Guanine nucleotide exchange factors (GEF) of the BRAG subfamily activate small Arf GTPases, which are pivotal regulators of intracellular membrane traffic and actin dynamics. Consequently, BRAG proteins have been implicated to regulate the surface levels of adhesive and signaling receptors. However, not much is known about the mechanism leading to the regulation of these surface proteins. In this study we found that the Drosophila BRAG GEF Schizo interacts physically with the Abl-interactor (Abi). schizo mutants display severe defects in myoblast fusion during syncytial muscle formation and show increased amounts of the cell adhesion protein N-cadherin. We demonstrate that the schizo myoblast fusion phenotype can be rescued by the expression of the Schizo GEF (Sec7) and membrane-binding (pleckstrin homology) domain. Furthermore, the expression of the Sec7-PH domain in a wild-type background decreases the amounts of N-cadherin and impairs myoblast fusion. These findings support the notion that the Sec7-PH domain serves as a constitutive-active form of Schizo. Using a yeast-two hybrid assay, we show that the SH3 domain of Abi interacts with the N-terminal region of Schizo. This region is also able to bind to the cytodomain of the cell adhesion molecule N-cadherin. To shed light on the function of Schizo and Abi in N-cadherin removal, we employed epistasis experiments in different developmental contexts of Drosophila. These studies point towards a new model for the regulation of Schizo. We propose that the binding of Abi to the N-terminal part of Schizo antagonizes Schizo function to inhibit N-cadherin removal.
Guanine nucleotide exchange factors (GEF) of the BRAG subfamily activate small Arf GTPases, which are pivotal regulators of intracellular membrane traffic and actin dynamics. Here, we demonstrate a novel interaction between the Abl-interactor (Abi) and the BRAG family member Schizo. We mapped the SH3 domain of Abi to interact with the N-terminal region of Schizo. This region is additionally involved in the binding of the cytodomain of the cell adhesion molecule N-cadherin. In schizo loss of function mutants, we detected increased amounts of N-cadherin. In contrast, the expression of the GEF (Sec7) and the membrane-binding (pleckstrin homology) domains decreased amounts of N-cadherin, indicating a crucial role of the Sec7-PH module in regulating N-cadherin levels. Unlike other Sec7 GEFs, where the catalytic Sec7 domain is autoinhibited, the Sec7 and PH domain of BRAG2 are constitutively accessible, raising the question how GEF activity is controlled in a spatial and temporal manner. Our genetic analyses demonstrate that the nature of the Abi Schizo interaction is to antagonize Schizo function and to restore wild-type amounts of N-cadherin.
Zusammenfassung Die Bildung eines synzytialen Muskels beruht auf der Fähigkeit von einkernigen Myoblasten zu fusionieren. Wesentliche Prozesse während der Myoblastenfusion von Drosophila und Säugetieren sind dabei die Zellerkennung und Zelladhäsion, die Bildung eines Zug‐ und Druckkräfte generierenden Zentrums durch Polymerisation von monomerem zu F‐Aktin, die Bildung einer Fusionspore sowie die Vermischung des Zytoplasmas und die Integration der Myoblasten in die wachsende Myotube. In den letzten 20 Jahren wurden viele Schlüsselgene der Myoblastenfusion am Modellorganismus Drosophila melanogaster identifiziert. Keines dieser Gene kodiert für ein Fusogen. Im Gegensatz dazu wurden in den letzten Jahren bei Mäusen zwei Proteine identifiziert, die direkt an der Umstrukturierung der Plasmamembran während der Bildung einer Fusionspore beteiligt sind und die fusogene Eigenschaften besitzen. Diese Proteine können uns zukünftig helfen zu verstehen, was während der Myoblastenfusion an der Zellmembran passiert.
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