A Non-sulfated Chondroitin Stabilizes Membrane Tubulation in Cnidarian Organelles

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 β-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.     

Length Is Associated with Pain: Jellyfish with Painful Sting Have Longer Nematocyst Tubules than Harmless Jellyfish

A large number of humans are stung by jellyfish all over the world. The stings cause acute pain followed by persistent pain and local inflammation. Harmful jellyfish species typically cause strong pain, whereas harmless jellyfish cause subtle or no pain. Jellyfish sting humans by injecting a tubule, contained in the nematocyst, the stinging organ of jellyfish. The tubule penetrates into the skin leading to venom injection. The detailed morphology of the nematocyst tubule and molecular structure of the venom in the nematocyst has been reported; however, the mechanism responsible for the difference in pain that is caused by harmful and harmless jellyfish sting has not yet been explored or explained. Therefore, we hypothesized that differences in the length of the nematocyst tubule leads to different degrees of epithelial damage. The initial acute pain might be generated by penetration of the tubule, which stimulates pain receptor neurons, whilst persistent pain might be caused by injection of venom into the epithelium. To test this hypothesis we compared the lengths of discharged nematocyst tubules from harmful and harmless jellyfish species and evaluated their ability to penetrate human skin. The results showed that the harmful jellyfish species, Chrysaora pacifica, Carybdea brevipedalia, and Chironex yamaguchii, causing moderate to severe pain, have nematocyst tubules longer than 200 μm, compared with a jellyfish species that cause little or no pain, Aurelia aurita. The majority of the tubules of harmful jellyfishes, C. yamaguchii and C. brevipedalia, were sufficiently long to penetrate the human epidermis and physically stimulate the free nerve endings of Aδ pain receptor fibers around plexuses to cause acute pain and inject the venom into the human skin epithelium to cause persistent pain and inflammation.     

Organelle survival in a foreign organism: Hydra nematocysts in the flatworm Microstomum lineare

Nematocysts are characteristic organelles of the phylum cnidaria. They are designated kleptocnidae when sequestered in animals that feed on cnidaria. Kleptocnidae are known for more than a century. Nevertheless it is still enigmatic how selected nematocyst types survive in the predator and how they reach their final destination in the foreign body. In the free-living Platyhelminth Microstomum lineare the fate of nematocysts of the prey Hydra oligactis was analyzed at the ultrastructural level and by fluorescence microscopy using hydra polyps that had been stained in vivo with the fluorescent dyes TROMI and TRITC. M. lineare digested hydra tissue in its intestine within 30?min and all nematocyst types were phagocytosed without adherent cytoplasm by intestinal cnidophagocytes. Desmoneme and isorhiza nematocysts were digested whereas cnidophagocytes containing the venom-loaded stenotele nematocysts started to migrate out of the intestinal epithelia through the parenchyma to the epidermis thereby traversing the subintestinal and subepidermal muscle layer. Within one to two days, M. lineare began to form a muscle layer basolateral around epidermal cnidophagocytes. Epidermal stenoteles survived in M. lineare for at least four weeks. The ability of epidermal stenotele nematocysts to discharge suggest that this hydra organelle preserved its physiological properties in the new host.     

Minicollagen-15, a novel minicollagen isolated from Hydra, forms tubule structures in nematocysts

Minicollagens constitute a family of unusually short collagen molecules isolated from cnidarians. They are restricted to the nematocyst, a cylindrical explosive organelle serving in defense and capture of prey. The nematocyst capsule contains a long tubule inside of its matrix, which is expelled and everted during an ultrafast discharge process. Here, we report the cloning and characterization of a novel minicollagen in Hydra, designated minicollagen-15 (NCol-15). NCol-15, like NCol-3 and NCol-4, shows deviations from the canonical cysteine pattern in its terminal cysteine-rich domains (CRDs). Minicollagens share common domain architectures with a central collagen sequence flanked by polyproline stretches and short N- and C-terminal CRDs. The CRDs are involved in the formation of a highly resistant cysteine network, which constitutes the basic structure of the nematocyst capsule. Unlike NCol-1, which is part of the capsule wall, NCol-15 is localized to tubules, arguing for a functional differentiation of minicollagens within the nematocyst architecture. NMR analysis of the altered C-terminal CRD of NCol-15 showed a novel disulfide-linked structure within the cysteine-containing region exhibiting similar folding kinetics and stability as the canonical CRDs. Our data provide evidence for evolutionary diversification among minicollagens, which probably facilitated alterations in the morphology of the nematocyst wall and tubule.     

Morphology and morphodynamics of the stenotele nematocyst of Hydra attenuata Pall. (Hydrozoa,Cnidaria)

This light- and electron-microscopic study has investigated the structure, the morphodynamics of discharge, and the impact of the stenotele cyst of Hydra attenuata (Hydrozoa, Cnidaria) on the prey's integument. The triggered capsule, which is ejected from the cell, discharges its tubular content (shaft, stylets and tubule) by a process of evagination. In doing so the three joined stylets punch a hole into the cuticle of the prey through which the long evaginating tubule penetrates into the interior of the target. The behaviour of the tubule is described in detail and the functional significances of the various parts of the capsule are discussed.     

THE INTERNAL ORGANIZATION OF MITOCHONDRIA

Sections of mitochondria in Paramecium and Euplotes present a consistent pattern. The mitochondrion in these cells can be conceived of as a twisted mass of closely compacted tubules. Two general kinds of substances can be recognized: the electron-dense that borders the lumen of the tubule, and the less dense that forms the continuum. In sections of mitochondria in rat kidney and snail oviduct, tubular internal organization can be recognized. In the same organs, mitochondria with lamellar internal structure can be demonstrated. The thesis is developed that the mitochondrion is a structure capable of differentiation and change, and that developmental continuity among the different kinds may exist. Mitochondria that appear to be different may be quite similar basically; mitochondria that appear to be similar in structure may be different in other ways. The tubule is proposed as the most basic of the presently recognized mitochondrial structures.     

Ultrastructure of a novel eurytele nematocyst of Carybdea alata Reynaud (Cubozoa,Cnidaria)

The ultrastructural characteristics of nematocysts from the cubozoan Carybdea alata Reynaud, 1830 (Hawaiian box jellyfish) were examined using light, scanning and transmission electron microscopy. We reclassified the predominant nematocyst in C. alata tentacles as a heterotrichous microbasic eurytele, based on spine, tubule and capsule measurements. These nematocysts exhibited a prominent and singular stylet, herein referred to as the lancet. Discharged nematocysts from fixed tentacle preparations displayed the following structures: a smooth shaft base, lamellae, a hemicircumferential fissure demarking the proximal end of a stratified lancet, and a gradually tapering tubule densely covered with large triangularly shaped spines. The lancet remained partially adjoined to the shaft base in a hinge-like fashion in rapidly fixed, whole-tentacle preparations. In contrast, this structure was not observed in discharged nematocyst preparations which involved multiple transfer steps prior to fixation. Various approaches were designed to detect this structure in the absence of fixative. Detached lancets were located in proximity to discharged tubules in undisturbed coverslip preparations of fresh tentacles. In addition, examination of embedded nematocysts from fresh tentacles laid on polyacrylamide gels revealed still-attached lancets. To examine the function of this structure in prey capture, Artemia sp. laden tentacles were prepared for scanning electron microscopy. While carapace exteriors exhibited structures proximal to the lancet, i.e., the nematocyst capsule and shaft base, neither tubule nor lancet structures were visible. Taken together, the morphological data suggested a series of events involved in the discharge of a novel eurytele from C. alata.     

Remodelling during the development of nematocysts in a siphonophore

Remodelling of a smoothly tapering internal tubule to form the enlarged shaft of microbasic mastigophores of the siphonophore Rosacea takes place very late in development. It occurs after deployment of the nematocyst in its final position, and after the pleats of the internal tubule have been completely obliterated in the shaft region by the growth of spine material. The optical and mechanical properties of the internal tubule at the earlier (bedspring) stage give no hint that a shaft will develop. The possible ways in which remodelling might occur inside the capsule and remote from the cytoplasm are discussed.     

Cellular basis for tentacle adherence in the Portuguese man-of-war (Physalia physalis)

The fishing tentacles of Physalia physalis (Portuguese man-of-war) adhere to prey and human victims by the penetration of a barbed tubule connected to an intracellular nematocyst. The nematocyst is surrounded by a fibrillar system of microtubules and microfilaments that terminate in hemidesmosomal processes which anchor the nematocyst to the acellular mesoglea of the tentacle.     

THE AXOSTYLE OF SACCINOBACULUS : I. Structure of the Organism and Its Microtubule Bundle

The axostyle of the flagellate Saccinobaculus is a motile ribbon composed of microtubules, cross-bridged to form interconnected rows. We find a centriole-related row of dark-staining tubules near the nucleus at the anterior end of the axostyle. Other tubule rows bind parallel to this primary row, acquire ordered relationships, and become the tubules of the axostyle proper. The number of tubule rows is constant in Saccinobaculus lata from the region near the nucleus to within a few micrometers of the posterior tip of the cell. In Saccinobaculus ambloaxostylus a few tubule rows are added to the axostyle posterior to the nucleus, giving this axostyle a leaf spring construction. The tubules of S. lata are held in rows by links with a 140 Å periodicity along the tubule axis; bridges between rows of tubules are also seen but are not apparently periodic. Each tubule in S. ambloaxostylus shows an axial periodicity of 150 Å due to pairs of arms, one of which is always part of the intrarow link. Interrow bridges in this species run either from tubule to tubule or from tubule to the free arm, but as in S. lata they do not display an obvious axial periodicity. An average unit cell is presented for the axostyle of each species, and the relation of the intertubule links to the microtubule substructure is discussed.     

Commitment during stenotele differentiation in Hydra is localized near the S/G2 boundary in the terminal cell cycle

The timing of commitment during stenotele differentiation in Hydra was determined. Regeneration of isolated distal regions of the body column induces stenotele differentiation. The kinetics of appearance of committed stenotele precursors was determined in such regenerating pieces. Using [³H]thymidine labeling and hydroxyurea sensitivity, the G1/S and the S/G2 boundaries of the precursor population was also determined. Comparison of these results indicates that stenotele commitment is localized near the S/G2 boundary in the terminal cell cycle of nests of precursor cells.     

Calcium- and voltage-dependence of nematocyst discharge in Hydra vulgaris

In Hydra vulgaris, discharge of stenotele nematocysts was induced by contact with prey, electrical stimuli, or increase in the external potassium concentration. In each case 10^-4 M calcium was required in the culture medium. The results indicated a voltage- and calcium-dependent mechanism different from mechano- or chemoreception allowing calcium influx from the external medium. A threshold for activation was suggested by the steep increase of the rate of electrically induced discharge in external fields of 3.5 kV/m. Although organic antagonists for vertebrate calcium channels were ineffective in blocking the calcium-induced nematocyst discharge, inorganic divalent and trivalent cations competitively inhibited the process, with a sequence (Co²⁺ < Ni²⁺ < Cd²⁺ < La³⁺ < Gd³⁺) similar to that seen for antagonism of calcium influx through voltage-dependent channels. Magnesium, an intracellular calcium antagonist, decreased nematocyst discharge, while strontium replacing calcium supported the discharge at a lowered rate. It is concluded that in the nematocyte a voltage-activated influx of calcium through apical ion channels initiates the discharge of the nematocyst in an exocytotic process.     

NMDA and GABA B receptors are involved in controlling nematocyst discharge in hydra

The role of chemical neurotransmission in nematocyst discharge was investigated by stimulating the cnidocils of nematocysts in ablated tentacles of Hydra vulgaris with a piezoelectrically-driven glass probe, in the presence of selected neurotransmitters. Acetylcholine, dopamine, epinephrine, glycine, and serotonin (10^− 4, 10^− 6, 10^− 8 M) per se, did not alter stenotele and desmoneme discharge. γ-Amino-butyric acid (GABA) significantly increased desmoneme discharge when the cnidocil of another desmoneme in the same or adjacent battery cell complex was stimulated without affecting the discharge rates of the directly stimulated desmonemes or stenoteles. Baclofen (GABA_B agonist) mimicked the increase; its antagonist, phaclofen, counteracted it. GABA_A agonists and antagonists did not alter discharge rates. Glutamate caused a dose-dependent increase in the discharge rate of directly stimulated stenoteles; distant stenotele and desmoneme discharge rates were unaffected. Kainate, AMPA, and NMDA, per se, did not alter discharge rates. Co-administration of NMDA and kainate mimicked glutamate's effects. AMPA plus NMDA increased discharge rates. DAP-5 (NMDA antagonist) and CNQX, (kainate/AMPA antagonist) counteracted the increase. The findings suggest that metabotropic GABA is involved in recruiting desmonemes by disinhibiting those previously inhibited, and that the NMDA/kainate–AMPA mechanism regulating Ca⁺⁺ entry in higher neuroeffector systems is an early-evolved process, which, in hydra, modulates nematocyst discharge.     

Proteome of Hydra nematocyst

Stinging cells or nematocytes of jellyfish and other cnidarians represent one of the most poisonous and sophisticated cellular inventions in animal evolution. This ancient cell type is unique in containing a giant secretory vesicle derived from the Golgi apparatus. The organelle structure within the vesicle comprises an elastically stretched capsule (nematocyst) to which a long tubule is attached. During exocytosis, the barbed part of the tubule is accelerated with >5 million g in <700 ns, enabling a harpoon-like discharge (Nüchter, T., Benoit, M., Engel, U., Ozbek, S., and Holstein, T. W. (2006) Curr. Biol. 16, R316-R318). Hitherto, the molecular components responsible for the organelle's biomechanical properties were largely unknown. Here, we describe the proteome of nematocysts from the freshwater polyp Hydra magnipapillata. Our analysis revealed an unexpectedly complex secretome of 410 proteins with venomous and lytic but also adhesive or fibrous properties. In particular, the insoluble fraction of the nematocyst represents a functional extracellular matrix structure of collagenous and elastic nature. This finding suggests an evolutionary scenario in which exocytic vesicles harboring a venomous secretome assembled a sophisticated predatory structure from extracellular matrix motif proteins.     

ULTRASTRUCTURAL ZONATION OF ADRENOCORTEX IN THE RAT

The fine structure of the different zones in the adrenal cortex of the adult rat has been studied under the electron microscope. Four regions mainly differentiated by the mitochondrial morphology, the lipid droplets, and the structure of the ground cytoplasm were recognized. In the glomerular zone mitochondria are thin and elongated with an abundant matrix. The inner structure is characterized by the presence of tubules of 300 A that are straight or bend at an angle and which may be grouped in parallel array giving a pseudocrystalline pattern. The wall of each tubule is a finger-like projection of the inner membrane and its cavity corresponds to the outer chamber of the mitochondrion. In the intermediary zone mitochondria are larger and irregular. The matrix is filled with convoluted tubules and vesicular elements. The lipid droplets are larger and irregular in the glomerulosa and and small in the intermedia. The ground substance is dense and contains free ribosomes in the glomerulosa and starts to be vacuolated in the intermedia. In the fasciculata mitochondria are round or oval and are filled with vesicular elements with a mean size of 450 A. Larger vesicles and more clear elements (vacuoles) are seen near the edge as if their content was diluted. Some of these vacuoles protrude on the surface. In the reticular zone mitochondria are also vesicular but frequently show signs of alteration and disruption. Dense elements recognized as microbodies are observed in the fasciculata but they increase in number in the reticularis. These results are discussed on the light of the so called zonal theory of the adrenal cortex. Two stages in the differentiation of the mitochondria are postulated. The tubular structure of the glomerulosa undergoes a process of disorientation and dilatation of the tubules to form the tubulo-vesicular elements of the intermediary zone. In a second stage of differentiation, by fragmentation of the tubules, the vesicular structure of fasciculata is formed. These findings are discussed from the viewpoint of the relationship between mitochondria and synthesis of steroid hormones. A secretory process that starts within mitochondria by the formation of vesicles and proceeds into the ground cytoplasm, as extruded and more clear vacuoles, is postulated.     

CELLULAR SPECIALIZATION IN THE EXCRETORY EPITHELIA OF AN INSECT, Macrosteles fascifrons STÅL (HOMOPTERA)

An electron microscopic investigation of the Malpighian tubules of a leaf hopper, Macrosteles fascifrons, shows that these organs comprise three quite distinct cell types, and the structure of these and of the mid- and hindgut epithelial cells is described. In particular, a comparison is made between the organization of the basal and apical surfaces of cells in the Malpighian tubule and in the vertebrate kidney, and it is suggested that similarities between these excretory epithelia reflect functional parallels between them. While the midgut and one region of the Malpighian tubule bear a typical microvillar brush border, elsewhere in the tubule and in the hindgut the apical surface bears cytoplasmic leaflets or lamellae. The sole solid excretory material of these insects consists of the brochosomes, secreted by cells of one region of the Malpighian tubule. The structure, geometry, and development of these unusual bodies, apparently formed within specialized Golgi regions, has been investigated, and histochemical tests indicate that they contain lipid and protein components.     

Structure/function analysis of spinalin, a spine protein of Hydra nematocysts

The nematocyst capsules of the cnidarians are specialized explosive organelles that withstand high osmotic pressures of approximately 15 MPa (150 bar). A tight disulfide network involving cysteine-rich capsule wall proteins, like minicollagens and nematocyst outer wall antigen, characterizes their molecular composition. Nematocyst discharge leads to the expulsion of a long inverted tubule that was coiled inside the capsule matrix before activation. Spinalin has been characterized as a glycine-rich, histidine-rich protein associated with spine structures on the surface of everted tubules. Here, we show that full-length Hydra spinalin can be expressed recombinantly in HEK293 cells and has the property to form disulfide-linked oligomers, reflecting its state in mature capsules. Furthermore, spinalin showed a high tendency to associate into dimers in vitro and in vivo. Our data, which show incomplete disulfide connectivity in recombinant spinalin, suggest a possible mechanism by which the spine structure may be linked to the overall capsule polymer.