Interfibrillar stiffening of echinoderm mutable collagenous tissue demonstrated at the nanoscale

The mutable collagenous tissue (MCT) of echinoderms (e.g., sea cucumbers and starfish) is a remarkable example of a biological material that has the unique attribute, among collagenous tissues, of being able to rapidly change its stiffness and extensibility under neural control. However, the mechanisms of MCT have not been characterized at the nanoscale. Using synchrotron small-angle X-ray diffraction to probe time-dependent changes in fibrillar structure during in situ tensile testing of sea cucumber dermis, we investigate the ultrastructural mechanics of MCT by measuring fibril strain at different chemically induced mechanical states. By measuring a variable interfibrillar stiffness (E(IF)), the mechanism of mutability at the nanoscale can be demonstrated directly. A model of stiffness modulation via enhanced fibrillar recruitment is developed to explain the biophysical mechanisms of MCT. Understanding the mechanisms of MCT quantitatively may have applications in development of new types of mechanically tunable biomaterials

Ancient role of vasopressin/oxytocin-type neuropeptides as regulators of feeding revealed in an echinoderm.

BACKGROUND: Vasopressin/oxytocin (VP/OT)-type neuropeptides are well known for their roles as regulators of diuresis, reproductive physiology and social behaviour. However, our knowledge of their functions is largely based on findings from studies on vertebrates and selected protostomian invertebrates. Little is known about the roles of VP/OT-type neuropeptides in deuterostomian invertebrates, which are more closely related to vertebrates than protostomes. RESULTS: Here, we have identified and functionally characterised a VP/OT-type signalling system comprising the neuropeptide asterotocin and its cognate G-protein coupled receptor in the starfish (sea star) Asterias rubens, a deuterostomian invertebrate belonging to the phylum Echinodermata. Analysis of the distribution of asterotocin and the asterotocin receptor in A. rubens using mRNA in situ hybridisation and immunohistochemistry revealed expression in the central nervous system (radial nerve cords and circumoral nerve ring), the digestive system (including the cardiac stomach) and the body wall and associated appendages. Informed by the anatomy of asterotocin signalling, in vitro pharmacological experiments revealed that asterotocin acts as a muscle relaxant in starfish, contrasting with the myotropic actions of VP/OT-type neuropeptides in vertebrates. Furthermore, in vivo injection of asterotocin had a striking effect on starfish behaviour-triggering fictive feeding where eversion of the cardiac stomach and changes in body posture resemble the unusual extra-oral feeding behaviour of starfish. CONCLUSIONS: We provide a comprehensive characterisation of VP/OT-type signalling in an echinoderm, including a detailed anatomical analysis of the expression of both the VP/OT-type neuropeptide asterotocin and its cognate receptor. Our discovery that asterotocin triggers fictive feeding in starfish provides important new evidence of an evolutionarily ancient role of VP/OT-type neuropeptides as regulators of feeding in animals

Ferritin Assembly in Enterocytes of Drosophila melanogaster

Ferritins are protein nanocages that accumulate inside their cavity thousands of oxidized iron atoms bound to oxygen and phosphates. Both characteristic types of eukaryotic ferritin subunits are present in secreted ferritins from insects, but here dimers between Ferritin 1 Heavy Chain Homolog (Fer1HCH) and Ferritin 2 Light Chain Homolog (Fer2LCH) are further stabilized by disulfide-bridge in the 24-subunit complex. We addressed ferritin assembly and iron loading in vivo using novel transgenic strains of Drosophila melanogaster. We concentrated on the intestine, where the ferritin induction process can be controlled experimentally by dietary iron manipulation. We showed that the expression pattern of Fer2LCH-Gal4 lines recapitulated iron-dependent endogenous expression of the ferritin subunits and used these lines to drive expression from UAS-mCherry-Fer2LCH transgenes. We found that the Gal4-mediated induction of mCherry-Fer2LCH subunits was too slow to effectively introduce them into newly formed ferritin complexes. Endogenous Fer2LCH and Fer1HCH assembled and stored excess dietary iron, instead. In contrast, when flies were genetically manipulated to co-express Fer2LCH and mCherry-Fer2LCH simultaneously, both subunits were incorporated with Fer1HCH in iron-loaded ferritin complexes. Our study provides fresh evidence that, in insects, ferritin assembly and iron loading in vivo are tightly regulated

Neuroblast and ganglion mother cells populations and neuronal axons derived from embryonic neurons are affected in ferritin mutants.

<p>Neuroblast populations, as marked by Dpn (A, control) are affected in ferritin mutants (B-D). Ganglion mother cells (E, control) are affected in ferritin mutants (F-H). Axons emanating from the brain and ventral nerve cord have a stereotyped pattern in normal development (I). In ferritin mutants, the axons form but are disorganized (J-L).</p

<i>Fer1HCH⁴⁵¹ lacZ</i> enhancer trap is expressed in the embryonic CNS.

<p>Using an antibody against Anti- Beta-Galactosidase (green) and an antibody against the neuronal marker Elav (red), colocalization is observed in (A) heterozygous <i>Fer1HCH^451/+</i> and (B) homozygous <i>Fer1HCH⁴⁵¹</i> embryos. Ferritin homozygous mutant embryo has abnormally shaped and separated nervous system.</p

Ferritin Is Required in Multiple Tissues during <i>Drosophila melanogaster</i> Development

<div><p>In <i>Drosophila melanogaster</i>, iron is stored in the cellular endomembrane system inside a protein cage formed by 24 ferritin subunits of two types (Fer1HCH and Fer2LCH) in a 1:1 stoichiometry. In larvae, ferritin accumulates in the midgut, hemolymph, garland, pericardial cells and in the nervous system. Here we present analyses of embryonic phenotypes for mutations in <i>Fer1HCH</i>, <i>Fer2LCH</i> and in both genes simultaneously. Mutations in either gene or deletion of both genes results in a similar set of cuticular embryonic phenotypes, ranging from non-deposition of cuticle to defects associated with germ band retraction, dorsal closure and head involution. A fraction of ferritin mutants have embryonic nervous systems with ventral nerve cord disruptions, misguided axonal projections and brain malformations. Ferritin mutants die with ectopic apoptotic events. Furthermore, we show that ferritin maternal contribution, which varies reflecting the mother’s iron stores, is used in early development. We also evaluated phenotypes arising from the blockage of COPII transport from the endoplasmic reticulum to the Golgi apparatus, feeding the secretory pathway, plus analysis of ectopically expressed and fluorescently marked Fer1HCH and Fer2LCH. Overall, our results are consistent with insect ferritin combining three functions: iron storage, intercellular iron transport, and protection from iron-induced oxidative stress. These functions are required in multiple tissues during <i>Drosophila</i> embryonic development.</p></div

Ferritin mutants result in CNS phenotypes, as revealed by ɑ-Elav staining.

<p>(A) Mutant CNS (B-H) appear twisted and irregular (E, G); often, holes are seen within the ventral nerve cord (white arrows). Holes can range in sizes from small, partially (B, C) or completely (H) interrupting the ventral nerve cord, to large (F). There can also be multiple holes (D), as compared to wild type. Embryonic brains are also disrupted (F, opposite white arrow). Embryos are photographed at stages 14–15. (I) Quantification and distribution of CNS defects in different ferritin mutants that present CNS defects; statistical difference compared to control following a Chi squared test, at p<0.0001, and is denoted by an asterisk (n = 32, 10, and 138 embryos, respectively).</p

Marked ferritin accumulation in embryos with different genetic backgrounds.

<p><i>Fer1HCH</i> protein was visualized in all embryos using the <i>Fer1HCH^G188</i> GFP trap line (green). In stage 17 <i>Fer1HCH^G188/+</i> embryos that successfully complete development, ferritin accumulates mainly in the midgut and hemocytes. (A) The GFP-Fer1HCH signal is found in hemocytes marked by <i>cg</i> driving nuclear RFP, but not (B) in the fat bodies marked by the fat body driver FB driving nuclear RFP. (C-F) <i>Fer1HCH^G188</i> homozygous embryos, which die like other ferritin mutants, also show intestinal ectopically localized ferritin accumulation (D) and much reduced hemocyte ferritin accumulation (F), compared to heterozygous controls (C, E). (G, H) Blocking the secretory pathway using a homozygous mutant <i>sec23^j13C8</i> background leads to a decrease of ferritin levels in the hemocytes (green arrowheads),; and ectopic accumulation in the intestine (white arrowheads). Intensity of GFP::Fer1HCH flurescence is shown in the left side of panels G and H using a fire scale. The fire scales are shown in the lower right side of each, with warmer colors (red-orange-yellow-white) denoting higher intensity of fluorescence, and colder colors (purple-blue) denoting lower levels of fluorescence for the green channel. Note higher fluorescence levels in the <i>sec23</i> homozygous mutant embryo (H). A merged image showing GFP::Fer1HCH fluorescence and control RFP staining (G) due to the balancer chromosome is shown to the right side of each panel.</p

Ferritin mutants cause apoptosis in the CNS and other tissues.

<p>(A-D) Whole embryos were stained with an α-CSP3act marking apoptotic cells (green), and an α-Elav marking neurons (red). Ectopic apoptosis was observed in ferritin mutants from stage 12 onwards; at this stage it was mostly restricted to the neurogenic region (B). At stage 15 apoptosis covers most mutant embryonic tissues (D). Quantification of the mean intensity value on CSP3act staining in control and ferritin mutants at stage 15 show a significant difference, with higher levels of staining in mutant embryos (n = 5; T-test, p = 0.0113).</p

Ferritin genes interact genetically with the DMT1 homolog <i>Mvl</i>.

<p>(A) The <i>Mvl^97f-LacZ</i> line shows a spatially restricted expression pattern for <i>Mvl</i>, mainly in the head region, the brain, and a segmentally repeated pattern. (B) In a ferritin homozygous mutant background, <i>Mvl^97f-LacZ</i> expression increases. Black arrows denote the head region, white asterisk the embryonic brain, red asterisk the ventral nerve cord, and red arrows mark the segmented expression pattern. Introduction of a <i>Mvl^97f</i> allele into a ferritin mutant background resulted in the appearance of necrotic patches in the cuticle (C, D). (E) Quantification of the total area covered by LacZ staining in control and ferritin mutant backgrounds, (p<0.0001; T-test, asterisk). (F) Quantification of the number of embryos showing necrotic patches with one or two <i>Mvl^97f</i> alleles; statistical difference using a Chi squared test with p<0.0001 is shown by an asterisk.</p