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.
Highlights
A key step in the dynamic remodeling of plasma membranes during tubulation processes are localized invaginations
We show that a non-sulfated chondroitin appears as a scaffold in early morphogenesis of all nematocyst types in Hydra and Nematostella
We report that membrane tubulation has an important function in the morphogenesis of a sophisticated secretory organelle, the nematocyst
Summary
Hydra animals were cultured in Hydra medium [18] at 18 °C and fed three to five times a week with Artemia nauplii. The samples were digested with a mixture of chondroitinases AC-I, AC-II, and ABC, and derivatized with a fluorophore 2-aminobenzamide (2AB), followed by the removal of the excess derivatization reagents by chloroform extraction and anion-exchange HPLC analysis on an amine-bound silica column [19]. The reactivity of the antibody was evaluated by incubation with the secondary antibody, alkaline phosphatase-conjugated goat-anti mouse IgA (3000-fold dilution with 0.1% BSA/TBS), followed by plate development with disodium p-nitrophenyl phosphate. Animals were washed several times with PBS and incubated for 1–2 h with adequate secondary antibody coupled to ALEXA fluorochrome (Molecular Probes) at 1:400 in PBS/0.1% BSA. Image processing was performed by using ImageJ and Adobe Photoshop CS3
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