Fluctuation Methods To Study Protein Aggregation in Live Cells: Concanavalin A Oligomers Formation

• Published in: Biophysical journal Vol. 100; no. 3; pp. 774 - 783
• Main Authors: Vetri, V., Ossato, G., Militello, V., Digman, M.A., Leone, M., Gratton, E.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 02.02.2011; Biophysical Society; The Biophysical Society
• Subjects: 2-Naphthylamine - analogs & derivatives; 2-Naphthylamine - metabolism; Agglomeration; Animals; Annexin A5 - metabolism; Biophysics; Biophysics - methods; Brightness; cell biology; Cell Membrane - drug effects; Cell Membrane - metabolism; Cell Shape - drug effects; Cell Survival - drug effects; Cells; Concanavalin A; Concanavalin A - chemistry; Concanavalin A - pharmacology; death; Diffusion - drug effects; diffusivity; Embryo, Mammalian - cytology; Fibroblasts - cytology; Fibroblasts - drug effects; Fibroblasts - metabolism; Fluorescein-5-isothiocyanate - metabolism; image analysis; Laurates - metabolism; Membranes; Mice; Morphology; Neurodegeneration; neurodegenerative diseases; Neurological disorders; Oligomers; Pathogens; Protein Structure, Quaternary; Proteins; Raster; spectroscopy; Spectroscopy, Imaging, and Other Techniques; Spectrum Analysis; Time Factors
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/j.bpj.2010.11.089

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.