title: The core of tau-paired helical filaments studied by scanning transmission electron microscopy and limited proteolysis.
abstract: In Alzheimer's disease and frontotemporal dementias the microtubule-associated protein tau forms intracellular paired helical filaments (PHFs). The filaments formed in vivo consist mainly of full-length molecules of the six different isoforms present in adult brain. The substructure of the PHF core is still elusive. Here we applied scanning transmission electron microscopy (STEM) and limited proteolysis to probe the mass distribution of PHFs and their surface exposure. Tau filaments assembled from the three repeat domain have a mass per length (MPL) of approximately 60 kDa/nm and filaments from full-length tau (htau40DeltaK280 mutant) have approximately 160 kDa/nm, compared with approximately 130 kDa/nm for PHFs from Alzheimer's brain. Polyanionic cofactors such as heparin accelerate assembly but are not incorporated into PHFs. Limited proteolysis combined with N-terminal sequencing and mass spectrometry of fragments reveals a protease-sensitive N-terminal half and semiresistant PHF core starting in the first repeat and reaching to the C-terminus of tau. Continued proteolysis leads to a fragment starting at the end of the first repeat and ending in the fourth repeat. PHFs from tau isoforms with four repeats revealed an additional cleavage site within the middle of the second repeat. Probing the PHFs with antibodies detecting epitopes either over longer stretches in the C-terminal half of tau or in the fourth repeat revealed that they grow in a polar manner. These data describe the physical parameters of the PHFs and enabled us to build a model of the molecular arrangement within the filamentous structures.
authors.affiliation: Max Planck Unit for Structural Molecular Biology, Notkestrasse 85, D-22607 Hamburg, Germany. vbergen@mpasmb.desy.de
authors.forename: Martin
authors.initials: M
authors.lastname: von Bergen
authors.forename: Stefan
authors.initials: S
authors.lastname: Barghorn
authors.forename: Shirley A
authors.initials: SA
authors.lastname: Müller
authors.forename: Marcus
authors.initials: M
authors.lastname: Pickhardt
authors.forename: Jacek
authors.initials: J
authors.lastname: Biernat
authors.forename: Eva-Maria
authors.initials: EM
authors.lastname: Mandelkow
authors.forename: Peter
authors.initials: P
authors.lastname: Davies
authors.forename: Ueli
authors.initials: U
authors.lastname: Aebi
authors.forename: Eckhard
authors.initials: E
authors.lastname: Mandelkow
mesh_terms: D000544:Alzheimer Disease / Q000378:metabolism / Q000473:pathology; D000595:Amino Acid Sequence; D001921:Brain / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D017348:Microscopy, Electron, Scanning Transmission; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D008954:Models, Biological; D008957:Models, Genetic; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016874:Neurofibrillary Tangles / Q000737:chemistry / Q000378:metabolism; D010447:Peptide Hydrolases; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016875:tau Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*
chemical_list: D011994:Recombinant Proteins; D016875:tau Proteins; D010447:Peptide Hydrolases