NATURE CELL BIOLOGY VOLUME 9 | NUMBER 1 | JANUARY 2007

1. NATURE CELL BIOLOGY VOLUME 9 | NUMBER 1 | JANUARY 2007 Presentation by: Christian Stern

2. Lipids and proteins are organized in domains (rafts)

3. Cell membrane

4. Cell membrane

5. Phospholipids

6. Sphingolipids These compounds play important roles in signal transmission and cell recognition

7. Glycolipids carbohydrate-attached lipids Role: to provide energy and also serve as markers for cellular recognition

8. Cholesterol • essential component of mammalian cell membranes, where it is required to establish proper membrane permeability and fluidity • Stabilizes the membrane • Important for ex- and import of signaling molecules • insoluble in water • 95 % intracellular

9. Cell membrane

10. Lipid raft organization scheme • A Intracellular space or cytosol • B Extracellular space or vesicle/Golgi apparatus lumen • Non-raft membrane • Lipid raft • Lipid raft associated transmembrane protein • Non-raft membrane protein • Glycosylation modifications (on glycoproteins and glycolipids) • GPI-anchored protein • Cholesterol • Glycolipid

11. Lipid raft organization scheme • Features: • Biggest difference from normal membrane • lipid composition • contains twice the amount of cholesterol than normal membrane • Cholesterol is the dynamic "glue" that holds the raft together • enriched in sphingolipids such as sphingomyelin (50% more than membrane)

12. Lipid rafts. What’s their job? • Jobs: • Organize cellular processes by: • Assembling of signaling molecules • Influencing membrane fluidity • Influencing membrane protein trafficking • Regulating neurotransmission and receptor trafficking

13. History Until 1982, it was widely accepted that phospholipids and membrane proteins were randomly distributed in cell membranes, according to the Singer-Nicolsonfluid mosaic model, published in 1972. 1982 Karnovsky et al. “Lipids in a more ordered way” or Concept of lipid domains 1997 Simons and Ikonen “Lipid rafts” Problems: Many theories and models existed: --from lipid ‘shells’ to the idea that the membrane is a collection of contiguous rafts with fluid inclusions— reflects the difficulty in structurally characterizing the cell membrane

14. History Until 1982, it was widely accepted that phospholipids and membrane proteins were randomly distributed in cell membranes, according to the Singer-Nicolsonfluid mosaic model, published in 1972. 1982 Karnovsky et al. “Lipids in a more ordered way” or Concept of lipid domains 1997 Simons and Ikonen “Lipid rafts” 2006 Keystone Symposium of Lipid rafts: Lipid Rafts: “small (10-200nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes. Small rafts can sometimes be stabilized to form larger platforms through protein-protein interactions" Latest Definition

15. Enough of history Let’s have a closer look on Lipid Rafts!

16. Organization of Lipid Rafts on the cell membrane Concentration of integral Proteins is very high: Quinn et al., 1984: density of integral membrane proteins in the ER and Golgi: ~30,000 molecules per μm2 A membrane is a lipid-protein composite, rather than a dilute solution of protein in an lipid solvent

17. Lipid Raftsare not all the same. They vary in composition, size, lifetime and functionality So, how to characterise and name them?

18. Lipid Rafts in Caveolae • invaginations of the plasma membrane in many vertebrate cell types • contain clusters of glycosphingolipids, GPI-anchored proteins and a high concentration of cholesterol • well characterized 50–100-nm nanodomain • detergent resistant • can be readily identified in most cells by the marker caveolin-1 • Function: • have several functions in signal transduction. • They are also believed to play • a role in endocytosis, • oncogenesis, and the uptake • of pathogenic bacteria and • certain viruses Bender et al., 2002

19. Shells • Smaller than 10 nm • specific classes of plasma-membrane proteins bind to preassembled complexes of cholesterol and sphingolipids • The dynamics of exchange may range from the short-lived classical boundary layer lipids (~1–10 s), in which shell lipids may rapidly interchange with non-shell lipids, to long-lived • lipids that are tightly bound through specific lipid–protein interactions “….lateral organization probably results from preferential packing of sphingolipids and cholesterol into moving platforms, or rafts, onto which specific proteins attach within the bilayer.” Simons and Ikonen, 1997

20. Nanodomains • 50–200 nm in dimension in the outer leaflet of the plasma membrane • Many proteins organized in cluster

21. Microdomains • ~1µm or bigger in dimension • Visible in Light and Electron Microscopy • MD of detergent-resistant transporters were stable in growing yeast for more than 10 min • MD in smooth muscle cells and macrophages were stable for tens of seconds • Exist in various cells but the stabilizing factors are largely unknown

22. Do they have Evidences?

24. Examples of lipid and protein domains in cell membranes Single domains, enriched in the fluorescent lipid analogue DMPECy5 Cholesterol-rich domains indirect immunofluorescence microscopy Lipid domains with greater relative order than the bulk membrane, visualized in living macrophages with the fluorescent probe, Laurdan. The warmer pseudo-colours represent more ordered regions Domains formed by the proton–argenine symporter transporter (Can1p–GFP) The scale bars represent 1 μm in a, and 5 μm in c and d.

25. Methods for detecting and characterizing membrane domains

26. Problems in Lipid Raft research: The lipid-raft research is at a technical impasse, largely because the tools to study biological membranes as liquids structured in space and time are rudimentary.

27. Biophysical tools to study membrane domains in biological membranes • Possibility to get significant results: • When domains have a minimum size of a few protein diameters and a • minimum lifetime of ~microseconds • Problems: • Small and transient domains  Nanoscale Level • Technology with sufficient simultaneous spatial and temporal resolution is not available • Best choice: • Fluorescense microscopy  high sesitivity, easy application to single, living cells • Secondary ion mass spectrometry (SIMS) - only with quick frozen specimens • Atomic force microscopy (AFM) - bad time resolution • Near field scanning microscopy (NSOM) - bad time resolution • Scattering techniques - only used in model-membrane studies

28. Other challenges: ‚energy suply‘ to biological membranes Lipids and proteins are constantly added and removed.  changes the whole membrane organization changes the interactions between lipids and proteins This lipid flux is necessary to understand the structure and function of nanodomains

29. Open Questions • What are the effects of membrane protein levels? • What is the physiological function of lipid rafts? • What effect does flux of membrane lipids have or raft formation? • What effect do diet and drugs have on lipid rafts? • What effect do proteins located at raft boundaries have on lipid rafts? • … • Needed: • Appropriate artificial membranes • to study domain properties such as formation, size, lifetime and morphology • Problem: complexity of bio membranes, energetics • Computational models •  simulations of the large assemblies of molecules found in domains

30. Take home message: A lipid raft is a cholesterol and sphingolipid-enriched domain or platform found in cell membranes. These specialized membrane microdomains compartmentalize cellular processes by serving as organizing centers for the assembly of signaling molecules, influencing membrane fluidity and membrane protein trafficking, and regulating neurotransmission and receptor trafficking. Lipid rafts are more ordered and tightly packed than the surrounding bilayer, but float freely in the membrane bilayer. Keystone symposium 2006: “small (10-200nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes. Small rafts can sometimes be stabilized to form larger platforms through protein-protein interactions” Organized in Shells (1-10 nm), Nanodomains (10-100 nm) and Microdomains (bigger than 100 nm) Caveolae: well characterized 50–100-nm invaginations of the plasma membrane. Contains clusters of glycosphingolipids, GPI-anchored proteins and a high concentration of cholesterol. Can be identified via the marker caveolin-1. Cavveolaes have several functions in signal transduction. They are also believed to play a role in endocytosis, oncogenesis, and the uptake of pathogenic bacteria and certain viruses.

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