Optical microscopy of growing insulin amyloid spherulites on surfaces in vitro.
Amyloid fibrils are often found arranged into large ordered spheroid structures, known as spherulites, occurring in vivo and in vitro. The spherulites are predominantly composed of radially ordered amyloid fibrils, which self-assemble from protein in solution. We have observed and measured amyloid spherulites forming from heat-treated solutions of bovine insulin at low pH. The spherulites form in large numbers as semispherical dome-shaped objects on the cell surfaces, showing that surface defects or impurities, or the substrates themselves, can provide good nucleation sites for their formation. Using optical microscopy, we have measured the growth of individual spherulites as a function of time and in various conditions. There is a lag time before nucleation of the spherulites. Once they have nucleated, they grow, each with a radius increasing linearly, or faster than linearly, with time. Remarkably, this growth period has a sudden end, at which all spherulites in the system suddenly stop growing. A model of spherulite formation based on the polymerization of oriented fibrils around a nucleus, from a precursor in solution, quantitatively accounts for the observed growth kinetics. Seeding of native insulin solutions with preformed spherulites led to the preformed spherulites growing without a lag time. This seeding behavior is evidence that the fibrils in the spherulites assemble from small protein species rather than fibrils. The density of the spherulites was also measured and found to be constant with respect to radius, indicating that the space fills as the spherulite grows.
Rogers
Salman S
SS
Cambridge University, Cambridge, United Kingdom.
Krebs
Mark R H
MR
Bromley
Elizabeth H C
EH
van der Linden
Erik
E
Donald
Athene M
AM
D000682:Amyloid / Q000737:chemistry*; D000818:Animals; D001681:Biological Assay; D001703:Biophysics / Q000379:methods*; D002417:Cattle; D003460:Crystallization; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007328:Insulin / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008853:Microscopy / Q000379:methods*; D018613:Microscopy, Confocal; D046912:Multiprotein Complexes / Q000737:chemistry; D011489:Protein Denaturation; D013499:Surface Properties; D013997:Time Factors
D000682:Amyloid; D007328:Insulin; D046912:Multiprotein Complexes