Combination of 3D Scanning, Modeling and Analyzing Methods around the Castle of Coatfrec Reconstitution

International audienceThe castle of Coatfrec is a medieval castle in Brittany constituting merely a few remaining ruins currently in the process of restoration. Beyond its great archeological interest, it has become, over the course of the last few years, the subject of experimentation in digital archeology. Methods of 3D scanning were implored in order to gauge comparisons between the remaining structures and their absent hypothetical ones, resulting in the first quantitative results of its kind. This paper seeks to introduce the methods which carried out said research, as well as to present the subsequent results obtained using these new digital tools

Modulation of the interaction and organization of Human Dystrophin repeats 11-15 subdomain with Membrane

Dystrophin is essential to skeletal muscle function and confers resistance to the sarcolemma by interacting with cytoskeleton and membrane. In the present work, we characterized the behavior of dystrophin 11–15 (DYS R11–15), five spectrin-like repeats from the central domain of human dystrophin, with lipids. DYS R11–15 displays an amphiphilic character at the liquid/air interface while maintaining its secondary α-helical structure. The interaction of DYS R11–15 with small unilamellar vesicles (SUVs) depends on the lipid nature, which is not the case with large unilamellar vesicles (LUVs). In addition, switching from anionic SUVs to anionic LUVs suggests the lipid packing as a crucial factor for the interaction of protein and lipid. The monolayer model and the modulation of surface pressure aim to mimic the muscle at work (i.e. dynamic changes of muscle membrane during contraction and relaxation) (high and low surface pressure). Strikingly, the lateral pressure modifies the protein organization. Increasing the lateral pressure leads the proteins to be organized in a regular network. Nevertheless, a different protein conformation after its binding to monolayer is revealed by trypsin proteolysis. Label-free quantification by nano-LC/MS/MS allowed identification of the helices in repeats 12 and 13 involved in the interaction with anionic SUVs. These results, combined with our previous studies, indicate that DYS R11–15 constitutes the only part of dystrophin that interacts with anionic as well as zwitterionic lipids and adapts its interaction and organization depending on lipid packing and lipid nature. We provide strong experimental evidence for a physiological role of the central domain of dystrophin in sarcolemma scaffolding through modulation of lipid-protein interactions

Spectrin-like repeats 11–15 of human dystrophin show adaptations to a lipidic environment

Dystrophin is essential to skeletal muscle function and confers resistance to the sarcolemma by interacting with cytoskeleton and membrane. In the present work, we characterized the behavior of dystrophin 11–15 (DYS R11–15), five spectrin-like repeats from the central domain of human dystrophin, with lipids. DYS R11–15 displays an amphiphilic character at the liquid/air interface while maintaining its secondary α-helical structure. The interaction of DYS R11–15 with small unilamellar vesicles (SUVs) depends on the lipid nature, which is not the case with large unilamellar vesicles (LUVs). In addition, switching from anionic SUVs to anionic LUVs suggests the lipid packing as a crucial factor for the interaction of protein and lipid. The monolayer model and the modulation of surface pressure aim to mimic the muscle at work (i.e. dynamic changes of muscle membrane during contraction and relaxation) (high and low surface pressure). Strikingly, the lateral pressure modifies the protein organization. Increasing the lateral pressure leads the proteins to be organized in a regular network. Nevertheless, a different protein conformation after its binding to monolayer is revealed by trypsin proteolysis. Label-free quantification by nano-LC/MS/MS allowed identification of the helices in repeats 12 and 13 involved in the interaction with anionic SUVs. These results, combined with our previous studies, indicate that DYS R11–15 constitutes the only part of dystrophin that interacts with anionic as well as zwitterionic lipids and adapts its interaction and organization depending on lipid packing and lipid nature. We provide strong experimental evidence for a physiological role of the central domain of dystrophin in sarcolemma scaffolding through modulation of lipid-protein interactions

Interaction of the actin binding domain 2 of dystrophin with lipid membrane is modulated by lipid packing

National audienceDystrophin is essential for skeletal muscle function and confers resistance to the sarcolemma by interacting with cytoskeletal and membrane partners. We previously showed that a large part of the rod domain of dystrophin interacts with membrane lipids. Amongst the central region, spectrin like repeats 11 to 15 (DYS R1115) constitutes an actin binding domain ABD2 as well as a lipid binding domain (Legardinier et al. J.Mol.Biol. 389:54658 2009). In this work, we aimed to characterize the specific proteinlipid interactions of DYS R1115. First, we show that DYS R1115 displays a strong amphiphilic character at the liquid/air interface while maintaining its αhelical secondary structure. We demonstrated that DYS R1115 strongly interacts with anionic small unilamellar vesicles, while only a weak binding is observed with zwitterionic small unilamellar vesicles and large anionic and zwitterionic vesicles, suggesting a binding dependent upon lipid charges and curvature or packing. Strikingly, it appears that the protein binds strongly to lipid monolayers with both anionic and zwiterrionic lipids; in addition, upon increase of surface pressure, regular protein networks are observed by AFM at the monolayer surfaces. This further indicates that the binding is dependent upon lipid packing. However, we observed that the accessibility to trypsin of DYSR1115 linked to monolayers depends on the phospholipid nature, suggesting a mode of proteinlipid binding also dependent on the lipid charges. This was confirmed by using trypsin proteolysis of the protein in the presence of vesicles with different radius. Labelfree quantification mass spectroscometry data showed that several regions of DYS R1115 are protected from trypsin action when the protein is in contact with anionic small unilamellar vesicles while another domain is found more subject to proteolysis in the presence of either anionic or zwitterionic vesicles. All together, our results indicate that DYS R1115 constitutes part of the dystrophin protein interacting with anionic as well as zwitterionic lipids, while the anchoring and interaction with membrane mostly depends on the lipid packing. Such behaviour, in addition to actin binding properties, provides a strong experimental support for a physiological role of dystrophin central domain on sarcolemma scaffolding upon contractionrelaxation cycles and dynamics of muscle cells