Lipid organisation and dynamics in the myelin membrane sheets
von Olena Steshenko
Datum der mündl. Prüfung:2013-10-21
Betreuer:Prof. Dr. Mikael Simons
Gutachter:Prof. Dr. Silvio Rizzoli
Gutachter:Prof. Dr. Jörg Enderlein
EnglischMyelin is a unique lipid-enriched membrane, produced by oligodendrocytes in the central nervous system, that is enwrapped around axons in the multilayered fashion to ensure a swift saltatory conduction of the neuronal impulses. Myelin has a unique composition. It is enriched in the cholesterol, sphingomyelin and myelin lipids possess high number of long-chain fatty acids in their structure. Previous results have shown that myelin basic protein (MBP) acts as a diffusion barrier within myelin sheath, preventing the majority of the proteins to enter the sheath region. Therefore, myelin sheaths are deprived of cytoplasm and proteins, and do not contain cortical actin cytoskeleton. This specific composition raises the question of how lipid diffusion might be affected in the myelin sheath. We used primary oligodendrocyte culture as a well-established model to study lipid diffusion in myelin sheath. To assess lipid diffusion modes, we used STED-FCS (stimulated emission depletion microscopy combined with fluorescence correlation spectroscopy). We showed that artificial analogs of phosphatidylethanolamine, sphingomyelin and galactosylcerebroside (PE, SM and GalC, respectively) introduced into the cellular membrane, have a faster diffusion within oligodendrocytes, when compared to the diffusion within control membrane of the fibroblasts cell line (PtK2 cells). Moreover, we could show a complete absence of trapping for SM and a decrease in the trapping for GalC in the membrane of oligodendrocytes. Additionally, using a model of the reconstituted oligodendrocyte sheets in PtK2 cells, we could demonstrate that MBP presence is sufficient to reorganize cellular membrane and change the mode of the lipids behaviour, from trapping to the free diffusion. In the second project we addressed the question of the polarised distribution of phosphatidylinositols in oligodendrocyte membrane. We could demonstrate a distribution of PIP2 and PIP3 into two distinct cellular regions: sheets for PIP2 and outer rim and processes for PIP3. We showed that MBP interacts with PIP2 in the sheets and masks it from antibody staining, but not with PIP3 in the processes. Furthermore, we found that oligodendrocytes are highly susceptible to the changes in the PIP3 levels: upon its augmentation the size of non-compact domains within oligodendrocytes increased. We found that an increase of actin accompanied an with enlargement of the non-compact regions. We speculate that this augmentation of the filamentous actin is a reason that would prevent MBP from zipping the non-compact regions. We suggest actin to be a downstream effector of the increased PIP3 levels.
Keywords: myelin; FCS-STED; oligodendrocytes; lipid diffusion; myelin membrane sheaths