title: Imaging amyloid fibrils within cells using a Se-labelling strategy.
abstract: The process of aggregation leading to amyloid formation by peptides and proteins is associated with diseases ranging from systemic amyloidoses to neurodegenerative disorders such as Alzheimer's disease. A key question in understanding the link between amyloid formation and its pathological consequences is the ultrastructural localisation and morphological form of amyloid species within the cellular environment. The acquisition of such information has proven to be challenging, but we report here a novel approach that enables amyloid fibrils to be visualised directly within a cell. First, fibrils are assembled from selenium analogues of the sulfur-containing cysteine peptides, and then, atomic number contrast transmission electron microscopy is used to detect the selenium doped species selectively within the carbon-rich background of the cell. We demonstrate the power of this approach by imaging human monocyte-derived macrophage cells that have been exposed to fibrils from an amyloidogenic fragment of the disease-associated protein transthyretin. The ready incorporation of seleno-cysteine and methionine instead of their natural sulfur-containing analogues, a feature that is already commonly used in X-ray diffraction studies of proteins, suggests that this method can be used as a general strategy to image specific peptides and proteins within the cellular environment using electron microscopy.
authors.affiliation: Department of Materials, Imperial College London, London SW7 2AZ, UK. a.porter@imperial.ac.uk
authors.forename: Alexandra E
authors.initials: AE
authors.lastname: Porter
authors.forename: Tuomas P J
authors.initials: TP
authors.lastname: Knowles
authors.forename: Karin
authors.initials: K
authors.lastname: Muller
authors.forename: Sarah
authors.initials: S
authors.lastname: Meehan
authors.forename: Eva
authors.initials: E
authors.lastname: McGuire
authors.forename: Jeremy
authors.initials: J
authors.lastname: Skepper
authors.forename: Mark E
authors.initials: ME
authors.lastname: Welland
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Dobson
mesh_terms: D000682:Amyloid / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure*; D002477:Cells / Q000378:metabolism*; D002478:Cells, Cultured; D003198:Computer Simulation; D003287:Contrast Media / Q000493:pharmacokinetics; D006801:Humans; D046529:Microscopy, Electron, Transmission / Q000379:methods; D055503:Protein Multimerization / Q000502:physiology; D018036:Selenium Compounds / Q000493:pharmacokinetics*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D000682:Amyloid; D003287:Contrast Media; D018036:Selenium Compounds