title: Fluctuation methods to study protein aggregation in live cells: concanavalin A oligomers formation.
abstract: Prefibrillar oligomers of proteins are suspected to be the primary pathogenic agents in several neurodegenerative diseases. A key approach for elucidating the pathogenic mechanisms is to probe the existence of oligomers directly in living cells. In this work, we were able to monitor the process of aggregation of Concanavalin A in live cells. We used number and brightness analysis, two-color cross number and brightness analysis, and Raster image correlation spectroscopy to obtain the number of molecules, aggregation state, and diffusion coefficient as a function of time and cell location. We observed that binding of Concanavalin A to the membrane and the formation of small aggregates paralleled cell morphology changes, indicating progressive cell compaction and death. Upon protein aggregation, we observed increased membrane water penetration as reported by Laurdan generalized polarization imaging.
authors.affiliation: Dipartimento di Scienze Fisiche ed Astronomiche, University of Palermo, Palermo, Italy.
authors.forename: V
authors.initials: V
authors.lastname: Vetri
authors.affiliation: Laboratory for Fluorescence Dynamics, University of California, Irvine, California.
authors.forename: G
authors.initials: G
authors.lastname: Ossato
authors.affiliation: Dipartimento di Scienze Fisiche ed Astronomiche, University of Palermo, Palermo, Italy.
authors.forename: V
authors.initials: V
authors.lastname: Militello
authors.affiliation: Laboratory for Fluorescence Dynamics, University of California, Irvine, California.
authors.forename: M A
authors.initials: MA
authors.lastname: Digman
authors.affiliation: Dipartimento di Scienze Fisiche ed Astronomiche, University of Palermo, Palermo, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Leone
authors.affiliation: Laboratory for Fluorescence Dynamics, University of California, Irvine, California. Electronic address: egratton22@yahoo.com.
authors.forename: E
authors.initials: E
authors.lastname: Gratton
mesh_terms: D015081:2-Naphthylamine / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D001703:Biophysics / Q000379:methods*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D048430:Cell Shape / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003208:Concanavalin A / Q000737:chemistry* / Q000494:pharmacology; D004058:Diffusion / Q000187:drug effects; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D007848:Laurates / Q000378:metabolism; D051379:Mice; D020836:Protein Structure, Quaternary; D013057:Spectrum Analysis; D013997:Time Factors
chemical_list: D017304:Annexin A5; D007848:Laurates; D003208:Concanavalin A; D015081:2-Naphthylamine; D016650:Fluorescein-5-isothiocyanate; C065580:laurdan