Reorganization of Lipid Diffusion by Myelin Basic Protein as Revealed by STED Nanoscopy

Olena Steshenko,Débora M Andrade,Alf Honigmann,Veronika Mueller,Falk Schneider,Erdinc Sezgin,Stefan W Hell,Mikael Simons,Christian Eggeling

doi:10.1016/j.bpj.2016.04.047

Abstract

Myelin is a multilayered membrane that ensheathes axonal fibers in the vertebrate nervous system, allowing fast propagation of nerve action potentials. It contains densely packed lipids, lacks an actin-based cytocortex, and requires myelin basic protein (MBP) as its major structural component. This protein is the basic constituent of the proteinaceous meshwork that is localized between adjacent cytoplasmic membranes of the myelin sheath. Yet, it is not clear how MBP influences the organization and dynamics of the lipid constituents of myelin. Here, we used optical stimulated emission depletion super-resolution microscopy in combination with fluorescence correlation spectroscopy to assess the characteristics of diffusion of different fluorescent lipid analogs in myelin membrane sheets of cultured oligodendrocytes and in micrometer-sized domains that were induced by MBP in live epithelial PtK2 cells. Lipid diffusion was significantly faster and less anomalous both in oligodendrocytes and inside the MBP-rich domains of PtK2 cells compared with undisturbed live PtK2 cells. Our data show that MBP reorganizes lipid diffusion, possibly by preventing the buildup of an actin-based cytocortex and by preventing most membrane proteins from entering the myelin sheath region. Yet, in contrast to myelin sheets in oligodendrocytes, the MBP-induced domains in epithelial PtK2 cells demonstrate no change in lipid order, indicating that segregation of long-chain lipids into myelin sheets is a process specific to oligodendrocytes.

Highlights

Myelin is a highly specialized membrane that forms a continuous, multilayered stack of tightly packed membrane, ensheathing the axons in the central and peripheral nervous systems [1,2]
We incorporated different lipid analogs labeled with the lipophilic organic dye Atto647N into the plasma membrane of living oligodendrocytes by incubation with lipid-bovine serum albumin complexes, and monitored their diffusion pattern using stimulated emission depletion (STED)-FCS
We previously showed that myelin basic protein (MBP) expression as a chimeric construct (green fluorescent protein (GFP) labeled (GFP-TM-MBP)) generates connections between the membrane of the endoplasmic reticulum (ER) and the plasma membrane in epithelial PtK2 cells (Fig. 2 A) [13]

Summary

INTRODUCTION

Myelin is a highly specialized membrane that forms a continuous, multilayered stack of tightly packed membrane, ensheathing the axons in the central and peripheral nervous systems [1,2]. Prominent examples include lipid microdomains, caveolae, coated pits, and actin cytoskeleton-induced subcompartments [24,25,26,27,28] Due to this patchwork organization of the plasma membrane, diffusion of molecules is in many cases not free, but anomalous [21,24,29,30,31,32]. Interactions with less mobile entities such as proteins lead to transient slowdowns in diffusion (trapping) [22,29,31,33], whereas actin-induced compartments result in temporary confinements within 30–300 nm compartments (hop diffusion) [24,30,34,35] All of these structural and diffusional heterogeneities rule the bioactivity of membrane molecules [36]. Our data showed no difference in order between inside and outside of the domains

MATERIALS AND METHODS

RESULTS

DISCUSSION