The mechanism of amyloid spherulite formation by bovine insulin

Abstract

The formation of amyloid-containing spherulite-like structures has been observed in some instances of amyloid diseases, as well as in amyloid fibril-containing solutions in vitro. In this article we describe the structure and kinetics of bovine insulin amyloid fibril spherulites formed in the presence and absence of different salts and at different salt concentrations. The general spherulite structure consists of radially oriented amyloid fibrils, as shown by optical microscopy and environmental scanning electron microscopy. In the center of each spherulite, a "core" of less regularly oriented material is observed, whose size decreases when the spherulites are formed in the presence of increasing concentrations of NaCl. Similarly, amyloid fibrils form faster in the presence of NaCl than in its absence. A smaller enhancement of the rate of formation with salt concentration is observed for spherulites. These data suggest that both amyloid fibril formation and random aggregation occur concurrently under the conditions tested. Changes in their relative rates result in the different-sized cores observed in the spherulites. This mechanism can be likened to that leading to the formation of spherulites of polyethylene, in agreement with observations that polypeptide chains under partially denaturing conditions can exhibit behavior not dissimilar to that of synthetic polymers.

FIGURE 1

Insulin amyloid spherulites formed in the presence of various salts. Optical microscopy images of solutions of 1 mM insulin, incubated at pH 2 and 65°C for 24 h. (A) No added salt. (B–F) Solutions of 0.1 M ionic strength using (B) NaCl, (C) NaI, (D) Na₃C₆H₅O₇ (sodium citrate), (E) MgCl₂, and (F) LiCl. Scale bar in all pictures, 100 μm. (G) Histogram of spherulite diameters, n = 575 in the absence of NaCl (black bars), n = 666 in the presence of 100 mM (shaded bars).

FIGURE 2

Core size at different NaCl concentrations and temperature. Core size as a percentage of spherulite diameter shown for spherulites formed by incubation of 1-mM insulin solutions at pH 2, with the indicated NaCl concentration and 65°C (black squares) and 55°C (shaded circles) for 24 h. Error bars are ± SD, based on a minimum of 30 measurements on several samples.

FIGURE 3

Confocal microscope analysis of insulin spherulites. Images of aliquots of solutions containing spherulites, formed by incubating 1 mM insulin at pH 2.0 and 65°C in the absence (A) and presence (B) of 100 mM NaCl. (A, left) Fluorescence confocal image, using ThT. The fluorescence is preferentially associated with those areas of the solution corresponding to spherulites, indicating the presence of amyloid fibrils in the spherulites. (Right) Same image as on left but in transmission mode using crossed polarizing filters. Maltese crosses indicating spherulites can clearly be seen. In B, the same images are shown of spherulites formed in the presence of 100 mM NaCl. Scale bar, 20 μm.

FIGURE 4

ESEM images of insulin spherulites. (A) Insulin spherulite formed by incubating 1 mM insulin at pH 2 and 65°C for 24 h. After rapid dehydration in the ESEM chamber, the spherulite shown has cracked and opened up, showing radial lines from the periphery to the center. The latter is not apparently regularly organized. (B) Insulin spherulite formed by incubation of 1 mM insulin at pH 2 and 65°C in the presence of 0.1 M NaCl for 24 h. Again the rapid dehydration has caused the spherulite to open and reveal radial lines. An apparently nonregular core can be seen at the center of the spherulite. The core is substantially smaller than that observed in A. Scale bar in both images, 30 μm.

FIGURE 5

Kinetics of fibril and spherulite formation in the absence of NaCl. Samples of 1 mM insulin were incubated at pH 2 and 65°C. Thick lines show the average kinetics, squares the spread of the data for fibril formation (shaded) and spherulite formation (black). An additional fibril kinetics time point taken at 24 h is also shown. The presence of spherulites was confirmed by optical microscopy after each experiment.

FIGURE 6

Kinetics of fibril and spherulite formation in the presence of 0.1 M NaCl. Samples of 1 mM insulin and 0.1 M NaCl were incubated at pH 2 and 65°C. Lines show the average kinetics, squares the spread of the data for fibril formation (shaded) and spherulite formation (black). An additional fibril kinetics time point taken at 24 h is also shown. Samples were checked by optical microscopy to confirm the presence of spherulites at the end of each experiment.