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Poly--glutamate synthesis during formation of nematocyst capsules in Hydra

Zoologisches Institut, Ludwig-Maximilians-Universität München, Luisenstr. 14, 80333 München, Germany

View larger version (74K): [in a new window]   Fig. 1. Acridine orange (red)- and DAPI (yellow)-stained nematocysts in tentacles of Hydra. (A) Overview of distal tentacle stained with AO. (B) High magnification of AO stained tentacle. The small green fluorescent structures are nuclei of the nematocytes. (C) High magnification of DAPI-stained tentacle. (D) High magnification of ethanol fixed AO stained tentacle showing exploded capsules. The matrix of exploded stenoteles and isorhizas is empty and unstained; the matrix and everted tubule of exploded desmonemes is filled with pG and stained by AO. (A-C) fluorescence micrograph; (D) phase contrast and fluorescence micrograph. Bar, 20 µm (A); 10 µm (B-D).

View larger version (22K): [in a new window]   Fig. 2. Separation of poly--glutamate (pG) from discharged nematocysts by native PAGE on 16% gel. Gel was fixed and stained with alcian blue (lane 1) and acridine orange (lane 2).

View larger version (79K): [in a new window]   Fig. 3. Influence of Na+ and Ca2+ ions on AO staining of nematocysts. Animals were fixed, stained and washed in the indicated ion concentrations. Bar, 10 µm.

View larger version (53K): [in a new window]   Fig. 4. Biosynthesis of pG in developing nematocysts stained with AO. (A) Nest of immature yellow/green stained stenoteles, a single red stained migrating desmoneme is also present; (B) nest of nearly mature red stained stenoteles. Bars, 10 µm.

View larger version (17K): [in a new window]   Fig. 5. Size distribution of AO-stained stenotele nematocysts in the body column and tentacles. (A) Nests of nematocytes with yellow fluorescent capsules in the body column. (B) Migrating nematocytes with red fluorescent capsules in the body column. (C) Stenotele nematocysts with red fluorescent capsules mounted in the tentacles. (D) Exploded stenotele nematocysts (empty) in tentacles.

View larger version (91K): [in a new window]   Fig. 6. Confocal images of DAPI-stained nematocysts (false colors). (A) Desmoneme, (B) isorhiza, (C,D) stenotele. The coloured images are single optical sections. The approximate positions of the sections shown in D are indicated by numbers next to the capsule in C. In A-C the black-and-white images are projections made from the full stack of optical sections. The colour scale has been inverted to permit better visualisation of the tubule within the capsule. (E,F) Fluorescence images of DAPI-stained stenoteles that have been extensively washed (see text for details). (E) View from the side of the capsule; (F) view down the long axis of the capsule. The three regions of DAPI-stained pG are located within the tubule lumen at the base of the stylets. Bars, 2 µm (A-B); 5 µm (C-F).

View larger version (41K): [in a new window]   Fig. 7. AO staining of nematocysts after fixation in the presence and absence of chelators of divalent ions. Animals were fixed in 4% formaldehyde in the presence of PBS (A), Tris buffer (B), or Tris buffer followed by incubation in PBS (C). Capsules in A stain bright red with AO. Capsules in B stain weakly with AO; the insert shows an exploded capsule that is empty of pG for comparison. Capsules in C also stain weakly with AO indicating that post-treatment cannot improve AO staining. Identical results were obtained when 10 mM EDTA (in Tris buffer) was used in place of PBS. Bar, 10 µm.

View larger version (33K): [in a new window]   Fig. 8. The stenotele explosion process (modified from Tardent and Holstein, 1982). PG in the matrix is shown in pale red, pG in the lumen of the tubule in dark red. The tubule containing the stylets and spines is ejected in the first phase of the explosion process. In the second phase of the explosion, the pG in the lumen swells, spreading the stylets and permitting the continued ejection of the tubule.