RESUMEN
Purpose and Aims: Sea urchin teeth consist of calcite and form in two stages with different magnesium contents. The first stage structures of independently formed plates and needle-prisms define the shape of the tooth, and the columns of the second stage mineral cements the first stage structures together and control the fracture behavior of the mature tooth. This study investigates the nucleation and growth of the second stage mineral. MATERIALS AND METHODS: Scanning electron microscopy (SEM) and synchrotron microComputed Tomography characterized the structures of the second phase material found in developing of Lytechinus variegatus teeth. RESULTS: Although the column development is a continuous process, defining four phases of column formation captures the changes that occur in teeth of L. variegatus. The earliest phase consists of small 1-2 µm diameter hemispheres, and the second of 5-10 µm diameter, mound-like structures with a nodular surface, develops from the hemispheres. The mounds eventually bridge the syncytium between adjacent plates and form hyperboloid structures (phase three) that appear like mesas when plates separate during the fracture. The mesa diameter increases with time until the column diameter is significantly larger than its height, defining the fourth phase of column development. Energy dispersive x-ray spectroscopy confirms that the columns contain more magnesium than the underlying plates; the ratios of magnesium to calcium are consistent with compositions derived from x-ray diffraction. CONCLUSION: Columns grow from both bounding plates. The presence of first phase columns interspersed among third stage mesas indicates very localized control of mineralization.
Asunto(s)
Lytechinus/química , Minerales/química , Animales , Electrones , Lytechinus/ultraestructura , Espectrometría por Rayos X , Microtomografía por Rayos XRESUMEN
The importance of proteins in shaping the membranes that define the perimeters of organelles is well documented. By forming cross-links, motors, or scaffolds or by inserting into membranes, proteins can harness energy to deform membranes, particularly when high degrees of curvature are necessitated-as in small membrane vesicles, tubules of the endoplasmic reticulum, the edges of endoplasmic reticulum sheets or Golgi apparatus cisternae, and membrane fusion intermediates (stalks). Here we propose that membrane lipids displaying negative curvature act in concert with membrane proteins to contribute to the alteration and maintenance of bending in biological membranes. We emphasize recent data from studies of sea urchin eggs and embryos and suggest how novel approaches can lead to future directions for investigating the roles of such lipids in vivo.
Asunto(s)
Retículo Endoplásmico/metabolismo , Lytechinus/metabolismo , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Óvulo/metabolismo , Animales , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Diglicéridos/metabolismo , Retículo Endoplásmico/ultraestructura , Enzimas/metabolismo , Lytechinus/ultraestructura , Microinyecciones , Microscopía Confocal , Oocitos/metabolismo , Oocitos/ultraestructura , Óvulo/ultraestructura , Liposomas Unilamelares/metabolismoRESUMEN
Sea urchin teeth grow continuously and develop a complex mineralized structure consisting of spatially separate but crystallographically aligned first stage calcitic elements of high Mg content (5-15 mol% mineral). These become cemented together by epitaxially oriented second stage very high Mg calcite (30-40 mol% mineral). In the tooth plumula, ingressing preodontoblasts create layered cellular syncytia. Mineral deposits develop within membrane-bound compartments between cellular syncytial layers. We seek to understand how this complex tooth architecture is developed, how individual crystalline calcitic elements become crystallographically aligned, and how their Mg composition is regulated. Synchrotron microbeam X-ray scattering was performed on live, freshly dissected teeth. We observed that the initial diffracting crystals lie within independent syncytial spaces in the plumula. These diffraction patterns match those of mature tooth calcite. Thus, the spatially separate crystallites grow with the same crystallographic orientation seen in the mature tooth. Mineral-related proteins from regions with differing Mg contents were isolated, sequenced, and characterized. A tooth cDNA library was constructed, and selected matrix-related proteins were cloned. Antibodies were prepared and used for immunolocaliztion. Matrix-related proteins are acidic, phosphorylated, and associated with the syncytial membranes. Time-of-flight secondary ion mass spectroscopy of various crystal elements shows unique amino acid, Mg, and Ca ion distributions. High and very high Mg calcites differ in Asp content. Matrix-related proteins are phosphorylated. Very high Mg calcite is associated with Asp-rich protein, and it is restricted to the second stage mineral. Thus, the composition at each part of the tooth is related to architecture and function.
Asunto(s)
Carbonato de Calcio/metabolismo , Lytechinus/crecimiento & desarrollo , Magnesio/metabolismo , Proteínas/metabolismo , Diente/crecimiento & desarrollo , Diente/metabolismo , Animales , Cristalización , Células Gigantes/metabolismo , Lytechinus/citología , Lytechinus/metabolismo , Lytechinus/ultraestructura , Coloración y Etiquetado , Cloruro de Tolonio/metabolismo , Diente/citología , Diente/ultraestructuraRESUMEN
Microstructure of the teeth of the sea urchin Lytechinus variegatus was investigated using optical microscopy, SEM (scanning electron microscopy) and SIMS (secondary ion mass spectroscopy). The study focused on the internal structure of the first-stage mineral structures of high Mg calcite (primary, secondary and carinar process plates, prisms) and on morphology of the columns of second-stage mineral (very high Mg calcite) that cement the first-stage material together. Optical micrographs under polarized light revealed contrast in the centers (midlines) of carinar process plates and in prisms in polished sections; staining of primary and carinar process plates revealed significant dye uptake at the plate centers. Demineralization with and without fixation revealed that the midlines of primary and carinar process plates (but not secondary plates) and the centers of prisms differed from the rest of the plate or prism, and SIMS showed proteins concentrated in these plate centers. SEM was used to study the morphology of columns, the fracture surfaces of mature teeth and the 3D morphology of prisms. These observations of internal structures in plates and prisms offer new insight into the mineralization process and suggest an important role for protein inclusions within the first-stage mineral. Some of the 3D structures not reported previously, such as twisted prisms and stacks of carinar process plates with nested wrinkles, may represent structural strengthening strategies.
Asunto(s)
Carbonato de Calcio/química , Lytechinus/anatomía & histología , Lytechinus/química , Diente/anatomía & histología , Diente/química , Animales , Lytechinus/ultraestructura , Microscopía Electrónica de Rastreo , Diente/ultraestructuraRESUMEN
The performance requirements of ciliary band feeding explain the convoluted forms of many marine invertebrate larvae. Convolutions increase surface area and therefore feeding rates per unit body volume. We review recent advances in morphology, neural development, and behavior at settlement of the echinoid Lytechinus pictus and provide new ultrastructural and expression data on larvae of its congener, L. variegatus. Larvae of the echinometrid Colobocentrotus atratus contain neurons identified by their expression of nitric oxide synthase (NOS), indicating that this character is not unique to Lytechinus. We hypothesize that in some echinoids the convoluted shape of the post-oral vibratile lobe (POVL) covaries with the distribution of identified sensory neurons to enable olfaction during settlement. An analysis of variation in structural elaboration of the post-oral transverse ciliary band (PTB) within Echinoida and in feeding larvae of other echinoderm classes indicates that only echinoids, but not all echinoids, possess this novel character; larvae that do are distributed heterogeneously within the class. In recognition of this specialized function for the POVL and surrounding ectoderm, and because it is lobate and grows toward the mouth, we propose naming this structure the adoral lobe.
Asunto(s)
Lytechinus/ultraestructura , Neuronas Receptoras Olfatorias/ultraestructura , Animales , Evolución Biológica , Larva/fisiología , Larva/ultraestructura , Lytechinus/fisiología , Óxido Nítrico Sintasa/metabolismo , Neuronas Receptoras Olfatorias/fisiologíaRESUMEN
The calcite plates and prisms in Lytechinus variegatus teeth form a complex biocomposite and employ a myriad of strengthening and toughening strategies. These crystal elements have macromolecule-containing internal cavities that may act to prevent cleavage. Transmission electron microscopy employing a small objective aperture was used to quantify several characteristics of these cavities. Cavity diameters ranged from 10 to 225 nm, the mean cavity diameter was between 50 and 60 nm, and cavities comprised approximately 20% of the volume of the crystal. Some cavities exhibited faceting and trace analysis identified these planes as being predominately of {1014} type. Through focus series of micrographs show the cavities were homogeneously distributed throughout the foil. The electron beam decomposed a substance within cavities and this suggests that these cavities are filled with a hydrated organic phase.