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First published online May 24, 2004
doi: 10.1242/10.1242/jcs.01116

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The sequences appended to the amyloid core region of the HET-s prion protein determine higher-order aggregate organization in vivo

¹ Laboratoire de Génétique Moléculaire des Champignons, Institut de Biochimie et de Génétique Cellulaires, UMR 5095 CNRS/Université de Bordeaux 2, 1 rue Camille St Saëns, 33077 Bordeaux, France
² Institut Européen de Chimie et Biologie CNRS FRE 2247 16, Avenue Pey Berland 33607 Pessac Cedex, France

View larger version (47K): [in a new window]   Fig. 1. Activity of HET-s deletion constructs. (A) On the left, various deletion constructs of HET-s are depicted. Numberings correspond to amino acid positions. The prion-forming domain is shown in gray. For each construct, the abilities to propagate [Het-s], to produce a barrage reaction toward het-S (incompatibility function) and to aggregate into amyloid fibrils in vitro are listed. For the 218-289 construct, the results are for the 218-289 peptide fused to GFP. (B) Barrage test with various HET-s deletion constructs: a het-S tester strain (central line) was confronted on solid medium with strains bearing various HET-s deletion constructs. Confrontation between incompatible strains leads to the formation of a dark, dense contact line termed the barrage (black, arrowed). Compatible strains display normal clear contact lines (white arrowed). The strains that do not produce a barrage reaction to het-S are grouped on the top line; the strains producing a barrage reaction to het-S are on the bottom line. Notice the attenuated barrage reaction obtained with the het-s(218-289)GFP construct. (C) Strains were cultivated for 5 days on solid medium. Notice that expression of HET-s(157-289) in a het-S background strongly affects growth, whereas strains expressing HET-s(157-289) in a het-s or het-s background display normal growth. (D) Microscopic examination of a transformant expressing HET-s(157-289) in a het-S background and of an untransformed het-S control strain. Notice the extensive vacuolization of the hyphae in strains expressing HET-s(157-289). Scale bar, 4 µm.

View larger version (37K): [in a new window]   Fig. 2. Aggregation and formation of a proteinase-K-resistant core in recombinant HET-s(157-289). (A) Recombinant HET-s(157-289) aggregates in vitro and catalyses the aggregation of full-length HET-s. (top) Time course of aggregation at 0.2 mg ml–1 at 20°C. (bottom) Aggregation of recombinant full-length HET-s protein at 3 mg ml–1 inoculated with an aliquot (0.1 mg ml–1) of aggregated HET-s(157-289) (triangles) or buffer alone (squares). (B) Circular dichroism spectra of 20 µM recombinant HET-s(157-289) at pH 8 in the soluble (black triangles) and aggregated (white squares) states. (C) Time course of limited proteinase-K digestion of soluble (top) and aggregated (bottom) recombinant HET-s(157-289) protein analysed by SDS-PAGE followed by Coomassie-Blue staining. Digestion times are given in minutes. The last lane (218-289) corresponds to the recombinant HET-s(218-289) peptide. Size of molecular weight markers (M lane) is given in kDa. (D) Mass spectrum of the proteinase-K-resistant material. The y axis gives intensity in arbitrary units, the x axis gives mass-to-charge (m/z) ratios in Daltons per unit charge. The peak at 8650.81 Da (*) corresponds to an internal control (recombinant 218-289 peptide with a six-histidine extension). The major peak at 8520.07 was identified as the 218-289 fragment (with six histidines). The 130.74 Da difference in the measured average mass between the control recombinant 218-289 peptide (*) and the major peak is caused by the recombinant peptide displaying an additional N-terminal methionine residue (theoretical mass difference 131.20 Da).

View larger version (72K): [in a new window]   Fig. 3. Amyloid fibrils of recombinant HET-s(157-289). (A-D) Electron microscopy of HET-s(157-289) aggregates. (B,C) Tightly compacted fibrils and more loosely organized bundles fringing out into individual fibrils. (D) The well-ordered lateral association of individual fibrils. Scale bars, 10 µm (A), 500 nm (B), 200 nm (C), 100 nm (D). (E) light microscopy of HET-s(157-289) fibrils. Scale bar, 10 µm. (F) Fluorescence microscopy of HET-s(157-289) stained with Congo red. Scale bar, 10 µm.

View larger version (90K): [in a new window]   Fig. 4. HET-s(157-289)/GFP forms elongated fibrillar aggregates in vivo upon transition to the prion state. (A) Strains expressing HET-s(157-289)/GFP were analysed by fluorescence microscopy. In the [Het-s*] state, the fluorescence signal is diffuse and cytoplasmic. (B) Upon transition to the [Het-s] prion state, HET-s(157-289) forms fibrillar aggregates. (C) In apical regions, in which septa are absent, fibrillar aggregates grow very large. (D,E) Aggregates are not rigid and are able to bend when blocked by septa. (F) Aggregates are able to pass through an anastomosis bridge. (G) Aggregate extending into an hyphal branch. (H) HET-s(157-289) are incorporated into microconidia. Scale bar, 4 µm.

View larger version (43K): [in a new window]   Fig. 5. Intracytoplasmic movement of HET-s(157-289)/GFP aggregates. (Triangle) A HET-s(157-289)/GFP aggregate as it moves within the filament through the analysed field. Notice the absence of septa in the analysed region. The sequence is taken from supplementary movie 1 (http://jcs.biologists.org/supplemental/).

View larger version (35K): [in a new window]   Fig. 6. Association of HET-s(157-289)/GFP aggregates. Notice the coalescence of the aggregates into a single fibrillar aggregate once at 4 minutes and again at 11 minutes. The sequence is taken from supplementary movie 2 (http://jcs.biologists.org/supplemental/) (see also supplementary movie 3).

View larger version (56K): [in a new window]   Fig. 7. Deterioration of HET-s(157-289)/GFP aggregates. (A) HET-s(157-289) aggregates retract into curly structures upon aging. The sequence is taken from supplementary movie 4 (http://jcs.biologists.org/supplemental/) (see also supplementary movies 5, 6). (B) Fibril deterioration as an aggregate passes through a septal pore. (C) Upon hyphal lysis, aggregates dissociated into many smaller fibrils. Scale bar, 4 µm.

View larger version (200K): [in a new window]   Fig. 8. HET-s(157-289) aggregates formed in vivo observed by electron microscopy. HET-s(157-289) aggregates are marked by arrowheads. Scale bar, 500 nm.

View larger version (84K): [in a new window]   Fig. 9. HET-s/GFP and HET-s(218-289)/GFP form elongated fibrillar aggregates when expressed with HET-s(157-289). In the prion state, HET-s/GFP and HET-s(218-289)/GFP form dot-like aggregates when expressed alone (left) but fibrillar aggregates when expressed with HET-s(157-289) (right). Notice that dot-like aggregates co-exist with fibrillar aggregates in strains expressing either HET-s/GFP and HET-s(157-289) or HET-s(218-289)/GFP and HET-s(157-289). Scale bar, 4 µm.

View larger version (81K): [in a new window]   Fig. 10. HET-S(157-289)/GFP aggregates upon transition to the [Het-s] state. Upon transition to the [Het-s] prion state, HET-S(157-289)/GFP forms fibrillar aggregates. Notice that fibrillar aggregates are often not as regular as HET-s(157-289)/GFP aggregates (left). In apical regions (right), donut-shaped circular structures are detected. Scale bar, 2 µm.

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