trouble bipolaire de type 1 chez l’enfant et l’adolescent

International audienc

Milk sphingomyelin domains in biomimetic membranes and the role of cholesterol: morphology and nanomechanical properties investigated using AFM and force spectroscopy

Milk sphingomyelin (MSM) and cholesterol segregate into domains in the outer bilayer membrane surrounding milk fat globules. To elucidate the morphology and mechanical properties of theses domains, supported lipid bilayers with controlled molar proportions of MSM, dioleoylphosphatidylcholine (DOPC) and cholesterol were produced in buffer mimicking conditions of the milk aqueous phase. Atomic force microscopy imaging showed that (i) for T < 35 \ub0C MSM segregated in gel phase domains protruding above the fluid phase, (ii) the addition of 20 mol % cholesterol resulted in smaller and more elongated l o phase domains than in equimolar MSM/DOPC membranes, (iii) the MSM/cholesterol-enriched lo phase domains were less salient than the MSM gel phase domains. Force spectroscopy measurements furthermore showed that cholesterol reduced the resistance of MSM/DOPC membrane to perforation. The results are discussed with respect to the effect of cholesterol on the biophysical properties of lipid membranes. The combination of AFM imaging and force mapping provides unprecedented insight into the structural and mechanical properties of milk lipid membranes, and opens perspectives for investigation of the functional properties of MSM domains during milk fat processing or digestion. \ua9 2014 American Chemical Society.Peer reviewed: YesNRC publication: Ye

A simple approach for controlled deposition of Prussian blue analogue nanoparticles on a functionalised plasmonic gold surface

International audienceWe report here a simple and efficient approach for the controlled deposition of a monolayer of Prussian blue analogue nanoparticles on a gold surface functionalised with amino groups and their characterisation by surface plasmon resonance spectroscopy and atomic force microscopy combined with theoretical modeling