The structure of mollusc larval shells formed in the presence of the chitin synthase inhibitor Nikkomycin Z

Background Chitin self-assembly provides a dynamic extracellular biomineralization interface. The insoluble matrix of larval shells of the marine bivalve mollusc Mytilus galloprovincialis consists of chitinous material that is distributed and structured in relation to characteristic shell features. Mollusc shell chitin is synthesized via a complex transmembrane chitin synthase with an intracellular myosin motor domain. Results Enzymatic mollusc chitin synthesis was investigated in vivo by using the small-molecule drug NikkomycinZ, a structural analogue to the sugar donor substrate UDP-N-acetyl-D-glucosamine (UDP-GlcNAc). The impact on mollusc shell formation was analyzed by binocular microscopy, polarized light video microscopy in vivo, and scanning electron microscopy data obtained from shell material formed in the presence of NikkomycinZ. The partial inhibition of chitin synthesis in vivo during larval development by NikkomycinZ (5 μM – 10 μM) dramatically alters the structure and thus the functionality of the larval shell at various growth fronts, such as the bivalve hinge and the shell's edges. Conclusion Provided that NikkomycinZ mainly affects chitin synthesis in molluscs, the presented data suggest that the mollusc chitin synthase fulfils an important enzymatic role in the coordinated formation of larval bivalve shells. It can be speculated that chitin synthesis bears the potential to contribute via signal transduction pathways to the implementation of hierarchical patterns into chitin mineral-composites such as prismatic, nacre, and crossed-lamellar shell types

The structure of mollusc larval shells formed in the presence of the chitin synthase inhibitor Nikkomycin Z

Background: Chitin self-assembly provides a dynamic extracellular biomineralization interface. The insoluble matrix of larval shells of the marine bivalve mollusc Mytilus galloprovincialis consists of chitinous material that is distributed and structured in relation to characteristic shell features. Mollusc shell chitin is synthesized via a complex transmembrane chitin synthase with an intracellular myosin motor domain. Results: Enzymatic mollusc chitin synthesis was investigated in vivo by using the small-molecule drug NikkomycinZ, a structural analogue to the sugar donor substrate UDP-N-acetyl-D-glucosamine (UDP-GlcNAc). The impact on mollusc shell formation was analyzed by binocular microscopy, polarized light video microscopy in vivo, and scanning electron microscopy data obtained from shell material formed in the presence of NikkomycinZ. The partial inhibition of chitin synthesis in vivo during larval development by NikkomycinZ (5 μM — 10 μM) dramatically alters the structure and thus the functionality of the larval shell at various growth fronts, such as the bivalve hinge and the shell's edges. Conclusion: Provided that NikkomycinZ mainly affects chitin synthesis in molluscs, the presented data suggest that the mollusc chitin synthase fulfils an important enzymatic role in the coordinated formation of larval bivalve shells. It can be speculated that chitin synthesis bears the potential to contribute via signal transduction pathways to the implementation of hierarchical patterns into chitin mineral-composites such as prismatic, nacre, and crossed-lamellar shell types

Dictyostelium discoideum als Expressionssystem für die transmembrane Myosin-Chitinsynthase Ar-CS1 aus Atrina rigida (Mollusca, Bivalvia) - einem Modellorganismus der Biomineralisation - und Charakterisierung der Myosindomäne

Chitin spielt möglicherweise eine funktionelle Rolle bei der Schalenentwicklung von Mollusken. Man vermutet, dass an der Grenzfläche zwischen den Mantelepithelzellen und der mineralisierenden Schale physikalische Kräfte auftreten. Es stellt sich die Frage, ob eine transmembrane Myosin-Chitinsynthase kolloidale Scherkräfte zwischen der wachsenden Schale und dem Cytoskelett der Mantelepithelzellen detektieren und ausgleichen kann, und damit die Assemblierung des Chitins und indirekt die Kristallisation von amorphen Calciumcarbonat beeinflusst werden könnte. Derzeit ist noch nicht geklärt, wie die hoch konservierten Myosin-Chitinsynthasen der Mollusken bezüglich der Familien der Myosine und der Glykosyltransferasen zu klassifizieren sind. Die Myosindomänen der Chitinsynthasen der Mollusken sind phylogenetisch von den bisher bekannten Myosinklassen getrennt. Die komplexe Transmembranarchitektur der Chitinsynthasen von Mollusken und Insekten wurde selbst bei Pilzen noch nicht beschrieben. Eine detaillierte Charakterisierung der Myosin-Chitinsynthasen von Mollusken am Beispiel der Ar-CS1 ist also eine wichtige Voraussetzung, um die Bildung von komplexen mineralisierten Strukturen, die auf Chitin basieren, wie z.B. Perlmutt verstehen zu können. In dieser Arbeit werden die ersten molekularen und biochemischen Studien der Myosindomäne der Ar-CS1 vorgestellt. Da die Übergangsregion niedriger Komplexität ionisch wechselwirken könnte, wurde die Myosindomäne der Ar-CS1 mit und ohne diese Region kloniert. Für die Untersuchung der Funktion einer mutmaßlichen Regulationsstelle wurde die Myosindomäne der Ar-CS1 auch durch ortsspezifische Mutationen verändert. Dictyostelium discoideum zeigte sich als geeignetes Expressionssystem. Die Verteilung der Myosindomänen der Ar-CS1 innerhalb der D. discoideum-Zellen wurde mittels Immunfluoreszenztechniken untersucht. Alle Varianten der Myosindomänen der Ar-CS1 waren im Cytoplasma lokalisiert. Standardreinigungsprotokolle für Myosine waren im Falle der Myosindomäne der Ar-CS1 nicht erfolgreich. Prinzipiell gelten die aus dem Cytoskelett extrahierten Myosindomänen als funktionell. Es zeigte sich, dass die Myosindomäne nur in Gegenwart von Harnstoff oder Kaliumiodid, die vermutlich auf hydrophobe Wechselwirkungen wirken, gelöst werden konnten. Folgendes Protokoll hat sich zur Aufreinigung rekombinanter Myosindomänen der Ar-CS1 bewährt: Zellen werden ohne Detergenz lysiert und mittels Ultrazentrifugation fraktioniert. Der lösliche Extrakt wurde gegen einen Puffer mit KI dialysiert und einer Ni-NTA-Chromatographie unterzogen. Ein 90 kDa Protein, das im Eluat Hauptbestandteil war, wurde mittels MALDI als Myosindomäne der Ar-CS1 identifiziert. Diese Arbeit mit der präparativen Isolierung der Myosindomänen legt den Grundstein für weitere biochemische und biophysikalische Untersuchungen der regulatorischen Funktionen und der Prozessivität und der Kraftentfaltung dieses molekularen Motors. Zweites Ziel dieser Arbeit war die Etablierung von D. discoideum-Zelllinien für die Untersuchung der von Ar-CS1 katalysierten Chitinsynthese in vivo ähnlich zu den Bedingungen in den Mantelepithelzellen. Bisher wurde in der Literatur noch nicht die heterologe Expression eines komplexen Membranproteins wie der Ar-CS1 beschrieben. Ar-CS1 mit einem Fluoreszenztag wurde in Ax3 orf+ sowie in Cellulossynthase defiziente dcsA- transformiert. In ersten Analysen wiesen die Ar-CS1 exprimierenden Zelllinien reproduzierbar Fluoreszenzsignale in zwei verschiedenen Mustern auf: über das gesamte Cytoplasma verteilt oder in globulären Strukturen akkumuliert. Ar-CS1-YFP kommt auch im Zellcortex und in Filopodien vor. Basierend auf diesen Beobachtungen kann man eine direkte Wechselwirkung mit den Aktincytoskelett annehmen. Zudem wurde Ar-CS1-YFP in Plasmamembranfraktionen nachgewiesen. Aktivität wurde in vitro in radioaktiv markierten Chitinpolymeren untersucht. Zudem wurden schwache Signale an der Oberfläche von in Gegenwart von UDP-GlcNAc kultivierten Zelllinien und in deren filtrierten Kulturüberstand mittels Calcofluor White und mit GFP-getaggten Chitinbindeproteinen detektiert. Dies deutet auf die Fähigkeit der Zelllinien, prinzipiell Chitinfragmente zu bilden, die vorwiegend in das Medium abgegeben werden, hin. AFM Studien der Oberfläche von Ar-CS1-YFP exprimierenden Zellen zeigte eine körnige Feinstruktur, die auf Wildtypzellen nicht beobachtet werden konnte. Insgesamt ist D. discoideum vermutlich für eine funktionelle Expression der Ar-CS1 geeignet. Gegenstand weiterer Studien bleibt, wie die Rate der Chitinsynthese reguliert wird und ob die Myosindomäne eher für die Ausbildung oder Detektion der Kräfte als für den Transport wie andere unkonventionelle Myosine dient. Die neuen Zelllinien, die in dieser Arbeit hergestellt wurden, sind ein vielversprechendes Modellsystem, um enzymatische Einblicke in chitin-abhängige Biomineralisationsprozesse zu gewinnen

The structure of mollusc larval shells formed in the presence of the chitin synthase inhibitor Nikkomycin Z

Background Chitin self-assembly provides a dynamic extracellular biomineralization interface. The insoluble matrix of larval shells of the marine bivalve mollusc Mytilus galloprovincialis consists of chitinous material that is distributed and structured in relation to characteristic shell features. Mollusc shell chitin is synthesized via a complex transmembrane chitin synthase with an intracellular myosin motor domain. Results Enzymatic mollusc chitin synthesis was investigated in vivo by using the small-molecule drug NikkomycinZ, a structural analogue to the sugar donor substrate UDP-N-acetyl-D-glucosamine (UDP-GlcNAc). The impact on mollusc shell formation was analyzed by binocular microscopy, polarized light video microscopy in vivo, and scanning electron microscopy data obtained from shell material formed in the presence of NikkomycinZ. The partial inhibition of chitin synthesis in vivo during larval development by NikkomycinZ (5 μM – 10 μM) dramatically alters the structure and thus the functionality of the larval shell at various growth fronts, such as the bivalve hinge and the shell's edges. Conclusion Provided that NikkomycinZ mainly affects chitin synthesis in molluscs, the presented data suggest that the mollusc chitin synthase fulfils an important enzymatic role in the coordinated formation of larval bivalve shells. It can be speculated that chitin synthesis bears the potential to contribute via signal transduction pathways to the implementation of hierarchical patterns into chitin mineral-composites such as prismatic, nacre, and crossed-lamellar shell types

The structure of mollusc larval shells formed in the presence of the chitin synthase inhibitor Nikkomycin Z-6

<p><b>Copyright information:</b></p><p>Taken from "The structure of mollusc larval shells formed in the presence of the chitin synthase inhibitor Nikkomycin Z"</p><p>http://www.biomedcentral.com/1472-6807/7/71</p><p>BMC Structural Biology 2007;7():71-71.</p><p>Published online 6 Nov 2007</p><p>PMCID:PMC2241824.</p><p></p>urface of a 5 day old larva grown in the presence of 5 μM NikkomycinZ for three days appears brittle along the borders of the flakes. . Holes were observed in the outer shell surfaces of larvae. Here, one 19 day old example is shown. The shell was extracted after the larva was treated with 10 μM NikkomycinZ from the 8day on

The structure of mollusc larval shells formed in the presence of the chitin synthase inhibitor Nikkomycin Z-2

<p><b>Copyright information:</b></p><p>Taken from "The structure of mollusc larval shells formed in the presence of the chitin synthase inhibitor Nikkomycin Z"</p><p>http://www.biomedcentral.com/1472-6807/7/71</p><p>BMC Structural Biology 2007;7():71-71.</p><p>Published online 6 Nov 2007</p><p>PMCID:PMC2241824.</p><p></p>Z. This lively animal synthesized its shell (S) much too small compared to the whole organism. Thus, the shell is not suitable to protect the body tissue. The velum (V, distal border marked by black arrowheads) with its characteristic cilia (C) can not be fully retracted into the shell. The rim of the shell is indicated by white arrowheads. Note that also the hinge (H) of this shell is malformed. . Scanning electron microscopy image of a 5 day old larval shell that consists of asymmetric valves due to 3 days of growth in the presence of 10 μM NikkomycinZ. Note that one valve (V1) is much smaller than the second valve (V2). These data suggest that the inhibition of chitin synthase influences the synthesis and biomineralization of larval shells on length scales of > 100 μm

The structure of mollusc larval shells formed in the presence of the chitin synthase inhibitor Nikkomycin Z-0

<p><b>Copyright information:</b></p><p>Taken from "The structure of mollusc larval shells formed in the presence of the chitin synthase inhibitor Nikkomycin Z"</p><p>http://www.biomedcentral.com/1472-6807/7/71</p><p>BMC Structural Biology 2007;7():71-71.</p><p>Published online 6 Nov 2007</p><p>PMCID:PMC2241824.</p><p></p>ndependent test series that were microscopically evaluated at 80× magnification. The mobility of the larvae prevented an exact quantification of individuals. Based on the comparison of values estimated by different experimenters, the uncertainty was defined as 20% deviation. NikkomycinZ was supplied in concentrations of 0 μM (black bars, control), 5 μM (grey bars), and 10 μM (white bars) at the earliest developmental stages as indicated on the x-axis (larval age in days after fertilization) of each graph. Each test series ended on the 15th day after fertilization. . Semi-quantitative estimation of survival rates as observed in larvae test cultures. . Less than 20% of the 2 day old larvae survived a treatment with > 5 μM NikkomycinZ for more than 6 days. . Larvae cultures treated with NikkomycinZ from day five on show a similar effect after three days: The survival rate is about 20% on the 8day after fertilization in cultures with > 5 μM NikkomycinZ. Exceptionally, this graph represents only three out of four test series as the respective control culture in the 4series was of low quality. Note that values of more than 100% (deviation bars) are due only to the mean value calculation (see also additional data file ). . Compared to the untreated larvae populations, about 50% more of the 8 day old individuals died during the following 7 days of NikkomycinZ treatment. Larvae were not as much affected at this age as in younger developmental stages. . The survival rate of 12 day old larvae was not significantly influenced by the addition of NikkomycinZ for three days

The structure of mollusc larval shells formed in the presence of the chitin synthase inhibitor Nikkomycin Z-1

<p><b>Copyright information:</b></p><p>Taken from "The structure of mollusc larval shells formed in the presence of the chitin synthase inhibitor Nikkomycin Z"</p><p>http://www.biomedcentral.com/1472-6807/7/71</p><p>BMC Structural Biology 2007;7():71-71.</p><p>Published online 6 Nov 2007</p><p>PMCID:PMC2241824.</p><p></p> independent test series that were microscopically evaluated at 80× magnification. The mobility of the larvae prevented an exact quantification of malformed individuals (see also supplementary material for video microscopy data). Based on the comparison of values estimated by different experimenters, the uncertainty was defined as 20% deviation. NikkomycinZ was supplied in concentrations of 0 μM (black bars, control), 5 μM (grey bars), and 10 μM (white bars) at the earliest developmental stages as indicated on the x-axis (larval age in days after fertilization) of each graph. Each test series ended on the 15th day after fertilization. . Semi-quantitative estimation of morphological abnormalities observed in living individuals of the respective larvae test cultures. The abnormal phenotype rate was defined as 100% once the whole population died. Note that only the most obvious malformations (asymmetric valves, broken shells, "half-naked" individuals) were detected at the scale of binocular microscopy at 80× magnification, whereas for example undulations or tiny fractures of the larval shells were hardly visible. . A steady increase in the abnormal phenotype rate was observed when larvae were grown in the presence of NikkomycinZ from the 2day on. About three times more individuals than in the control culture were malformed on the 8day. . When NikkomycinZ treatment started on the 5day, the fraction of the abnormal larvae also increased with time. In comparison to the control population, about 50% more individuals were affected after three days. . No obvious increase in the malformation rate was observed at 80× magnification for the duration of the test in cultures treated with NikkomycinZ from the 8day (c) or the 12day (d) on

Fibroblast-like cells change gene expression of bone remodelling markers in transwell cultures

Introduction Periprosthetic fibroblast-like cells (PPFs) play an important role in aseptic loosening of arthroplasties. Various studies have examined PPF behavior in monolayer culture systems. However, the periprosthetic tissue is a three-dimensional (3D) mesh, which allows the cells to interact in a multidirectional way. The expression of bone remodeling markers of fibroblast-like cells in a multilayer environment changes significantly versus monolayer cultures without the addition of particles or cytokine stimulation. Gene expression of bone remodeling markers was therefore compared in fibroblast-like cells from different origins and dermal fibroblasts under transwell culture conditions versus monolayer cultures. Methods PPFs from periprosthetic tissues (n = 12), osteoarthritic (OA) synovial fibroblast-like cells (SFs) (n = 6), and dermal fibroblasts (DFs) were cultured in monolayer (density 5.5 × 103/cm2) or multilayer cultures (density 8.5 × 105/cm2) for 10 or 21 days. Cultures were examined via histology, TRAP staining, immunohistochemistry (anti-S100a4), and quantitative real-time PCR. Results Fibroblast-like cells (PPFs/SFs) and dermal fibroblasts significantly increased the expression of RANKL and significantly decreased the expression of ALP, COL1A1, and OPG in multilayer cultures. PPFs and SFs in multilayer cultures further showed a higher expression of cathepsin K, MMP-13, and TNF-α. In multilayer PPF cultures, the mRNA level of TRAP was also found to be significantly increased. Conclusion The multilayer cultures are able to induce significant expression changes in fibroblast-like cells depending on the nature of cellular origin without the addition of any further stimulus. This system might be a useful tool to get more in vivo like results regarding fibroblast-like cell cultures