A non-sulfated chondroitin stabilizes membrane tubulation in cnidarian organelles

Abstract

Membrane tubulation is generally associated with rearrangements of the cytoskeleton and other cytoplasmic factors. Little is known about the contribution of extracellular matrix components to this process. Here, we demonstrate an essential role of proteoglycans in the tubulation of the cnidarian nematocyst vesicle. The morphogenesis of this extrusive organelle takes place inside a giant post-Golgi vesicle, which topologically represents extracellular space. This process includes the formation of a complex collagenous capsule structure that elongates into a long tubule, which invaginates after its completion. We show that a non-sulfated chondroitin appears as a scaffold in early morphogenesis of all nematocyst types in Hydra and Nematostella. It accompanies the tubulation of the vesicle membrane forming a provisional tubule structure, which after invagination matures by collagen incorporation. Inhibition of chondroitin synthesis by beta-xylosides arrests nematocyst morphogenesis at different stages of tubule outgrowth resulting in retention of tubule material and a depletion of mature capsules in the tentacles of hydra. Our data suggest a conserved role of proteoglycans in the stabilization of a membrane protrusion as an essential step in organelle morphogenesis.

FIGURE 1.

Tubulation during nematocyst morphogenesis illustrated by thin sections. A, schematic representation of nematocyst morphogenesis. B, differential interference contrast image of nematocyst nest at early stage. The nematocyst vesicle is visible as a rounded vesicle (box) in the cytoplasm of the nematocyte. Scale bar, 2 mm. C, onset of vesicle membrane tubulation (arrow). A premature capsule wall (cw) is lining the inner surface of the nematocyst vesicle. The area of tubule outgrowth adjacent to the TGN is free of capsule wall material. D, growing tubule is stabilized by microtubules at the cytoplasmic surface (yellow arrows). The tubule tip is closed by the vesicle membrane only (red arrow) whereas the tubule wall shows an additional inner layer (see also inset in E). E, tubule invagination. Cross sections of the invaginated and coiled tubule inside the vesicle matrix are marked by arrows. The inset shows the vesicle membrane at the tubule tip and the double-layered tubule with an inner tubule wall (tw) along the shaft. TGN, trans-Golgi network, mt, microtubules. Scale bars, 1 μm.

FIGURE 2.

Immunocytochemical detection of chondroitin in Hydra and Nematostella whole mounts. To present a more comprehensible view, some images show co-immunostainings with anti-NCol-1, which stains the capsule body (red signals). A–N, whole mounts of H. magnipapillata (A–L) or primary polyps of N. vectensis (M–N) were incubated with anti-chondroitin antibody and Alexa 488 as secondary antibody (A, D, F, H, J, L), followed by incubation with anti-NCol-1 antibody and Alexa 568 as second secondary antibody (B, C, E, G, I, K, M, N). A, whole mount of Hydra. Scale bar, 50 μm. B-B′, nests of differentiating nematocytes in the gastric region of Hydra. Scale bar, 20 μm. C–L, close-up view of differentiating Hydra nematocytes at different morphological stages stained with anti-chondroitin antibody. C, E, G, I, and K show co-immunostainings of developing nests with anti-NCol-1. D, F, H, J, and L show enlarged chondroitin immunostainings at similar developmental stages. Each scale bar represents 10 μm. M and N, co-immunostained (anti-NCol-1 and anti-chondroitin) nematocytes in Nematostella in the tentacle bulb of a primary polyp with (M) and without (N) differential interference contrast DIC image overlay. Scale bars, 5 μm.

FIGURE 3.

GAG analysis of isolated nematocysts and determination of antibody specificity. A, gel-filtration chromatography of the Hydra GAG fraction on a Superdex 200 column. 1.2-ml fractions were collected, digested individually with chondroitinases, derivatized with 2AB, and then analyzed by anion-exchange HPLC on an amine-bound silica column. The amount of the 2AB-derivative of unsaturated disaccharides from chondroitin of Hydra nematocysts was calculated based on the fluorescence intensity of the peaks. V₀ and V_t were determined using hyaluronan from human umbilical cord and NaCl, respectively. The inset shows the calibration curve giving a linear relationship between log M_r and elution volume, which was generated using size-defined commercial dextran preparations (average M_r: 60,000, 37,500, and 18,100). B, analysis of the reducing terminal structure of the Hydra chondroitin chains. The Hydra GAG fraction was subjected to derivatization with 2AB before (upper panel) or after (lower panel) treatment with mild alkali (LiOH). After removal of the excess 2AB reagent, the 2AB-derivative was digested with chondroitinases. Each digest was analyzed by HPLC on an amine-bound silica column with a linear NaH₂PO₄ gradient as indicated by dashed lines. The positions of authentic 2AB-disaccharides are indicated by numbered arrows: 1, ΔHexUA-GalNAc; 2, ΔHexUA-GalNAc(6-sulfate); 3, ΔHexUA-GalNAc(4-sulfate); 4, ΔHexUA(2-sulfate)-GalNAc(6-sulfate); 5, ΔHexUA(2-sulfate)-GalNAc(4-sulfate); 6, ΔHexUA-GalNAc(4,6-disulfate), where ΔHexUA represents 4,5-unsaturated hexuronic acid. C, reactivity of the antibody 473A12 to immobilized biotinylated GAG variants. Experiments were performed in duplicate. Values are expressed as means ± ranges. Chn, chondroitin; Hep, heparin. The antibody specifically recognized chondroitin.

FIGURE 4.

Immunostaining with anti-NCol-1 propeptide and anti-chondroitin antibodies. A, sequence of NCol-1 with the signal peptide in blue and the propeptide in red. The propeptide is cleaved in trans-Golgi vesicles after the conserved KR dipeptide. B–D, whole mounts of H. magnipapillata were treated with anti-NCol-1 propeptide antibody (red), followed by anti-chondroitin antibody (D, green), or anti-NCol-1 antibody (C, green). B, nests of differentiating nematocytes in the gastric region of hydra. Scale bar, 10 μm. C, arriving vesicles from the Golgi apparatus are stained with anti-NCol-1 propeptide antibody (red) at the onset of tubule development, while the capsule body is marked by anti-NCol-1 antibody (green). Scale bar, 10 μm. D, vesicles stained with anti-chondroitin antibody (green) and anti-NCol-1 propeptide (red) antibodies are clearly separated in the trans-Golgi network surrounding the growing tubule. Scale bar, 10 μm.

FIGURE 5.

Co-immunostaining with anti-chondroitin and anti-NCol-15 antibodies. A–G, whole mounts of H. magnipapillata stained with anti-chondroitin antibody (green) and anti-NCol-15 antibody (red). Scale bars, 10 μm. A, nests of differentiating nematocytes in the gastric region. B–D, differentiating stenoteles at different morphological stages. E–G, differentiating isorhizas at different morphological stages. D and G show stages after tubule invagination.

FIGURE 6.

Co-immunostaining with anti-chondroitin and anti-nematogalectin antibodies. A, whole mount of H. magnipapillata stained with anti-nematogalectin antibody (green) and anti-chondroitin antibody (red). B–J, close views of differentiating nests of desmonemes (B–D), isorhizas (E–G), and stenoteles (H–J) at different morphological stages. Scale bars represent 10 μm.

FIGURE 7.

Effect of xyloside treatment on nematocyst morphogenesis. Animals were cultured in Hydra medium containing 2 mm p-nitrophenyl-β-d-xylopyranoside and fixed after different timepoints, followed by immunostaining with anti-NCol-15 antibody. Scale bars represent 20 μm. A–D, tentacles after 3 (A), 6 (B), 13 (C), and 17 (D) days of incubation with xyloside. Left: DIC images; right: corresponding fluorescence microscopy.

FIGURE 8.

Effect of xyloside treatment on differentiating nematocytes in the body column. Animals were cultured in Hydra medium containing 2 mm p-nitrophenyl-β-d-xylopyranoside and fixed after different time points, followed by immunostaining with anti-NCol-15 antibody (red) and anti-chondroitin antibody (green). A, normal differentiating nests stained with anti-NCol-15 antibody after 3 days of xyloside treatment. Bar, 20 μm. B, disordered differentiating nests stained with anti-NCol-15 antibody after 17 days of xyloside treatment. Bar, 20 μm. C, normal differentiating late stage nest stained with anti-chondroitin and anti-NCol-15 antibodies without xyloside treatment. Bar, 10 μm. D, disordered late-stage nematocysts after 17 days of xyloside treatment. Inset shows magnification of aggregated material positive for both antigens. E, late-stage stenotele nest with invaginated tubules stained with anti-chondroitin and anti-NCol-15 antibodies. F and G, mature stenoteles after 17 days of xyloside treatment showing reduced and disordered tubules. Bars, 10 μm.

FIGURE 9.

Schematic drawing of chondroitin, nematogalectin, NCol-15, NCol-1, and spinalin antigen distribution during nematocyst morphogenesis (stenotele). A, early tubule development at the apical side of the nematocyst. The capsule grows inside a giant post-Golgi vesicle by addition of NCol-1-filled vesicles from the TGN. The capsule wall shows NCol-1 immunostaining (yellow) at this stage. B, onset of membrane tubulation is accompanied by the secretion of chondroitin and nematogalectin into the nematocyst vesicle. C, during tubule growth the outer layer is formed by chondroitin (blue) and the tubule inner layer by nematogalectin (red). Dense globular NCol-15 particles (brown) migrate through the tubule into the vesicle matrix. D, during tubule invagination the inner tubule layer turns inside out and now nematogalectin (red) is present at the surface and obscures the subjacent chondroitin (blue). NCol-15 (brown) appears in this stage in few large protein agglomerates together with the spine protein spinalin (green). E, shortly before final maturation the tubule is completely coiled up inside the capsule matrix, and NCol-15 is then incorporated into the tubule structure.