Mitochondrial creatine kinase (mtCK) membrane interaction studied on biomimetic systems and new fluorescent probes conception for a better characterization of lipids membrane lateral organization

L'étude de l'interaction de la CKmt avec des membranes biomimétiques par des techniques microscopiques a permis de mettre en évidence et de visualiser la formation de complexesspécifiques CKmt-cardiolipine (CL). Sur monocouches de Langmuir et par microscopie à l'angle de Brewster, il a pu être montré que la CKmt est capable d'organiser latéralement laCL au sein des monocouches et que la forme des domaines protéine-lipides dépend de la composition en chaines acyles. Une réorientation du moment dipolaire des hélices α de laprotéine a pu être mise en évidence par spectroscopie infrarouge de réflexion-absorption par modulation de la polarisation (PM-IRRAS) ; cette réorientation dépend de la composition des différentes monocouches de CL, et notamment du degré de saturation des chaines acyles. L'interaction entre la CKmt et la CL a été visualisée sur liposomes géants (GUVs) par microscopie confocale avec excitation biphotonique. La CKmt est capable de s'organiser à la surface des GUVs, de créer des contacts inter-liposomes et de modifier la morphologie des membranes contenant la CL. Cette modification de morphologie induite par le réarrangement de la CKmt à la surface des liposomes PC-PE-CL a lieu avec des lipides en phase fluide et, comme montré par les mesures de GP du laurdan et du C-laurdan sur les GUVs, ne modifie que très peu la fluidité membranaire. Les résultats sont discutés au regard des résultats indiquant que, via son interaction avec la membrane, la CKmt joue un rôle dans le maintien de la morphologie mitochondriale. Le laurdan est une sonde fluorescente largement utilisée pour apprécier la fluidité membranaire dans différents modèles d'étude. De nouvelles sondes dérivées du laurdan par modification de la polarité de leur tête polaire ont été synthétisées et caractérisées de façon à développer de nouveaux outils pour l'étude de l'organisation latérale des lipides membranairesSpecific mtCK-cardiolipin (CL) complexes were studied on biomimetic membranes by way of several complementary techniques. The shape of protein-lipids domains changes with CL acyl chain composition as visualized on Langmuir monolayer by Brewster Angle Microscopy (BAM). MtCK promotes lateral organization within the monolayer when CL is present. Using Polarization Modulation Infrared Reflection Adsorption Spectroscopy (PM-IRRAS) we were able to show that mtCK α helix dipole moment orientation depends on CL acyl chains length and on the number of double bonds within these chains. MtCK-CL interaction with giant liposomes (GUVs) was also visualized using confocal microscopy with biphotonic excitation. In the presence of CL, mtCK has the capacity to induce the formation of clusters on the GUVs surface. It can also provoke vesicles clamping and give rise to membrane morphology changes. This phenomenon occurs with a membrane in a liquid phase with local and minimal changes in its fluidity as determined by laurdan and C-laurdan GP measurements. Results are discussed in view of previous results which indicate that, through its interaction with membrane, mtCK is involved in mitochondrial morphology. Laurdan is a fluorescent probe largely used to study membrane fluidity in several models. New lauroylnaphthalene derivative probes with a diversity of polar heads were synthesized and characterized in order to develop new tools for lipids membrane lateral organization studie

Morphology modifications in negatively charged lipid monolayers upon mitochondrial creatine kinase binding

International audienceMitochondrial creatine kinase (mtCK) may participate to membrane organization at the mitochondrial level by modulating lipid state and fluidity. The effect of the protein on lipid phase behaviour of different acyl chain length phosphatidylglycerol monolayers was analyzed from pressure – area isotherms and from the compressional modulus variation with respect to the surface pressure. Monolayer morphology was visualized by Brewster angle microscopy. No condensation effect was visible on dimyristoylphosphatidylglycerol (DMPG). For the other PG monolayers tested, dipalmitoylphosphatidylglycerol (DPPG) and distearoylphosphatidylglycerol (DSPG), mtCK facilitated the formation of a liquid condensed phase. The effect depended on the surface pressure at which transition phase occurred. The effect of mtCK was more pronounced for tetramyristoylcardiolipin (TMCL) monolayers, as liquid condensed regions appeared 10 mN/m below the transition phase of the pure TMCL monolayer. The observed domains were circular and rather uniform, indicating a stabilization of the condensed phase. The same effect, namely an overall condensation of the monolayer with formation of circular domains, was observed upon protein injection beneath TMCL monolayers in different condensation states at constant area. MtCK ability to induce and stabilize a LC phase on monolayers could have important consequences in membrane organization and emphasize its structural role at mitochondrial level

Acyl chain composition determines cardiolipin clustering induced by mitochondrial creatine kinase binding to monolayers

International audienceIt has been recently shown that mitochondrial creatine kinase (mtCK) organizes mitochondrial model membrane by modulating the state and fluidity of lipids and by promoting the formation of protein–cardiolipin clusters. This report shows, using Brewster angle microscopy, that such clustering is largely dependent on the acyl chain composition of phospholipids. Indeed, mtCK-cardiolipin domains were observed not only with unsaturated cardiolipins, but also with the cardiolipin precursor phosphatidylglycerol. On the other hand, in the case of saturated dimyristoylphosphatidylglycerol and tetramyristoylcardiolipin, mtCK was homogeneously distributed underneath the monolayer. However, an overall decrease in membrane fluidity was indicated by infrared spectroscopy as well as by extrinsic fluorescence spectroscopy using Laurdan as a fluorescent probe, both for tetramyristoylcardiolipin and bovine heart cardiolipin containing liposomes. The binding mechanism implicated the insertion of protein segments into monolayers, as evidenced from alternative current polarography, regardless of the chain unsaturation for the phosphatidylglycerols and cardiolipins tested