Mechanical properties of the isolated catch apparatus of the sea urchin spine joint: muscle fibers do not contribute to passive stiffness changes

The catch apparatus (CA) is the collagenous ligament at the spinal joint of sea urchins. It maintains spine posture by stiffening and allows spine movement by softening. A CA preparation, which was isolated from ossicles, was used to test the hypothesis that frictional forces between collagen fibers and ossicles are the source of stiffness changes. Isolated preparations of the CA changed in stiffness, thus falsifying the hypothesis. Another hypothesis proposes that muscle fibers, which represent a relatively small component of the CA, cause stiffening of the CA by contraction. Chemicals that evoked contraction in spine muscles did not always stiffen the CA: the CA of Heterocentrotus mammillatus softened in response to artificial seawater with potassium concentration elevated to 100 mM. This provided evidence against the muscle-based hypothesis. The present results suggest that the stiffness changes of the CA are based on changes in the mechanical properties of the extracellular components of the connective tissue and are therefore related to the connective tissue catch that is widespread in other echinoderms.     

Some properties of the action potentials conducted in the spines of the sea urchin Diadema antillarum

Brief (2-5 msec) electrical pulses applied to the primary spines of the sea urchin Diadema antillarum elicit graded action potentials (ap's). These ap's can be attributed to the electrical activity of a set of 14-21 bundles of neurites, each comprising 1000 processes near the spine base and tapering towards the spine tip. The shape of the ap's varies from a simple diphasic deflection to a complex waveform with 6 or more components. Peak-to-peak amplitude is less than 1mV. The ap's are conducted at a uniform speed of ca. 27 cm/sec. The ap's are not affected by tetrodotoxin (1 microgram/ml) and continue to be produced in Na-free artificial sea water (ASW). The amplitude of the ap's is greatly reduced or totally abolished in Ca-free ASW. However, some electrical activity may continue in the absence of external Ca, due to release of Ca2+ ions from the calcium carbonate crystals of the spine shaft. Replacing the Ca content of ASW by barium ions causes an irreversible blockade of the ap's. Spines equilibrated with ASW containing Sr2+ ions instead of Ca2+ produce ap's of increased amplitude (up to X 2). The ap's are blocked by La3+, Co2+, Cd2+ (2-5 mM) and by the organic Ca channel blocker Bepridil (2 mM). We conclude that the spinal ap's are due to the summation of Ca spikes produced by the activation of Ca channels which are blocked by barium and have a high affinity for, or permeability to Sr vs Ca.     

The ultrastructure of spines in sea urchins of the family Strongylocentrotidae

The ultrastructure of primary spines (microscopic relief of the surface of radial wedges on the spines and cross-sections of the spines) was studied by scanning electron microscopy in seven sea urchin species of the family Strongylocentrotidae. The spines were taken from the ambitus area of equally sizes individuals with a test diameter of 50 ± 5 mm. According to the number of wedges on their spines, the studied species can be divided into three groups: Strongylocentrotus intermedius, S. pallidus (18–25 wedges), S. droebachiensis, S. polyacanthus, Allocentrotus fragilis (24–32), and Mesocentrotus franciscantus, S. nudus (45–70). The species visibly differ in the microrelief of the wedges, which can be longitudinally streaked, with protuberances, with cross-dentate or non-dentate wedges, or with cross-series of denticles; in some species, the relief is absent. In S. intermedius, spines with smooth surface of the wedges, longitudinally streaked, with sparse protuberances, and with numerous cross-series of denticles only distally, only proximally, or over the entire length of the spine have been found. Wedge surface is convex or flattened in cross-sections; wedge shape in cross-section is rectangular (S. droebachiensis, S. intermedius, S. polyacanthus), triangular (S. pallidus), trapezoid (S. fragilis), or ansiform (M. franciscanus, M. nudus). Species of the genus Mesocentrotus are readily distinguished from the other species by the stereome of their spines: wider than the height of the wedges and more homogeneous, without regular concentric circles. Data on the ultrastructure of primary spines confirm the generic status of Mesocentrotus Tatarenko et Poltaraus, 1993 and do not support the recognition of Allocentrotus Mortensen, 1942 as a distinct genus.     

Collagen and proteoglycan in a sea urchin ligament with mutable mechanical properties

Summary The problematic ligament of sea urchins is a connective tissue which crosses the ball-and-socket joint between spine and body wall. The problem of this ligament is that it is composed of parallel collagen fibrils, yet normally undergoes rapid and dramatic alterations in mechanical properties and in length. Previous work has suggested that the collagen fibrils of the ligament are able to slide past one another during length changes but are inhibited from sliding when the ligament is in catch. In this model of the ligament both the collagen fibrils and the interfibrillar matrix are mechanically important. We have found that the collagen fibrils of the spine ligament of the pencil urchin Eucidaris tribuloides are discontinuous and end by tapering within the body of the ligament. Intact fibrils that have been isolated from the ligament vary by more than an order of magnitude in length and in radius but have a constant length/radius (aspect) ratio of about 5300. This is the first determination of the aspect ratio of collagen fibrils from any source. The constant aspect ratio of the fibrils is consistent with their functioning as the discontinuous fiber phase in a fiber-reinforced composite material, while the high value of the aspect ratio indicates that the nonfibrillar matrix, which must act to transfer stress between fibrils, can produce a stiff and strong ligament even if it is several orders of magnitude weaker and more compliant than the fibrils. Moreover, the tensile properties of the ligament may be determined by the properties of the matrix. A prominent component of the interfibrillar matrix is a proteoglycan which associates with specific bands at the surface of the collagen fibrils through noncovalent binding of its core protein. The glycosaminoglycan moiety of this proteoglycan is partly comprised of chondroitin sulfate/dermatan sulfate polymers. These results are consistent with the sliding fibril hypothesis and suggest that the proteoglycan may be an important component of the stress-transfer matrix.     

Barium spikes are generated in the spines of the sea urchin Diadema antillarum

In the presence of calcium, Ba2+ ions, at concentrations as low as 1-2 mM, block the action potentials (a.p.'s) elicited by the electrical stimulation of the primary spines of Diadema antillarum. Lower concentrations of barium (0.1 mM) potentiate the a.p.'s recorded from spines equilibrated with Ca-Free artificial sea water (ASW). Exposure of the spines to a saturated solution of EDTA in Ca-free, unbuffered ASW reversibly blocks their electrical activity. Spines blocked by EDTA continue to generate a.p.'s following their equilibration with Ca-free ASW containing 1 mM of Ba2+ ions. The time course of the a.p.'s recorded from spines equilibrated with normal ASW is only slightly affected by the combined action of 15 mM of tetraethylammonium (TEA) and 5 mM of 4-aminopyridine (4-AP). In contrast, the duration of the a.p.'s recorded from spines blocked by EDTA and placed in 1 mM barium is markedly increased by the combined actions of TEA and 4-AP at the above concentrations. We conclude that the Ca-channels of the neurites present in Diadema spines are not, at least qualitatively, an exception with regard to their permeation by Ba2+. The blocking action of low concentrations of these ions, particularly in the presence of calcium, may be explained by the extremely high surface to volume ratio prevailing in neurites with a diameter of only less than 0.1-0.3 micron.     

Effects of cations on guanylate cyclase of sea urchin sperm.

Mn2+ and to some degree Fe2+, but not Mg+, Ca2+, ba2+, Sr2+, Co2+, Ni2+, La3+, or Fe3+ were able to serve as effective metal cofactors for sea urchin sperm guanylate cyclase. The apparent Michaelis constant for Mn2+ in the presence of 0.25 mM MnGTP was 0.23 mM. In the presence of a fixed free mn2+ concentration, variation in mngTP resulted in sigmoid velocity-substrate plots and in reciprocal plots that were concave upward. These positive cooperative patterns were observed at both pH 7.0 and 7.8 and in the presence or absence of Triton X-100. When Mn2+ and GTP were equimolar, Ca2+, Ba2+, Sr2+, and Mg2+ increased apparent guanylate cyclase activity. This increase in enzyme activity at least could be accounted for partially by an increase in free Mn2+ concentration caused by the complex formation of GTP with the added metals. However, even at relatively low GTP concentrations and with Mn2+ concentrations in excess of GTP, Ca2+, Sr2+, and Ba2+ significantly increased guanosine 3':5'-monophosphate production. As the total GTP concentration was increased, the degree of stimulation in the presence of Ca2+ decreased, despite maintenance of a fixed total concentration of Ca2+ and a fixed free concentration of Mn2+, suggesting that the concentration of CaGTP and MnGTP were determining factors in the observed response. The concave upward reciprocal plots of velocity against MnGTP concentration were changed to linear plots in the presence of CaGTP or SrGTP. These results suggest that sea urchin sperm guanylate cyclase contains multiple nucleotide binding sites and that stimulation of guanosine 3':5'-monophosphate synthesis by Ca2+, Sr2+, and perhaps other metals may reflect interaction of a metal-GTP complex with enzyme as either an effector or a substrate.     

Properties,morphogenesis, and effect of acidification on spines of the cidaroid sea urchin Phyllacanthus imperialis

Cidaroid sea urchins are the sister clade to all other extant echinoids and have numerous unique features, including unusual primary spines. These lack an epidermis when mature, exposing their high‐magnesium calcite skeleton to seawater and allowing the settlement of numerous epibionts. Cidaroid spines are made of an inner core of classical monocrystalline skeleton and an outer layer of polycrystalline magnesium calcite. Interestingly, cidaroids survived the Permian‐Triassic crisis, which was characterized by severe acidification of the ocean. Currently, numerous members of this group inhabit the deep ocean, below the saturation horizon for their magnesium calcite skeleton. This suggests that members of this taxon may have characteristics that may allow them to resist ongoing ocean acidification linked to global change. We compared the effect of acidified seawater (pH 7.2, 7.6, or 8.2) on mature spines with a fully developed cortex to that on young, growing spines, in which only the stereom core was developed. The cortex of mature spines was much more resistant to etching than the stereom of young spines. We then examined the properties of the cortex that might be responsible for its resistance compared to the underlying stereomic layers, namely morphology, intramineral organic material, magnesium concentration, intrinsic solubility of the mineral, and density. Our results indicate that the acidification resistance of the cortex is probably due to its lower magnesium concentration and higher density, the latter reducing the amount of surface area in contact with acidified seawater. The biofilm and epibionts covering the cortex of mature spines may also reduce its exposure to seawater.     

The ultrastructure of the sea urchin egg cortex isolated before and after fertilization

The three-dimensional organization of cortices isolated from unfertilized and fertilized Strongylocentrotus purpuratus eggs has been examined by several techniques of light and electron microscopy. It has been found that when moderate shear forces are used, the isolated unfertilized egg cortex, in addition to cortical granules, contains acidic vesicles and an elaborate network of rough endoplasmic reticulum. This network provides a physical link between the cell surface and several kinds of cytoplasmic organelles (mitochondria, yolk granules, acidic vesicles) which are retained as part of the isolated cortex when gentle shear forces are applied. Furthermore a good visualization of actin in the cortex is provided: it is present as short filaments and mostly within the stubby microvilli of the egg. Finally, it has been noted that plaques exist on the inside face of the plasma membrane ready to assemble into typical clathrin coats that prefigure the burst of coated vesicle endocytosis that takes place after fertilization. The cortex isolated soon after fertilization is shown to contain coated pits and a scaffolding of filaments (mostly actin) in which many acidic vesicles are embedded.     

Dynamics of the endoplasmic reticulum and golgi apparatus during early sea urchin development

The endoplasmic reticulum (ER) and Golgi were labeled by green fluorescent protein chimeras and observed by time-lapse confocal microscopy during the rapid cell cycles of sea urchin embryos. The ER undergoes a cyclical microtubule-dependent accumulation at the mitotic poles and by photobleaching experiments remains continuous through the cell cycle. Finger-like indentations of the nuclear envelope near the mitotic poles appear 2-3 min before the permeability barrier of the nuclear envelope begins to change. This permeability change in turn is approximately 30 s before nuclear envelope breakdown. During interphase, there are many scattered, disconnected Golgi stacks throughout the cytoplasm, which appear as 1- to 2-microm fluorescent spots. The number of Golgi spots begins to decline soon after nuclear envelope breakdown, reaches a minimum soon after cytokinesis, and then rapidly increases. At higher magnification, smaller spots are seen, along with increased fluorescence in the ER. Quantitative measurements, along with nocodazole and photobleaching experiments, are consistent with a redistribution of some of the Golgi to the ER during mitosis. The scattered Golgi coalesce into a single large aggregate during the interphase after the ninth embryonic cleavage; this is likely to be preparatory for secretion of the hatching enzyme during the following cleavage cycle.     

Actin-like filaments in the acrosomal apparatus of spermatozoa of a sea urchin

The acrosomal apparatus of a sea urchin, Echinocardium cordatum, consists of an acrosomal vesicle and a post-acrosomal rod. The rod is 2.5 μm long and extends from the acrosomal vesicle to the bottom of a nuclear invagination. The rod consists of a bundle of longitudinally disposed, 60 Å thick, actin-like filaments which bind heavy meromyosin to form arrowhead complexes. The actin-like filaments may have a dual function in the fertilization process: (1) extension of the acrosomal process through the egg investments; (2) incorporation of the sperm nucleus.     

Comparative ultrastructure of catch tentacles and feeding tentacles in the sea anemone Haliplanella

TEM observations of catch tentacles revealed that the tentacle tip epidermis is filled with two size classes of mature holotrich nematocysts and a gland cell filled with electron-dense vesicles. Vesicle production is restricted to upper-middle and tentacle tip regions, whereas holotrich development occurs in the lower-middle and tentacle base regions. Thus, catch tentacles have a maturity gradient along their length, with mature tissues concentrated at the tentacle tip. Occasional feeding tentacle cnidae (microbasic p-mastigophores and basitrichs) and mucus gland cells occur in proximal portions of catch tentacles, but are phagocytized by amoeboid granulocytes and transported to the gastrodermis for further degradation. No feeding tentacle cnidae or mucus cells occur distally in catch tentacles. Unlike catch tentacles, feeding tentacles are homogeneous in structure along their length with enidocytes containing mature spirocysts, microbasic p-mastigophore or basitrich nematocysts distributed along the epithelial surface. Cnidoblasts are recessed beneath cnidocytes, occurring along the nerve plexus. Mucus gland cells and gland cells filled with electron-dense vesicles are present in feeding tentacles, distributed at the epithelial surface. Granular phagocytes are rare in the feeding tentacle tip, but common in the tentacle base.     

TAME stabilizes the cortex and mitotic apparatus of the sea urchin egg during isolation

The synthetic substrate p-tosyl-L-arginine methyl ester (TAME) has been included in buffered EGTA media used for the isolation of the mitotic apparatus from clam eggs and also for the isolation of the cortex from sea urchin eggs. In the course of an investigation of the role of actin-fascin and actin-myosin interactions in cytokinesis, the isolation of the sea urchin egg cortex was re-examined and the stability of the cortex to lysis in a buffered EGTA medium near neutrality found to depend directly on the presence of TAME. Lysis of eggs at metaphase in this medium yielded a mixture of cortices and mitotic apparatuses (MA); MA stability under these conditions also required the presence of TAME, although a reduced pH allowed MA isolation in its absence. The action of TAME in stabilizing the actin-based structure of the cortex and the microtubule-based structure of the MA is not duplicated by other proteolysis inhibitors and this compound will also induce actin polymerization and gelation in extracts of the soluble cytoplasmic proteins of the egg under conditions where these are normally inhibited.