Microfibrils at basement membrane zones interact with perlecan via fibrillin-1

Mutational defects in fibrillin-rich microfibrils give rise to a number of heritable connective tissue disorders, generally termed microfibrillopathies. To understand the pathogenesis of these microfibrillopathies, it is important to elucidate the supramolecular composition of microfibrils and their interaction properties with extracellular matrix components. Here we demonstrate that the proteoglycan perlecan is an associated component of microfibrils typically close to basement membrane zones. Double immunofluorescence studies demonstrate colocalization of fibrillin-1, the major backbone component of microfibrils, with perlecan in fibroblast cultures as well as in dermal and ocular tissues. Double immunogold labeling further confirms colocalization of perlecan to microfibrils in various tissues at the ultrastructural level. Extraction studies revealed that perlecan is not covalently associated with microfibrils. High affinity interactions between fibrillin-1 and perlecan were found by kinetic binding studies with dissociation constants in the low nanomolar range. A detailed mapping study of the interaction epitopes by solid phase binding assays primarily revealed interactions of perlecan domains I and II with a central region of fibrillin-1. Analysis of perlecan null embryos showed less microfibrils at the dermal-epidermal junction as compared with wild-type littermates. The data presented indicate a functional significance for perlecan in anchoring microfibrils to basement membranes and in the biogenesis of microfibrils.     

Molecular structure and interaction of recombinant human type XVI collagen

Collagen XVI is a minor component of at least two different extracellular fibrillar networks of specialized regions of skin and cartilage. In skin, collagen XVI is integrated into particular fibrillin-rich microfibrils lacking an amorphous elastin core. In cartilage, collagen XVI is a component of small heterotypic D-banded fibrils, mainly occurring in the territorial matrix of chondrocytes. Here, we present the first direct evidence for the molecular structure and functional properties of these fibril-associated collagens with interrupted triple helices (FACIT). We have expressed recombinantly the full-length alpha1 chain of human collagen XVI in HEK 293 EBNA cells in large quantities using an episomal expression system. Secreted full-length recombinant collagen XVI forms stable disulfide-bonded homotrimers and is rapidly proteolytically processed to distinct fragments at specific protease sequence motifs, one resembling an aggrecanase recognition site. Limited trypsin digestion assays and thermal transition curves imply sequential thermal denaturation of individual triple helical domains of this recombinant collagen, similar to authentic collagen XVI. Molecular images of collagen XVI reveal rod-like molecules which harbor multiple sharp kinks attributing a highly flexible structure presumably introduced by non-collagenous (NC) regions. Terminally located cloverleaf-shaped nodules correspond to the large NC NC11 domain of trimeric collagen XVI. The total length of individual trimeric recombinant collagen XVI molecules constitutes about 240 nm as calculated by atomic force and negative staining electron microscopy. Recombinant collagen XVI interacts with fibrillin-1 and with fibronectin indicating multiple molecular interactions in which this ubiquitously expressed and versatile FACIT-collagen can participate. In vitro generated collagen XVI provides an indispensable tool for future determination of its function during supramolecular assembly of matrix aggregates and its role in maintenance, organization and interaction of fibrillar structures.     

Wirkung von Sulfhydrylverbindungen auf embryonale Induktionsfaktoren

Zusammenfassung Rohfraktionen aus 9 Tage alten Hühnerembryonen, die neuralisierenden und mesodermalisierenden Induktionsfaktor enthielten, sowie angereicherter mesodermalisierender Faktor wurden mit Thioglykolsäure sowie mit 2-Mercaptoäthanol behandelt. Die Fraktionen wurden an Gastrulen vonTriturus alpestris oderAmbystoma nach der Implantationsmethode getestet. Der mesodermalisierende Faktor wird inaktiviert. Die Aktivität des neuralisierenden Faktors bleibt dagegen erhalten.

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Action of sulfhydryl compounds on embryonic inducing factors

Summary Crude extracts from 9 days old chicken embryos containing neuralizing and mesodermalizing inducing factors as well as purified mesodermalizing factor were incubated with thioglycolic acid and with 2-Mercaptoethanol. The fractions were tested by implanting into early gastrulae ofTriturus orAmbystoma. The mesodermalizing factor is inactivated whereas the neuralizing factor does not lose its activity.

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Der Deutschen Forschungsgemeinschaft danken wir für Unterstützung der Arbeit.     

Affinity chromatography of embryonic inducing factors on heparin-Sepharose

Mesoderm-inducing factors were extracted from chicken embryos and partially purified by chromatography on DEAE-cellulose. The DEAE-cellulose eluate was applied to heparin-Sepharose. Most of the proteins are not bound to heparin. The adsorbed proteins were eluted with a linear NaCl gradient. In totipotent gastrula ectoderm of amphibians the eluted proteins induce the differentiation of muscle and notochord as well as of large masses of renal tubules and blood cells. A possible relationship to fibroblast growth factors and angiogenesis factor is discussed.     

Isolation of plasma membranes from Xenopus embryos

Summary Plasma membranes were isolated in high yield from Xenopus gastrulae by repeated sedimentation in discontinuous sucrose gradients. Most of the yolk was separated by lowspeed sedimentation before centrifugation on the discontinuous sucrose gradients. The isolation of plasma membranes was followed by covalent labelling of the surface of dissociated gastrula cells with diazoniobenzene sulphonate, by electron microscopy and the distribution of enzymatic markers. The isolated plasma membranes have a low neural inducing activity as compared to other cell constituents.     

Neural-inducing activity of nuclei and nuclear fractions from Xenopus laevis embryos

Summary From embryos (Xenopus laevis) of different developmental stages nuclei were isolated which exert neural inducing activity in the biological test. The active material could partly be extracted from the nuclei. Experiments for the isolation of nuclear ribonucleoprotein (RNP) particles have shown that the activity is localized at least in part in these particles. On the other hand, some neural inducer is not detached from chromatin and the nuclear matrix even with ionic detergents. Inducing activity was found in germinal vesicles and to a higher degree in the cytoplasm of oocytes, but in a masked, biologically inactive state.