Joe Sarkis

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.

The influence of lipids on MGD1 membrane binding highlights novel mechanisms for galactolipid biosynthesis regulation in chloroplasts

Mono‐ and digalactosyldiacylglycerol (MGDG and DGDG) are the most abundant lipids of photosynthetic membranes (thylakoids). In Arabidopsis green tissues, MGD1 is the main enzyme synthesizing MGDG. This monotopic enzyme is embedded in the inner envelope membrane of chloroplasts. DGDG synthesis occurs in the outer envelope membrane. Although the suborganellar localization of MGD1 has been determined, it is still not known how the lipid/glycolipid composition influences its binding to the membrane. The existence of a topological relationship between MGD1 and “embryonic” thylakoids is also unknown. To investigate MGD1 membrane binding, we used a Langmuir membrane model allowing the tuning of both lipid composition and packing. Surprisingly, MGD1 presents a high affinity to MGDG, its product, which maintains the enzyme bound to the membrane. This positive feedback is consistent with the low level of diacylglycerol, the substrate of MGD1, in chloroplast membranes. By contrast, MGD1 is excluded from membranes highly enriched in, or made of, pure DGDG. DGDG therefore exerts a retrocontrol, which is effective on the overall synthesis of galactolipids. Previously identified activators, phosphatidic acid and phosphatidylglycerol, also play a role on MGD1 membrane binding via electrostatic interactions, compensating the exclusion triggered by DGDG. The opposite effects of MGDG and DGDG suggest a role of these lipids on the localization of MGD1 in specific domains. Consistently, MGDG induces the self‐organization of MGD1 into elongated and reticulated nanostructures scaffolding the chloroplast membrane.—Sarkis, J., Rocha, J., Maniti, O., Jouhet, J., Vié, V., Block, M. A., Breton, C., Maréchal, E., Girard‐Egrot, A. The influence of lipids on MGD1 membrane binding highlights novel mechanisms for galactolipid biosynthesis regulation in chloroplasts. FASEB J. 28, 3114–3123 (2014). www.fasebj.org

Resisting sarcolemmal rupture: dystrophin repeats increase membrane-actin stiffness

Dystrophin is an essential part of a membrane protein complex that provides flexible support to muscle fiber membranes. Loss of dystrophin function leads to membrane fragility and muscle‐wasting disease. Given the importance of cytoskeletal interactions in strengthening the sarcolemma, we have focused on actin‐binding domain 2 of human dystrophin, constituted by repeats 11 to 15 of the central domain (DYS R11–15). We previously showed that DYS R11–15 also interacts with membrane lipids. We investigated the shear elastic constant (μ) and the surface viscosity (ηs) of Langmuir phospholipid monolayers mimicking the inner leaflet of the sarcolemma in the presence of DYS R11–15 and actin. The initial interaction of 100 nM DYS R11–15 with the monolayers slightly modifies their rheological properties. Injection of 0.125 μM filamentous actin leads to a strong increase of μ and ηs from 0 to 5.5 mN/m and 2.4 × 10–4 N · s/m, respectively. These effects are specific to DYS R11–15, require filamentous actin, and depend on phospholipid nature and lateral surface pressure. These findings suggest that the central domain of dystrophin contributes significantly to the stiffness and the stability of the sarcolemma through its simultaneous interactions with the cytoskeleton and lipid membrane. This mechanical link is likely to be a major contributing factor to the shock absorber function of dystrophin and muscle sarcolemmal integrity on mechanical stress.—Sarkis, J., Vié, V., Winder, S. J., Renault, A., Le Rumeur, E., Hubert, J.‐F. Resisting sarcolemmal rupture: dystrophin repeats increase membrane‐actin stiffness. FASEB J. 27, 359–367 (2013). www.fasebj.org

Carrier-inside-carrier: polyelectrolyte microcapsules as reservoir for drug-loaded liposomes

Abstract Conventional liposomes have a short life-time in blood, unless they are protected by a polymer envelope, most often polyethylene glycol. However, these stabilizing polymers frequently interfere with cellular uptake, impede liposome-membrane fusion and inhibit escape of liposome content from endosomes. To overcome such drawbacks, polymer-based systems as carriers for liposomes are currently developed. Conforming to this approach, we propose a new and convenient method for embedding small size liposomes, 30–100 nm, inside porous calcium carbonate microparticles. These microparticles served as templates for deposition of various polyelectrolytes to form a protective shell. The carbonate particles were then dissolved to yield hollow polyelectrolyte microcapsules. The main advantage of using this method for liposome encapsulation is that carbonate particles can serve as a sacrificial template for deposition of virtually any polyelectrolyte. By carefully choosing the shell composition, bioavailability of the liposomes and of the encapsulated drug can be modulated to respond to biological requirements and to improve drug delivery to the cytoplasm and avoid endosomal escape.

Human Dystrophin Rod 11-15 Sub-Domain: A Membrane Interacting Zone Modulated by Lipid Packing

Dystrophin is essential for skeletal muscle function and confers resistance to the sarcolemma by interacting with cytoskeletal and membrane partners. We investigated here protein-lipid interaction of a five repeat from the central domain of dystrophin (DYS R11-15) also referred as an actin binding domain. In a first step, we demonstrated that DYS R11-15 interacts more strongly with anionic than with zwitterionic small unilamellar vesicles. Using large unilamellar vesicles with different radius and trypsin accessibility assays, we showed that the protein presents different conformation depending on vesicle curvature and lipid nature. Using label-free quantification mass spectroscometry, a protein domain protected from proteolysis in presence of anionic vesicles was observed while a protein domain more accessible to trypsin in the presence of either anionic or zwitterionic vesicles was identified. In a second step, we studied the adsorption behavior of the protein at the air-liquid and lipid-liquid interface in a Langmuir trough. DYS R11-15 displayed a surface activity while maintaining its α-helical secondary structure as shown by PM-IRRAS. At 16mN/m lateral pressure of monolayer lipid film, few protein clusters were observed by AFM, while at 20 and 30mN/m, a striking protein network was formed with both negative and zwitterionic phospholipids. However, image analysis and behaviour of the networks towards trypsination in the trough as revealed by AFM showed that trypsin accessibility to the protein network depends on the surface pressure as well as on the nature of the phospholipid. These results indicate that DYS R11-15 constitutes part of the dystrophin protein for which anchoring and interaction with membrane depend on the packing and the nature of lipids. Such behaviour provides a strong experimental support for a physiological role of dystrophin central domain in contraction-relaxation cycles and dynamics of muscle cells.