The EMBO Journal (2001) 20, 3063–3073, doi:10.1093/emboj/20.12.3063
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| Figures and tables |
| A switch in disulfide linkage during minicollagen assembly in Hydra nematocysts |
| Ulrike Engel, Olivier Pertz, Charlotte Fauser, Jürgen Engel, Charles N. David and Thomas W. Holstein |
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| Figures |
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Figure 1 Minicollagen-1 sequence and domain organization. Signal peptide (gray); propeptide (light blue); polyproline stretches (blue); Gly-X-Y, collagenous domain (red); Cys-rich regions with Cys residues indicated in green.
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 | Figure 2 Minicollagen-1 expression and biochemical properties. (A) Design of minicollagen-1 and minicollagen-1–MBP fusions. (B) Recombinant expression. Conditioned medium of untransfected, minicollagen-1- and minicollagen-1–MBP-transfected cells was resolved by SDS–PAGE (Schägger and von Jagow, 1987) under reducing conditions. Mock, conditioned medium from untransfected cells. Lane 1, minicollagen-1 expression in the absence of ascorbic acid; lanes 2 and 3, minicollagen-1 expression in the presence of 50 and 200  g/ml ascorbic acid; lane 4, minicollagen-1–MBP expression in the absence of ascorbic acid; lanes 5 and 6, minicollagen-1–MBP expression in the presence of 50 and 200 g/ml ascorbic acid. (C) Effects of disulfide bonds on mobility of minicollagen-1. Equal amounts of non-reduced (lane 1) and reduced (lane 2) minicollagen-1 resolved by SDS–PAGE (Lämmli et al., 1970). Proteins were visualized by silver staining.
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Figure 3 Electron micrographs of minicollagen-1 and minicollagen-1–MBP. (A) Scanning transmission electron micrographs of negatively stained minicollagen-1 in 0.1% acetic acid. Arrows indicate fine protrusions of 6–8 nm in length, which are enlarged in one case five times for better visibility (lower right panel). (B) Field of transmission electron micrographs of rotary-shadowed minicollagen-1 in 0.1% acetic acid. (C) Representative gallery of rotary-shadowed minicollagen-1–MBP molecules in 0.1% acetic acid as revealed by transmission electron microscopy. Bars: 50 nm.
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 | Figure 4 Western blot analysis of minicollagens in Hydra tissue and in purified capsules. Proteins were separated by SDS–PAGE (Lämmli et al., 1970) and transferred to nitrocellulose membrane before immunodetection with minicollagen antibody. (A) Recombinant minicollagen-1 under non-reducing (lane 1) and reducing conditions (lane 2). (B) Chemical deglycosylation of minicollagens from mature capsules with anhydrous TFMS. Recombinant minicollagen-1 is shown as a control, before deglycosylation (lane 1) and after deglycosylation (lane 2). Untreated capsule material (lane 3) is compared with deglycosylated (lane 4) material. (C) Distribution of minicollagens in Hydra tissue. A flow chart depicts the sample preparation from animals cut into body column (b.) and head including tentacles (h.). Two body columns (lane 1) and two heads (lane 2) were completely solubilized in reducing sample buffer. For comparison 200 000 mature isolated capsules solubilized in reducing sample buffer are shown in lane 3. Ten Hydra heads and body columns were extracted with a buffer containing 2% CHAPS and 6 M urea but no reducing agent. The extractable protein was separated under non-reducing conditions (lane 4, body columns; lane 5, heads) or under reducing conditions (lane 6, body column; lane 7, heads).
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Figure 5 Immunolocalization of minicollagen in a whole mount labeled with minicollagen antibody (red) and mAb H22 (green). A fully developed bud is shown using confocal microscopy. Bar: 100 m.
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 | Figure 6 Early stages of nematocyst morphogenesis. (A–B) Nest of developing nematocytes in the gastric region viewed using confocal microscopy. (A) Optical section of a nest of nematocytes double labeled with minicollagen antibody (red) and mAb H22 (green). The cyst (arrow) is already clearly distinguishable by mAb H22 staining in the wall. (B) Same optical section showing minicollagen staining only. Note the presence of minicollagen in the ER and the capsule (arrow). (C) Electron micrograph of a single nematocyte with growing capsule at a similar stage (iw, inner wall; N, nucleus; ow, outer wall). Bars: (A and B) 5 m; (C) 1 m.
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Figure 7 Developing nematocytes before and during formation of the external tubule. Nematocytes in the gastric region viewed using confocal (A–D) and electron (E) microscopy. (A) Optical section of a nematocyst (isorhiza) double labeled with mAb H22 (green) and minicollagen antibody (red). H22 is detected in the outer wall, minicollagen is detected in the inner wall and to a lesser extent in the matrix. (B) Projection view of a nematocyst (stenotele) labeled with minicollagen antibody (red). At the apex of the cyst (arrow), minicollagen-filled vesicular structures are observed. (C) Three-dimensional reconstruction of the capsule shown in (B) in simulated fluorescence process mode. (D) Optical section of a nematocyst (desmoneme) with wall and external tubule labeled with minicollagen antibody (red). Vesicles fuse at the tip of the tubule (arrows). (E) Electron micrograph of a nematocyst (desmoneme or isorhiza) forming its external tubule (et, external tubule; iw, inner wall; ow, outer wall). Bars: (A, B and D) 2 m; (E) 1 m.
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 | Figure 8 Nematocysts undergoing wall maturation. Nematocyst in the gastric region viewed using confocal (A–C) and electron microscopy (D). (A) Projection view of a nematocyst nest (stenoteles) triple labeled with minicollagen antibody (red), mAb H22 (green) and DAPI (blue). The H22 antibody stains all capsules in the nest. Only the irregularly shaped capsules that have not yet undergone maturation are stained with the minicollagen antibody. (B) Minicollagen staining in a single optical section through the same nest. (C) Minicollagen staining in a nematocyst nest (stenoteles) in which capsules are undergoing compaction (projection view). Capsules with regular shape and weak minicollagen antibody staining are marked by arrows. (D) Electron micrograph of a nematocyst (stenotele) with invaginated tubule (iw, inner wall; ow, outer wall; s, stylets; t, invaginated tubule). This nematocyst is almost mature, and represents the same stages as the ones marked by arrows in (C). Bars: (A–C) 5 m; (D) 2 m.
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Figure 9 Schematic drawing of minicollagen and H22 antigen distribution during nematocyst morphogenesis. The stages of morphogenesis are outlined for a stenotele but also apply to a desmoneme or an isorhiza. Minicollagen is shown in red, the outer wall antigen H22 in green and membranes are drawn as blue lines. (A) A newly developing nematocyst grows in a post-Golgi vacuole surrounded by an extensive ER network and several Golgis. Minicollagen is synthesized in the ER and gets sorted via the Golgi to the cyst vesicle. (B) After accumulation inside the growing cyst, minicollagen is deposited on the pre-existing outer wall to form the inner wall layer (only one of several Golgis is shown). (C) By fusion of minicollagen-filled vesicles at one end of the capsule a long external tubule starts to grow and elongates, while the cyst itself still grows in diameter. (D) After the cyst has reached its final size, the tubule invaginates into the cyst (not shown), and is no longer stained with the minicollagen-1 antibody (tubule now shown as gray structure). Spines are then formed in the lumen of the inverted tubule (Koch et al., 1998). (E) During the final step of maturation, a gradual loss of minicollagen antibody staining (now shown as red thin line) reflects the wall compaction and hardening process. c, capsule; G, Golgi; N, nucleus; rER, rough endoplasmic reticulum; s, stylets; t, tubule.
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 | Figure 10 Structural model for minicollagen assembly. (A) Intrachain linkage in the soluble precursor. Two alternative arrangements of the intramolecular disulfide bonds within the N-terminal Cys-rich domain of minicollagen-1 are shown. (B) Model of assembly. Minicollagens are synthesized as soluble precursors that have an intrachain disulfide linkage. During the final maturation step, disulfide isomerization from intra- to intermolecular disulfide linkage leads to minicollagen polymerization. Polymeric minicollagen fibers stretch elastically to store spring-like forces that are released during explosive exocytosis (see Discussion). For simplicity, the conformational change of only one disulfide bridge per Cys-rich assembly region is shown.
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