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Super-resolution fluorescence of huntingtin reveals growth of globular species into short fibers and coexistence of distinct aggregates.

Duim WC, Jiang Y, Shen K, Frydman J, Moerner WE - ACS Chem. Biol. (2014)

Bottom Line: The fibers further aggregated with one another into aggregate assemblies of increasing size.Seeds created by sonication, which were comparable in shape and size to the globular species in the pathway, were observed to grow through multidirectional elongation into fibers, suggesting a mechanism for growth of globular species into fibers.The single-molecule sensitivity of our approach made it possible to characterize the aggregation pathway across a large range of size scales, from monomers to fiber assemblies, and revealed the coexistence of different aggregate species (globular species, fibers, fiber assemblies) even at late time points.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, ‡Department of Applied Physics, and §Department of Biology, Stanford University , Stanford, California 94305, United States.

ABSTRACT
Polyglutamine-expanded huntingtin, the protein encoded by HTT mutations associated with Huntington's disease, forms aggregate species in vitro and in vivo. Elucidation of the mechanism of growth of fibrillar aggregates from soluble monomeric protein is critical to understanding the progression of Huntington's disease and to designing therapeutics for the disease, as well as for aggregates implicated in Alzheimer's and Parkinson's diseases. We used the technique of multicolor single-molecule, super-resolution fluorescence imaging to characterize the growth of huntingtin exon 1 aggregates. The huntingtin exon 1 aggregation followed a pathway from exclusively spherical or globular species of ∼80 nm to fibers ∼1 μm in length that increased in width, but not length, over time with the addition of more huntingtin monomers. The fibers further aggregated with one another into aggregate assemblies of increasing size. Seeds created by sonication, which were comparable in shape and size to the globular species in the pathway, were observed to grow through multidirectional elongation into fibers, suggesting a mechanism for growth of globular species into fibers. The single-molecule sensitivity of our approach made it possible to characterize the aggregation pathway across a large range of size scales, from monomers to fiber assemblies, and revealed the coexistence of different aggregate species (globular species, fibers, fiber assemblies) even at late time points.

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Htt-ex1 seed growth into fibers. AFM of(a) Htt-ex1:Htt-ex1-AX647= 10:1 seeds created by sonicating mature aggregates and (b) the fibersresulting from addition of Htt-ex1:Htt-ex1-Cy3B = 10:1 to the seeds.The seeds indicated by arrows are 35 nm (white), 48 nm (green), 73nm (black), and 132 nm (blue) in length. (c) Schematic of two-colorgrowth experiment and possible outcomes. (d) SR overlay of Htt-ex1:Htt-ex1-AX647= 10:1 seeds (red) and Htt-ex1:Htt-ex1-Cy3B = 10:1 growth (green)after 22 h of aggregation. The localizations are plotted with Gaussiansof constant amplitude and σ = 50 nm (reflecting the smallernumber of photons emitted per blinking event, and thus the lower localizationprecision, of Cy3B). The TetraSpeck fiduciary beads used to alignthe channels are marked with gray boxes. (e–g) Enlarged imagesof selected fibers. Scale bars are (a) 500 nm, (b) 1 μm, (d)2 μm, and (e–g) 500 nm.
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fig6: Htt-ex1 seed growth into fibers. AFM of(a) Htt-ex1:Htt-ex1-AX647= 10:1 seeds created by sonicating mature aggregates and (b) the fibersresulting from addition of Htt-ex1:Htt-ex1-Cy3B = 10:1 to the seeds.The seeds indicated by arrows are 35 nm (white), 48 nm (green), 73nm (black), and 132 nm (blue) in length. (c) Schematic of two-colorgrowth experiment and possible outcomes. (d) SR overlay of Htt-ex1:Htt-ex1-AX647= 10:1 seeds (red) and Htt-ex1:Htt-ex1-Cy3B = 10:1 growth (green)after 22 h of aggregation. The localizations are plotted with Gaussiansof constant amplitude and σ = 50 nm (reflecting the smallernumber of photons emitted per blinking event, and thus the lower localizationprecision, of Cy3B). The TetraSpeck fiduciary beads used to alignthe channels are marked with gray boxes. (e–g) Enlarged imagesof selected fibers. Scale bars are (a) 500 nm, (b) 1 μm, (d)2 μm, and (e–g) 500 nm.

Mentions: First, Htt-ex1 seeds werecreated by probe sonicating18,25,26 Htt-ex1:Htt-ex1-AX647 = 10:1, 24-h-old aggregation reactions (Figure 6a). This procedure was reproducible and ensuredthat a sufficient number of fluorophores was incorporated into theseeds. The mean length (maximum distance between two points in theseed), area, and volume of the seeds measured by AFM were 50 nm ±30 nm, 1.5 × 10–3 μm2, and1.2 × 10–5 μm3, respectively(n = 1955). From the mean volume of the seeds andan estimate of the packing density of globular proteins (1.35 g cm–3),57 the approximate averagenumber of Htt-ex1 proteins in a seed was 650. The size of the seedsmeasured by SR was the same as the size of the globular species inthe time-course experiments above, with a mean fwhm of 78 nm ±6nm when reconstructed with σ = 30 nm, and 122 nm ± 5 nmwith σ = 50 nm (as represented in Figure 6d–g, Supporting Information Table S2). The SR size also agreed well with measurements by AFM: the 2Dhistogram of localizations (16 × 16 nm pixels) yielded a meanlength (maximum distance between two points in the seed, Feret’sdiameter) of 64 nm ±27 nm.


Super-resolution fluorescence of huntingtin reveals growth of globular species into short fibers and coexistence of distinct aggregates.

Duim WC, Jiang Y, Shen K, Frydman J, Moerner WE - ACS Chem. Biol. (2014)

Htt-ex1 seed growth into fibers. AFM of(a) Htt-ex1:Htt-ex1-AX647= 10:1 seeds created by sonicating mature aggregates and (b) the fibersresulting from addition of Htt-ex1:Htt-ex1-Cy3B = 10:1 to the seeds.The seeds indicated by arrows are 35 nm (white), 48 nm (green), 73nm (black), and 132 nm (blue) in length. (c) Schematic of two-colorgrowth experiment and possible outcomes. (d) SR overlay of Htt-ex1:Htt-ex1-AX647= 10:1 seeds (red) and Htt-ex1:Htt-ex1-Cy3B = 10:1 growth (green)after 22 h of aggregation. The localizations are plotted with Gaussiansof constant amplitude and σ = 50 nm (reflecting the smallernumber of photons emitted per blinking event, and thus the lower localizationprecision, of Cy3B). The TetraSpeck fiduciary beads used to alignthe channels are marked with gray boxes. (e–g) Enlarged imagesof selected fibers. Scale bars are (a) 500 nm, (b) 1 μm, (d)2 μm, and (e–g) 500 nm.
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fig6: Htt-ex1 seed growth into fibers. AFM of(a) Htt-ex1:Htt-ex1-AX647= 10:1 seeds created by sonicating mature aggregates and (b) the fibersresulting from addition of Htt-ex1:Htt-ex1-Cy3B = 10:1 to the seeds.The seeds indicated by arrows are 35 nm (white), 48 nm (green), 73nm (black), and 132 nm (blue) in length. (c) Schematic of two-colorgrowth experiment and possible outcomes. (d) SR overlay of Htt-ex1:Htt-ex1-AX647= 10:1 seeds (red) and Htt-ex1:Htt-ex1-Cy3B = 10:1 growth (green)after 22 h of aggregation. The localizations are plotted with Gaussiansof constant amplitude and σ = 50 nm (reflecting the smallernumber of photons emitted per blinking event, and thus the lower localizationprecision, of Cy3B). The TetraSpeck fiduciary beads used to alignthe channels are marked with gray boxes. (e–g) Enlarged imagesof selected fibers. Scale bars are (a) 500 nm, (b) 1 μm, (d)2 μm, and (e–g) 500 nm.
Mentions: First, Htt-ex1 seeds werecreated by probe sonicating18,25,26 Htt-ex1:Htt-ex1-AX647 = 10:1, 24-h-old aggregation reactions (Figure 6a). This procedure was reproducible and ensuredthat a sufficient number of fluorophores was incorporated into theseeds. The mean length (maximum distance between two points in theseed), area, and volume of the seeds measured by AFM were 50 nm ±30 nm, 1.5 × 10–3 μm2, and1.2 × 10–5 μm3, respectively(n = 1955). From the mean volume of the seeds andan estimate of the packing density of globular proteins (1.35 g cm–3),57 the approximate averagenumber of Htt-ex1 proteins in a seed was 650. The size of the seedsmeasured by SR was the same as the size of the globular species inthe time-course experiments above, with a mean fwhm of 78 nm ±6nm when reconstructed with σ = 30 nm, and 122 nm ± 5 nmwith σ = 50 nm (as represented in Figure 6d–g, Supporting Information Table S2). The SR size also agreed well with measurements by AFM: the 2Dhistogram of localizations (16 × 16 nm pixels) yielded a meanlength (maximum distance between two points in the seed, Feret’sdiameter) of 64 nm ±27 nm.

Bottom Line: The fibers further aggregated with one another into aggregate assemblies of increasing size.Seeds created by sonication, which were comparable in shape and size to the globular species in the pathway, were observed to grow through multidirectional elongation into fibers, suggesting a mechanism for growth of globular species into fibers.The single-molecule sensitivity of our approach made it possible to characterize the aggregation pathway across a large range of size scales, from monomers to fiber assemblies, and revealed the coexistence of different aggregate species (globular species, fibers, fiber assemblies) even at late time points.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, ‡Department of Applied Physics, and §Department of Biology, Stanford University , Stanford, California 94305, United States.

ABSTRACT
Polyglutamine-expanded huntingtin, the protein encoded by HTT mutations associated with Huntington's disease, forms aggregate species in vitro and in vivo. Elucidation of the mechanism of growth of fibrillar aggregates from soluble monomeric protein is critical to understanding the progression of Huntington's disease and to designing therapeutics for the disease, as well as for aggregates implicated in Alzheimer's and Parkinson's diseases. We used the technique of multicolor single-molecule, super-resolution fluorescence imaging to characterize the growth of huntingtin exon 1 aggregates. The huntingtin exon 1 aggregation followed a pathway from exclusively spherical or globular species of ∼80 nm to fibers ∼1 μm in length that increased in width, but not length, over time with the addition of more huntingtin monomers. The fibers further aggregated with one another into aggregate assemblies of increasing size. Seeds created by sonication, which were comparable in shape and size to the globular species in the pathway, were observed to grow through multidirectional elongation into fibers, suggesting a mechanism for growth of globular species into fibers. The single-molecule sensitivity of our approach made it possible to characterize the aggregation pathway across a large range of size scales, from monomers to fiber assemblies, and revealed the coexistence of different aggregate species (globular species, fibers, fiber assemblies) even at late time points.

Show MeSH
Related in: MedlinePlus