Characterization of Microfibrillar-associated Protein 4 (MFAP4) as a Tropoelastin- and Fibrillin-binding Protein Involved in Elastic Fiber Formation

MFAP4 (microfibrillar-associated protein 4) is an extracellular glycoprotein found in elastic fibers without a clearly defined role in elastic fiber assembly. In the present study, we characterized molecular interactions between MFAP4 and elastic fiber components. We established that MFAP4 primarily assembles into trimeric and hexameric structures of homodimers. Binding analysis revealed that MFAP4 specifically binds tropoelastin and fibrillin-1 and -2, as well as the elastin cross-linking amino acid desmosine, and that it co-localizes with fibrillin-1-positive fibers in vivo. Site-directed mutagenesis disclosed residues Phe(241) and Ser(203) in MFAP4 as being crucial for type I collagen, elastin, and tropoelastin binding. Furthermore, we found that MFAP4 actively promotes tropoelastin self-assembly. In conclusion, our data identify MFAP4 as a new ligand of microfibrils and tropoelastin involved in proper elastic fiber organization

Fibrillin-1 and -2 differentially modulate endogenous TGF-β and BMP bioavailability during bone formation

Extracellular microfibrils composed of fibrillin-1 and -2 regulate bone formation through modulation of TGF-β and BMP signaling

AMACO is a component of the basement membrane-associated fraser complex

Fraser syndrome (FS) is a phenotypically variable, autosomal recessive disorder characterized by cryptophthalmus, cutaneous syndactyly, and other malformations resulting from mutations in FRAS1, FREM2, and GRIP1. Transient embryonic epidermal blistering causes the characteristic defects of the disorder. Fras1, Frem1, and Frem2 form the extracellular Fraser complex, which is believed to stabilize the basement membrane. However, several cases of FS could not be attributed to mutations in FRAS1, FREM2, or GRIP1, and FS displays high clinical variability, suggesting that there is an additional genetic, possibly modifying contribution to this disorder. An extracellular matrix protein containing VWA-like domains related to those in matrilins and collagens (AMACO), encoded by the VWA2 gene, has a very similar tissue distribution to the Fraser complex proteins in both mouse and zebrafish. Here, we show that AMACO deposition is lost in Fras1-deficient zebrafish and mice and that Fras1 and AMACO interact directly via their chondroitin sulfate proteoglycan (CSPG) and P2 domains. Knockdown of vwa2, which alone causes no phenotype, enhances the phenotype of hypomorphic Fras1 mutant zebrafish. Together, our data suggest that AMACO represents a member of the Fraser complex

Modeling autosomal recessive cutis laxa type 1C in mice reveals distinct functions for Ltbp-4 isoforms

Recent studies have revealed an important role for LTBP-4 in elastogenesis. Its mutational inactivation in humans causes autosomal recessive cutis laxa type 1C (ARCL1C), which is a severe disorder caused by defects of the elastic fiber network. Although the human gene involved in ARCL1C has been discovered based on similar elastic fiber abnormalities exhibited by mice lacking the short Ltbp-4 isoform (Ltbp4S(-/-)), the murine phenotype does not replicate ARCL1C. We therefore inactivated both Ltbp-4 isoforms in the mouse germline to model ARCL1C. Comparative analysis of Ltbp4S(-/-) and Ltbp4-null (Ltbp4(-/-)) mice identified Ltbp-4L as an important factor for elastogenesis and postnatal survival, and showed that it has distinct tissue expression patterns and specific molecular functions. We identified fibulin-4 as a previously unknown interaction partner of both Ltbp-4 isoforms and demonstrated that at least Ltbp-4L expression is essential for incorporation of fibulin-4 into the extracellular matrix (ECM). Overall, our results contribute to the current understanding of elastogenesis and provide an animal model of ARCL1C.Peer reviewe

Modeling autosomal recessive cutis laxa type 1C in mice reveals distinct functions for Ltbp-4 isoforms

Recent studies have revealed an important role for LTBP-4 in elastogenesis. Its mutational inactivation in humans causes autosomal recessive cutis laxa type 1C (ARCL1C), which is a severe disorder caused by defects of the elastic fiber network. Although the human gene involved in ARCL1C has been discovered based on similar elastic fiber abnormalities exhibited by mice lacking the short Ltbp-4 isoform (Ltbp4S(-/-)), the murine phenotype does not replicate ARCL1C. We therefore inactivated both Ltbp-4 isoforms in the mouse germline to model ARCL1C. Comparative analysis of Ltbp4S(-/-) and Ltbp4-null (Ltbp4(-/-)) mice identified Ltbp-4L as an important factor for elastogenesis and postnatal survival, and showed that it has distinct tissue expression patterns and specific molecular functions. We identified fibulin-4 as a previously unknown interaction partner of both Ltbp-4 isoforms and demonstrated that at least Ltbp-4L expression is essential for incorporation of fibulin-4 into the extracellular matrix (ECM). Overall, our results contribute to the current understanding of elastogenesis and provide an animal model of ARCL1C.Peer reviewe

(EIN)FACH? : Komplexität, Wissen, Fortschritt und die Grenzen der Germanistik

Spätestens seit den gesellschaftlichen Modernisierungsschüben in den sechziger Jahren identifiziert auch die Germanistik Erkenntnis- und Wissenszuwachs, ja allgemeiner den "Fortschritt" ihres Fachs, mit Komplexitätserhöhung. Vor diesem Hintergrund erscheint es mir wenig plausibel, die seitdem erfolgten inneren Ausdifferenzierungen und interdisziplinären Grenzüberschreitungen als durch Identitätsverlust, Zerstreuung und Desintegration gekennzeichnete Niedergangsszenarien zu beschreiben. Die Veränderungen gehorchen der immanenten Logik germanistischer Forschung, einer "disziplinierten", auf Leistung ausgerichteten, an kooperativen Großforschungsvorhaben partizipierenden Wissensproduktion

The C313Y Piedmontese mutation decreases myostatin covalent dimerisation and stability

<p>Abstract</p> <p>Background</p> <p>Myostatin is a key negative regulator of muscle growth and development, whose activity has important implications for the treatment of muscle wastage disorders. Piedmontese cattle display a double-muscled phenotype associated with the expression of C313Y mutant myostatin. <it>In vivo</it>, C313Y myostatin is proteolytically processed, exported and circulated extracellularly but fails to correctly regulate muscle growth. The C313Y mutation removes the C313-containing disulphide bond, an integral part of the characteristic TGF-β cystine-knot structural motif.</p> <p>Results</p> <p>Here we present <it>in vitro </it>analysis of the structure and stability of the C313Y myostatin protein that reveals significantly decreased covalent dimerisation for C313Y myostatin accompanied by a loss of structural stability compared to wild type. The C313Y myostatin growth factor, processed from full length precursor protein, fails to inhibit C2C12 myoblast proliferation in contrast to wild type myostatin. Although structural modeling shows the substitution of tyrosine causes structural perturbation, biochemical analysis of additional disulphide mutants, C313A and C374A, indicates that an intact cystine-knot motif is a major determinant in myostatin growth factor stability and covalent dimerisation.</p> <p>Conclusions</p> <p>This research shows that the cystine-knot structure is important for myostatin dimerisation and stability, and that disruption of this structural motif perturbs myostatin signaling.</p

Micro-computed tomography and histology to explore internal morphology in decapod larvae

Traditionally, the internal morphology of crustacean larvae has been studied using destructive techniques such as dissection and microscopy. The present study combines advances in microcomputed tomography (micro-CT) and histology to study the internal morphology of decapod larvae, using the common spider crab (Maja brachydactyla Balss, 1922) as a model and resolving the individual limitations of these techniques. The synergy of micro-CT and histology allows the organs to be easily identified, revealing simultaneously the gross morphology (shape, size, and location) and histological organization (tissue arrangement and cell identification). Micro-CT shows mainly the exoskeleton, musculature, digestive and nervous systems, and secondarily the circulatory and respiratory systems, while histology distinguishes several cell types and confirms the organ identity. Micro-CT resolves a discrepancy in the literature regarding the nervous system of crab larvae. The major changes occur in the metamorphosis to the megalopa stage, specifically the formation of the gastric mill, the shortening of the abdominal nerve cord, the curving of the abdomen beneath the cephalothorax, and the development of functional pereiopods, pleopods, and lamellate gills. The combination of micro-CT and histology provides better results than either one alone.Financial support was provided by the Spanish Ministry of Economy and Competitiveness through the INIA project (grant number RTA2011-00004-00-00) to G.G. and a pre-doctoral fellowship to D.C. (FPI-INIA)

Activation of latent human GDF9 by a single residue change (Gly(391)Arg) in the mature domain

Growth differentiation factor 9 (GDF9) controls granulosa cell growth and differentiation during early ovarian folliculogenesis and regulates cumulus cell function and ovulation rate in the later stages of this process. Similar to other TGF-β superfamily ligands, GDF9 is secreted from the oocyte in a noncovalent complex with its prodomain. In this study, we show that prodomain interactions differentially regulate the activity of GDF9 across species, such that murine (m) GDF9 is secreted in an active form, whereas human (h) GDF9 is latent. To understand this distinction, we used site-directed mutagenesis to introduce nonconserved mGDF9 residues into the pro- and mature domains of hGDF9. Activity-based screens of the resultant mutants indicated that a single mature domain residue (Gly³⁹¹) confers latency to hGDF9. Gly³⁹¹ forms part of the type I receptor binding site on hGDF9, and this residue is present in all species except mouse, rat, hamster, galago, and possum, in which it is substituted with an arginine. In an adrenocortical cell luciferase assay, hGDF9 (Gly³⁹¹Arg) had similar activity to mGDF9 (EC₅₀ 55 ng/ml vs. 28 ng/ml, respectively), whereas wild-type hGDF9 was inactive. hGDF9 (Gly³⁹¹Arg) was also a potent stimulator of murine granulosa cell proliferation (EC₅₀ 52 ng/ml). An arginine at position 391 increases the affinity of GDF9 for its signaling receptors, enabling it to be secreted in an active form. This important species difference in the activation status of GDF9 may contribute to the variation observed in follicular development, ovulation rate, and fecundity between mammals.Courtney M. Simpson, Peter G. Stanton, Kelly L. Walton, Karen L. Chan, Lesley J. Ritter, Robert B. Gilchrist, and Craig A. Harriso