title: A two-step path to inclusion formation of huntingtin peptides revealed by number and brightness analysis.
abstract: Protein aggregation is a hallmark of several neurodegenerative diseases including Huntington's disease. We describe the use of the recently developed number and brightness method (N&B) that uses confocal images to monitor aggregation of Huntingtin exon 1 protein (Httex1p) directly in living cells. N&B measures the molecular brightness of protein aggregates in the entire cell noninvasively based on intensity fluctuations at each pixel in an image. N&B applied to mutant Httex1p in living cells showed a two-step pathway leading to inclusion formation that is polyQ length dependent and involves four phases. An initial phase of monomer accumulation is followed by formation of small oligomers (5-15 proteins); as protein concentration increases, an inclusion is seeded and forms in the cytoplasm; the growing inclusion recruits most of the Httex1p and depletes the cell leaving only a low concentration of monomers. The behavior of Httex1p in COS-7 and ST14A cells is compared.
authors.affiliation: Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, California, USA.
authors.forename: Giulia
authors.initials: G
authors.lastname: Ossato
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Digman
authors.forename: Charity
authors.initials: C
authors.lastname: Aiken
authors.forename: Tamas
authors.initials: T
authors.lastname: Lukacsovich
authors.forename: J Lawrence
authors.initials: JL
authors.lastname: Marsh
authors.forename: Enrico
authors.initials: E
authors.lastname: Gratton
mesh_terms: D000818:Animals; D019556:COS Cells; D002138:Calibration; D002522:Chlorocebus aethiops; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D000071058:Huntingtin Protein; D018613:Microscopy, Confocal; D057054:Molecular Imaging; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism*; D009687:Nuclear Proteins; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D051381:Rats
chemical_list: C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D010455:Peptides; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C097188:polyglutamine