Sequence-structure and structure-function analysis in cysteine-rich domains forming the ultrastable nematocyst wall

The nematocyst wall of cnidarians is a unique biomaterial that withstands extreme osmotic pressures, allowing an ultrafast discharge of the nematocyst capsules. Assembly of the highly robust nematocyst wall is achieved by covalent linkage of cysteine-rich domains (CRDs) from two main protein components, minicollagens and nematocyst outer wall antigen (NOWA). The bipolar minicollagens have different disulfide patterns and topologies in their N and C-terminal CRDs. The functional significance of this polarity has been elusive. Here, we show by NMR structural analysis that all representative cysteine-rich domains of NOWA are structurally related to N-terminal minicollagen domains. Natural sequence insertions in NOWA CRDs have very little effect on the tightly knit domain structures, nor do they preclude the efficient folding to a single native conformation. The different folds in NOWA CRDs and the atypical C-terminal minicollagen domain on the other hand can be directly related to different conformational preferences in the reduced states. Ultrastructural analysis in conjunction with aggregation studies argues for an association between the similar NOWA and N-terminal minicollagen domains in early stages of the nematocyst wall assembly, which is followed by the controlled association between the unusual structures of C-terminal minicollagen domains.     

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.     

Luminal particles within cellular microtubules

The regulation of microtubule dynamics is attributed to microtubule-associated proteins that bind to the microtubule outer surface, but little is known about cellular components that may associate with the internal side of microtubules. We used cryoelectron tomography to investigate in a quantitative manner the three dimensional structure of microtubules in intact mammalian cells. We show that the lumen of microtubules in this native state is filled with discrete, globular particles with a diameter of 7 nm and spacings between 8 and 20 nm in neuronal cells. Cross-sectional views of microtubules confirm the presence of luminal material in vitreous sections of brain tissue. Most of the luminal particles had connections to the microtubule wall, as revealed in tomograms. A higher accumulation of particles was seen near the retracting plus ends of microtubules. The luminal particles were abundant in neurons, but were also observed in other cells, such as astrocytes and stem cells.     

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.     

Small angle neutron scattering studies of C8 and C9 and their interactions in solution.

Small angle neutron scattering (SANS) results revealed that contrary to most reports C9 is not a globular protein. Its radius of gyration (Rg) at pH 8 and an ionic strength of 0.5 is 32.2 +/- 1.4 A increasing to 35 A at physiologic ionic strength. In contrast, C8, which has a 2.2-fold larger mass, has a similar Rg value [34.6 +/- 1.6 A]. Calibration plots of Rg vs. M(r) indicate that native C8 is a spherical protein whereas native C9 is elongated. From previous reports it was known that native C8 and C9 associate in solutions of low ionic strength. SANS results confirmed this observation but also demonstrated that C8-C9 heterodimers are already formed at physiologic ionic strength. The dimeric complex is globular [Rg = 40 +/- 0.8 A] indicating that the proteins associate side-by-side rather than end-to-end. In contrast, in presence of the drug Suramin, a potent inhibitor of the assembly of the C5b-9 complex, C9 forms a complex with twice the molecular mass that is still elongated (Rg = 48.8 +/- 0.8 A), suggesting that in this case the protein dimerizes end-to-end via a bridging Suramin molecule.     

Mapping the interaction surface of linker histone H1(0) with the nucleosome of native chromatin in vivo

H1 linker histones stabilize the nucleosome, limit nucleosome mobility and facilitate the condensation of metazoan chromatin. Here, we have combined systematic mutagenesis, measurement of in vivo binding by photobleaching microscopy, and structural modeling to determine the binding geometry of the globular domain of the H1(0) linker histone variant within the nucleosome in unperturbed, native chromatin in vivo. We demonstrate the existence of two distinct DNA-binding sites within the globular domain that are formed by spatial clustering of multiple residues. The globular domain is positioned via interaction of one binding site with the major groove near the nucleosome dyad. The second site interacts with linker DNA adjacent to the nucleosome core. Multiple residues bind cooperatively to form a highly specific chromatosome structure that provides a mechanism by which individual domains of linker histones interact to facilitate chromatin condensation.     

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.     

Model membrane morphology and crosslinking of oxidized lipids with proteins

Model membranes composed of thion-phosphatidylcholine, cardiolipin, and cytochrome c have been studied by 31P NMR, polyacrylamide gel electrophoresis, gel filtration, fluorescence, and freeze-fracturing. Covalent binding of oxidized phospholipids to cytochrome c was shown to result in the formation of high-molecular-weight oligomeric complexes via Schiff base formation between a protein molecule and aldehydes produced upon peroxidation of phospholipids. The initial steps of the protein oligomerization lead to the appearance of intramembranous particles (IMPs) of various size and distribution on freeze-fractured faces of these model membranes. In the final phase of the crosslinking between cytochrome c and oxidized products of cardiolipin there is a breakdown of membrane vesicles and formation of globular lipoprotein complexes which are seen as globular particles. It is believed that the covalent linking between the products of phospholipid peroxidation and membrane proteins causes the oligomerization of membrane proteins and structural alteration in the hydrophobic region of other models also and, perhaps, in biological membranes.     

Critical Examination of the Colloidal Particle Model of Globular Proteins

Recent studies of globular protein solutions have uniformly adopted a colloidal view of proteins as particles, a perspective that neglects the polymeric primary structure of these biological macromolecules, their intrinsic flexibility, and their ability to sample a large configurational space. While the colloidal perspective often serves as a useful idealization in many cases, the macromolecular identity of proteins must reveal itself under thermodynamic conditions in which the native state is no longer stable, such as denaturing solvents and high protein concentrations where macromolecules tend to have screened excluded volume, charge, and hydrodynamic interactions. Under extreme pH conditions, charge repulsion interactions within the protein chain can overcome the attractive hydrogen-bonding interactions, holding it in its native globular state. Conformational changes can therefore be expected to have great significance on the shear viscosity and other rheological properties of protein solutions. These changes are not envisioned in conventional colloidal protein models and we have initiated an investigation of the scattering and rheological properties of model proteins. We initiate this effort by considering bovine serum albumin because it is a globular protein whose solution properties have also been extensively investigated as a function of pH, temperature, ionic strength, and concentration. As we anticipated, near-ultraviolet circular dichroism measurements and intrinsic viscosity measurements clearly indicate that the bovine serum albumin tertiary structure changes as protein concentration and pH are varied. Our findings point to limited validity of the colloidal protein model and to the need for further consideration and quantification of the effects of conformational changes on protein solution viscosity, protein association, and the phase behavior. Small-angle Neutron Scattering measurements have allowed us to assess how these conformational changes influence protein size, shape, and interprotein interaction strength.     

Analysis of the structure of intramembrane particles of the mammalian urinary bladder

The luminal and discoid vacuole membranes of the superficial cell layer of the transitional epithelium of the mammalian urinary bladder have been studied by thin-sectioning and freeze-fracture-etch (FFE) electron microscope methods. For the FFE studies membranes were deposited on a cationized glass surface, covered by a thin copper disc, and fractured under liquid N2. Specimens were etched at -100 degrees C and replicated at -190 degrees C. A model of the lattice membrane derived from thin sections was used to predict the heights of the fracture faces above the glass surface. A hexagonal pattern of globular intramembrane particles spaced 160 A apart was seen in the external fracture (EF) face plaques as previously described and regarded as the dominant structure. However, very extensive areas of another pattern, seen before in only limited areas, have beeen found in the EF faces. The pattern consists of a smooth hexagonal lattice with the same space constant as the globular one but a different structure. By image analysis it consists of overlapping domains bordered by shared but incomplete metal rims. Each domain has a central spot of metal encircled by a shadow. The surface of the smooth lattice is partly complementary to the corresponding protoplasmic fracture (PF) face which shows a similar hexagonal lattice with the same space constant. The height of the smooth EF lattice above the glass substrate is the same as the plane of the center of the lipid bilayer predicted by the model. The mean heights of the particles of the globular EF lattice are greater than the total thickness of the membrane as predicted by the model and confirmed by measurements. The globular EF lattice is not complementary and it is concluded that the globular particles do not exist in the native membrane but arise artifactually during the preparatory procedures.     

Xylan deposition on secondary wall of Fagus crenata fiber

Summary. Delignified and/or xylanase-treated secondary walls of Fagus crenata fibers were examined by field emission scanning electron microscopy. Microfibrils with a smooth surface were visible in the innermost surface of the differentiating fiber secondary wall. There was no ultrastructural difference between control and delignified sections, indicating that lignin deposition had not started in the innermost surface of the cell wall. There was no ultrastructural difference between control and xylanase-treated sections. Microfibrils on the outer part of the differentiating secondary wall surface had globular substances in delignified sections. These globular substances disappeared following xylanase treatment, indicating that these globules are xylan. The globular substances were not visible near the inner part of the differentiating secondary wall but gradually increased toward the outer part of the secondary wall, indicating that xylan penetrated into the cell wall and continuously accumulated on the microfibrils. Mature-fiber secondary walls were also examined by field emission scanning electron microscopy. Microfibrils were not apparent in the secondary wall in control specimens. Microfibrils with many globular substances were observed in the delignified specimens. Following xylanase treatment, the microfibrils had a smooth surface without any globules, indicating that the globular substance is xylan. These results suggest that cellulose microfibrils synthesized on the plasma membrane are released into the innermost surface of the secondary wall and coated with a thin layer of xylan. Successive deposition of xylan onto the cell wall increases the microfibril diameter. The large amounts of xylan that accumulated on microfibrils appear globular but are covered with lignin after they are deposited. Received February 20, 2001/Accepted September 1, 2001     

Bacteriorhodopsin/amphipol complexes: structural and functional properties

The membrane protein bacteriorhodopsin (BR) can be kept soluble in its native state for months in the absence of detergent by amphipol (APol) A8-35, an amphiphilic polymer. After an actinic flash, A8-35-complexed BR undergoes a complete photocycle, with kinetics intermediate between that in detergent solution and that in its native membrane. BR/APol complexes form well defined, globular particles comprising a monomer of BR, a complete set of purple membrane lipids, and, in a peripheral distribution, ∼2 g APol/g BR, arranged in a compact layer. In the absence of free APol, BR/APol particles can autoassociate into small or large ordered fibrils.     

Investigation of protein refolding: a special feature of native structure responsible for refolding ability

A possible origin of the refolding ability of globular proteins is discussed. It is shown that the structure of native proteins has a special feature, namely, that this is the only structure in which the short overlapping segments of the polypeptide chain are in one of the significantly stable conformations of the oligopeptides with the same amino acid sequences as segments themselves. It is shown that this special feature is responsible for the refolding ability of proteins. A simple formula is given for the estimation of the time, t, necessary for the spontaneous formation of a refolding nucleus by a certain segment and it is shown that the segment which has the smallest t value, will serve as a refolding nucleus. It is suggested that natural selection which ensures the maintenance of the native structure of globular proteins automatically results in the refolding ability of proteins regardless of the biological relevance of this ability.     

Isolation and properties of the plasmalemma in yeast

Summary A method is described for the isolation of fragments of the plasmalemma based on differential and density gradient centrifugation using cell free extracts from anaerobically grown Saccharomyces cerevisiae. Electron microscopically investigated frozen-etched specimens of isolated plasmalemma revealed the presence of globular particles attached to the outer surface of the membrane; these particles correspond to those observed in situ.In isolated plasmalemma a high specific activity of Mg⁺⁺-dependent ATPase, which is not sensitive to Oligomycin, is present. Yeast plasmalemma contains protein, lipids (including phospholipids) and an appreciable amount of polysaccharide. Hydrolysis of this polysacharide yields only mannose.The treatment of the isolated plasmalemma with detergents liberates the globular particles which can be isolated by density gradient centrifugation. Protein and polysaccharide occur in the respective fraction; therefore the globular particle represents a mannan-protein. It is concluded that the particles, which cover the plasma-membrane of plant cells, represent glycoproteins, that is, building stones to be incorporated into the fibrillar network of the cell walls.     

Response in nematocyst uptake by the nudibranch Flabellina verrucosa to the presence of various predators in the Southern Gulf of Maine

Aeolid nudibranchs maintain nematocysts sequestered from their cnidarian prey for protection against predators. Selection for nematocyst incorporation is a function of diet and prey choice, but ratios vary among nudibranchs feeding on a given diet, indicating that other factors may be involved. It is proposed that the presence of predators influences nematocyst incorporation. Nematocyst uptake in the nudibranch Flabellina verrucosa collected from the southern Gulf of Maine was examined in response to various potential predators, including Crossaster papposus, Tautogolabrus adspersus, and Carcinus maenas. Nudibranchs in individual flow-through containers feeding on a diet of the hydroids Tubularia spp. and Obelia geniculata were subjected to tanks containing a predator, then their nematocyst distribution was examined. Although most of the changes over the experimental period were attributable to diet, F. verrucosa responded to both T. adspersus and C. papposus by significantly increasing microbasic mastigophore incorporation. No differential uptake was seen with C. maenas. Response was evident in the nudibranchs both for predators present in the collection area and for those with which they had no previous exposure, indicating that F. verrucosa modulates nematocyst incorporation in response to the presence of predators as well as to diet. A coevolution of nudibranchs and potential predators may govern changes in nematocyst uptake.     

The consistency principle in protein structure and pathways of folding

• 1.i) It is pointed out that various energy terms contributing to stabilize the native state of globular proteins are consistent in the first approximation with each other in the native state. This means that each energy term is individually minimized at the minimum point of the total energy. I proposed (1) to call this fact “the consistency principle in protein structure.”
• 2.ii) The fair success of various methods of prediction of the secondary structures in globular proteins from their amino acid sequence is often interpreted as indicating the dominance of the short-range interactions in determining the local structures of the polypeptide chains. Partly from such a point of view, the hierarchic condensation model has been popular for the process of protein folding. However the consistency principle indicates that the short-range interactions are just one type of intramolecular interaction which contributes to stabilization of the native structure together with other mutually consistent types of intramolecular interactions. Therefore the hierarchic condensation model is not necessarily a unique model of protein folding.
• 3.iii) Roles of a possible nonspecific globular state, stabilized by nonspecific long-range intramolecular interactions, in the folding process are discussed. It is expected that this nonspecific globular state is observed either as an equilibrium or a kinetic intermediate state between the unfolded and the folded native states. Observation as a kinetic intermediate state is expected to occur in experiments done under strongly refolding conditions. In this case the polypeptide chain in the unfolded state collapses into a nonspecific globule by the action of nonspecific long-range intramolecular interactions. Two possible mechanisms of the transition from the nonspecific globular state to the specific native folded state are discussed.
• 4.iv) In an experiment done under weakly refolding conditions, folding is expected to occur according to the embryo-nucleus model. This model is a refined version of the hierarchic condensation model. Refinement is done by taking into account the fact that the intermediate structures assumed in the hierarchic condensation model are unstable against both the native folded state and the unfolded state. A nucleus is an ordered structure of a certain size. Ordered structures of a size larger than a nucleus tend to fold further to become the native specific globule. Ordered structures of a size smaller than a nucleus tend to unfold. Embryos are intrinsically unstable ordered structures smaller than a nucleus. Folding occurs when embryos grow in size to become a nucleus. The intrinsic instability of embryos is the built-in mechanism to overcome the low resolving power of the short-range interactions in determining local conformations of the polypeptide chain.

     

Different nematocytes have different synapses in the sea anemone Aiptasia pallida (Cnidaria,Anthozoa)

Sea anemones feed by discharging nematocysts into their prey, but the pathway for control of nematocyst discharge is unknown. The purpose of this study was to investigate the ultrastructural evidence of neuro-nematocyte synapses and to determine the types of synaptic vesicles present at different kinds of nematocyst-containing cells. The tip and middle of tentacles from small specimens of Aiptasia pallida were prepared for electron microscopy and serial micrographs were examined. We found clear vesicles in synapses on mastigophore-containing nematocytes and dense-cored vesicles in synapses on basitrich-containing nematocytes and on one cnidoblast with a developing nematocyst. In addition, we found reciprocal neuro-neuronal and sequential neuro-neuro-nematocyte synapses in which dense-cored vesicles were present. It was concluded that : (1) neuro-nematocyte synapses are present in sea anemones, (2) different kinds of synaptic vesicles are present at cells containing different types of nematocysts, (3) synapses are present on cnidoblasts before the developing nematocyst can be identified and these synapses may have a trophic influence on nematocyst differentiation, and (4) both reciprocal and sequential synapses are present at the nematocyte, suggesting a complex pathway for neural control of nematocyst discharge. J. Morphol. 238:53–62, 1998. © 1998 Wiley-Liss, Inc.     

The Development of the Cnidoblasts of Hydra : An Electron Microscope Study of Cell Differentiation

The general histological organization of Hydra is reviewed and electron microscopic observations are presented which bear upon the nature of the mesoglea, the mode of attachment of the contractile processes of the musculo-epithelial cells, and the cytomorphosis of the cnidoblasts. Particular attention is devoted to the changes in form and distribution of the cytoplasmic organelles in the course of nematocyst formation. The undifferentiated interstitial cell is characterized by a small Golgi complex, few mitochondria, virtual absence of the endoplasmic reticulum, and a cytoplasmic matrix crowded with fine granules presumed to be ribonucleoprotein. These cytological characteristics persist through the early part of the period of interstitial cell proliferation which leads to formation of clusters of cnidoblasts. With the initiation of nematocyst formation in the cnidoblasts, numerous membrane-bounded vesicles appear in their cytoplasm. These later coalesce to form a typical endoplasmic reticulum with associated ribonucleoprotein granules. During the ensuing period of rapid growth of the nematocyst the reticulum becomes very extensive and highly organized. Finally, when the nematocyst has attained its full size, the reticulum breaks up again into isolated vesicles. The Golgi complex remains closely applied to the apical pole of the nematocyst throughout its development and apparently contributes to its enlargement by segregating formative material in vacuoles whose contents are subsequently incorporated in the nematocyst. The elaboration of this complex cell product appears to require the cooperative participation of the endoplasmic reticulum and the Golgi complex. Their respective roles in the formative process are discussed.     

Toxinological studies of the venom from Cassiopea xamachana nematocysts isolated by flow cytometry

The tentacle epithelial tissue of Cassiopea xamachana contains nematocysts and symbiotic algal particles. These two structures were dissociated, analyzed and sorted by flow cytometry. A simple separating method was developed utilizing the algal chlorophyll autofluorescence and the nematocysts' fluorescence after the uptake of fluorescent stains. A five-fold increase in mouse lethality; significantly more potent hemolytic and cytosensing activities; as well as a cleanup in the capillary electropherogram and SDS gel profiles for the crude nematocyst venom preparations prepared by fluorescence activated cell sorting (FACS), was observed relative to alternative methods. Because the hemolytic potency of pre-sorting nematocyst venom was minimal and the post-sorting counterpart was significantly positive, the possibility that algae inhibited the venom's toxinological activity was considered.     

The utilization of cnidarian nematocysts by aeolid nudibranchs: Nematocyst maintenance and release in spurilla

Some nudibranchs that feed on cnidarians are known to store nematocysts within cnidophage cells and use them for their own defense. Most of the nematocysts are in direct contact with the cytoplasm of the cnidophage. Nematocysts are not subjected to lysosomal enzymes because any phagocytic membrane that surrounded the nematocyst after engulfment does not persist. Cnidophage organelles are restricted to regions surrounding the nematocysts and may aid in the maintenance and development of the nematocysts. The release of cnidophages is initiated by a contraction of a dense muscle complex surrounding the cnidosac. Nematocysts do not discharge if the cnidophage membrane does not rupture upon release. A comparison of nematocyst maintenance in Spurilla neapolitana and nematocyst retention in other organisms is presented.