Optimization of fluorescence-based techniques to study the distribution and function of lipid ra fts in oligodendrocytes

 By Ellen Gielen

 Until fifteen years ago, the fluid mosaic model of Singer and Nicolson was the textbook model of how the cell membrane is organised. This model proposes that both lipids and proteins are free to diffuse in the membrane bilayer, implying a random organisation of protein in lipid (Singer and Nicolson, 1972). More recently, however, researchers have shifted to a view in which lipids are not randomly distributed within the membrane, but instead show local heterogeneity (Lai, 2003). The study of lipid domains - currently referred to as rafts - has been exploded since the formulation of the raft hypothesis by van Meer and Simons in 1987. Despite the extensive research in the field of rafts, almost all parameters of these lipid domains – their size, composition, lifetime, and biological significance – remain controversial. Evidence is accumulating that lipid rafts are involved in signal transduction, membrane sorting, cell adhesion, and in the pathogenesis of several diseases. The aim of this study is to investigate whether rafts do exist in oligodendrocytes, the myelin-producing cells of the central nervous system, and to determine their function in these cells.

Myelinating oligodendrocytes elaborate a complex array of thin processes (Fig. 1), which project outward from the cell body and terminate in highly specialized membrane sheaths of compact myelin. The ensheathment of axons with myelin is essential for the fast saltatory conduction of action potentials along the nerve cells and thus for the proper functioning of the nervous system. Abnormalities in myelin development or perturbation and degradation of its structure have severe pathological consequences, causing diseases such as multiple sclerosis (Bartlett and Mackay, 1983; de Vries and Hoekstra, 2000, Baumann and Pham-Dinh, 2001). Myelin is an architecturally complex membrane structure. While the cytoplasmic compartment is continuous from the OLG cell body to the myelin sheath, distinct membrane subdomains are evident with unequal distribution of lipids and proteins (Fig. 2).  In contrast to the oligodendroglial cell body and its processes, myelin is enriched in glycosphingolipids and cholesterol. The myelin membrane is further segregated into distinct subdomains with unequal protein distributions. 80% of proteins in purified myelin is made up by proteolipid protein (PLP), its isoform DM20, and myelin basic protein (MBP). The non-compacted regions of myelin, which include abaxonal loops, periaxonal loops, cytoplasmic incisures, and paranodal loops, contain 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), myelin-associated glycoprotein (MAG) and myelin/oligodendrocyte glycoprotein (MOG). Oligodendrocyte-specific protein/claudin-11 (OSP) is an essential component of a tight junctional array, which marks the border between compact and non-compact myelin and may serve as a diffusion barrier between these myelin subdomains (Krämer et al., 2001; Bronstein et al., 2000; Gow et al., 1999; Morita et al., 1999). 

In recent years, studies have been performed to explore the asymmetric localization of oligodendroglial membrane proteins and the role of lipid rafts in this unequal protein distribution. These studies have used a variety of techniques, including biochemical detergent extractions (Krämer et al., 1997; Kim and Pfeiffer, 1999; Simons et al., 2000), expression of apical and basolateral markers in oligodendroglial cells (de Vries et al., 1998) and transfections of oligodendroglial membrane proteins (MAG, MOG and PLP) into polarized MDCK epithelial cells (Minuk and Braun, 1996; Kroepfl and Gardinier, 2001 a,b). Besides the fact that conflicting data have been reported, the above methods might not be ideal to study the presence and function of lipid rafts in oligodendrocytes.

We are currently optimizing a fluorescence resonance energy transfer (FRET) approach to study lipid rafts in in vitro oligodendrocyte cultures derived from neonatal rat brain and/or spinal cord (Fig. 3). FRET detects energy transfers between two molecules only a few nm apart. It is a process by which a fluorophore (donor) in an excited state may transfer its excitation energy to a neighbouring fluorophore (acceptor) non-radiatively through dipole-dipole interactions. The usefulness of this technique derives from the fact that the efficiency of the energy transfer process varies as the inverse of the sixth power of the distance separating donor and acceptor, resulting in the ability to measure interactions between cellular components on a scale of 10-80 Å (Fig. 4). The aim of our FRET study is to investigate whether rafts indeed exist in oligodendrocytes, how they are distributed and whether PLP and MOG are associated with these microdomains in the myelin sheets. Furthermore, the effect of cholesterol depletion – either directly via cyclodextrin or indirectly by means of TNF-a – on the distribution of and the association with rafts will be analyzed. 

Besides FRET measurements, we will also perform fluorescence recovery after photobleaching (FRAP) measurements to verify the possible interaction of PLP and MOG with lipid rafts. These measurements will be performed on primary oligodendrocyte cultures by means of antibodies. As a control, MDCK (madine darby canine kidney) and HOG (human oligodendroglioma) cell lines, transfected with either PLP-EGFP or MOG-EGFP, will be used (Fig. 5 and 6). 

FRAP measurements allow us to determine the mobile fraction of PLP-EGFP or MOG-EGFP and to extract the diffusion coefficient of these proteins. Both FRET and FRAP measurements are performed on a Zeiss inverted Laser Scanning Confocal Microscope 510 META (Fig. 7).

Besides this research project, I also assist the following course and practical training:

  * 3^d year Biomedical students: Methods and techniques in cell physiology and cell biology.

LIST OF REFERENCES:

1. Bartlett, P. F. and Mackay, I. R. The oligodendroglial cell: biology and immunology and relationship to multiple sclerosis. J.Clin.Lab Immunol. 1983 May; 11(1): 1-7.

2. Baumann, N. and Pham-Dinh, D. Biology of oligodendrocyte and myelin in the mammalian central nervous system. Physiol Rev. 2001 Apr; 81(2): 871-927.

3. Bronstein, J. M.; Tiwari-Woodruff, S.; Buznikov, A. G., and Stevens, D. B. Involvement of OSP/claudin-11 in oligodendrocyte membrane interactions: role in biology and disease. J Neurosci Res. 2000 Mar 15; 59(6): 706-11.

4. de Vries, H.; Schrage, C., and Hoekstra, D. An apical-type trafficking pathway is present in cultured oligodendrocytes but the sphingolipid-enriched myelin membrane is the target of a basolateral-type pathway. Mol.Biol.Cell. 1998 Mar; 9(3): 599-609.

5. de Vries, H. and Hoekstra, D. On the biogenesis of the myelin sheath: cognate polarized trafficking pathways in oligodendrocytes. Glycoconj.J. 2000 Mar; 17(3 -4): 181-190.

6. Gow, A.; Southwood, C. M.; Li, J. S.; Pariali, M.; Riordan, G. P.; Brodie, S. E.; Danias, J.; Bronstein, J. M.; Kachar, B., and Lazzarini, R. A. CNS myelin and sertoli cell tight junction strands are absent in Osp/claudin-11 null mice. Cell. 1999 Dec 10; 99(6): 649-59.

7. Kim, T. and Pfeiffer, S. E. Myelin glycosphingolipid/cholesterol-enriched microdomains selectively sequester the non-compact myelin proteins CNP and MOG. J Neurocytol. 1999 Apr-1999 May 31; 28(4-5): 281-93.

8. Kramer, E. M.; Koch, T.; Niehaus, A., and Trotter, J. Oligodendrocytes direct glycosyl phosphatidylinositol-anchored proteins to the myelin sheath in glycosphingolipid-rich complexes. J Biol Chem. 1997 Apr 4; 272(14): 8937-45.

9. Kramer, E. M. ; Schardt, A., and Nave, K. A. Membrane traffic in myelinating oligodendrocytes. Microsc.Res.Tech. 2001 Mar 15; 52(6): 656-671.

10. Kroepfl, J. F. and Gardinier, M. V. Mutually exclusive apicobasolateral sorting of two oligodendroglial membrane proteins, proteolipid protein and myelin/oligodendrocyte glycoprotein, in Madin-Darby canine kidney cells. J Neurosci Res. 2001 Dec 15; 66(6): 1140-8.

11. Lai, E. C. Lipid rafts make for slippery platforms. J Cell Biol. 2003 Aug 4; 162(3): 365-70.

12. Minuk, J. and Braun P.E. Differential intracellular sorting of the myelin-associated glycoprotein isoforms. J Neurosci Res. 1996 Jun 1; 44(5): 411-420.

13. Morita, K.; Sasaki, H.; Fujimoto, K.; Furuse, M., and Tsukita, S. Claudin-11/OSP-based tight junctions of myelin sheaths in brain and Sertoli cells in testis. J Cell Biol. 1999 May 3; 145(3): 579-88.

14. Simons, M.; Kramer, E. M.; Thiele, C.; Stoffel, W., and Trotter, J. Assembly of myelin by association of proteolipid protein with cholesterol- and galactosylceramide-rich membrane domains. J.Cell Biol. 2000 Oct 2; 151(1): 143-154.

15. Singer, S. J. and Nicolson, G. L. The fluid mosaic model of the structure of cell membranes. Science. 1972 Feb 18; 175(23): 720-31.

16. van Meer, G.; Stelzer, E. H.; Wijnaendts-van-Resandt, R. W., and Simons, K. Sorting of sphingolipids in epithelial (Madin-Darby canine kidney) cells. J Cell Biol. 1987 Oct; 105(4): 1623-35.