Direct evidence of lipid rafts by in situ atomic force microscopy.

Lipid rafts are membrane microdomains enriched with cholesterol, glycosphingolipids, and proteins. Although they are broadly presumed to play a pivotal role in various cellular functions, there are still fierce debates about the composition, functions, and even existence of lipid rafts. Here high-resolution and time-lapse in situ atomic force microscopy is used to directly confirm the existence of lipid rafts in native erythrocyte membranes. The results indicate some important aspects of lipid rafts: most of the lipid rafts are in the size range of 100-300 nm and have irregular shape; the detergent-resistant membranes consist of cholesterol microdomains and are not likely the same as the lipid rafts; cholesterol contributes significantly to the formation and stability of the protein domains; and Band III is an important protein of lipid rafts in the inner leaflet of erythrocyte membranes, indicating that lipid rafts are exactly the functional domains in plasma membrane. This work provides direct evidence of the presence, size, and main constitutive protein of lipid rafts at a resolution of a few nanometers, which will pave the way for studying their structure and functions in detail.

[1]  Raphael Zidovetzki,et al.  Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies. , 2007, Biochimica et biophysica acta.

[2]  E. Sackmann,et al.  Structure of an adsorbed dimyristoylphosphatidylcholine bilayer measured with specular reflection of neutrons. , 1991, Biophysical journal.

[3]  L. Pike The challenge of lipid rafts This work was supported by National Institutes of Health grants RO1 GM064491 and R01 GM082824 to LJP. Published, JLR Papers in Press, October 27, 2008. , 2009, Journal of Lipid Research.

[4]  A. Shaw Lipid rafts: now you see them, now you don't , 2006, Nature Immunology.

[5]  S. Orkin,et al.  Anion Exchanger 1 (Band 3) Is Required to Prevent Erythrocyte Membrane Surface Loss but Not to Form the Membrane Skeleton , 1996, Cell.

[6]  Kai Simons,et al.  Lipid rafts and signal transduction , 2000, Nature Reviews Molecular Cell Biology.

[7]  T. Kerppola,et al.  Visualization of molecular interactions by fluorescence complementation , 2006, Nature Reviews Molecular Cell Biology.

[8]  I. Mikhalyov,et al.  Lipid raft detecting in membranes of live erythrocytes. , 2011, Biochimica et biophysica acta.

[9]  A. Buttafava,et al.  Resistance of Human Erythrocyte Membranes to Triton X-100 and C12E8 , 2008, Journal of Membrane Biology.

[10]  K. El Kirat,et al.  Membrane resistance to Triton X-100 explored by real-time atomic force microscopy. , 2006, Langmuir : the ACS journal of surfaces and colloids.

[11]  D. Lohr,et al.  Glutaraldehyde modified mica: a new surface for atomic force microscopy of chromatin. , 2002, Biophysical journal.

[12]  S. Hell,et al.  Direct observation of the nanoscale dynamics of membrane lipids in a living cell , 2009, Nature.

[13]  R. Zeng,et al.  The differential protein and lipid compositions of noncaveolar lipid microdomains and caveolae , 2009, Cell Research.

[14]  Y. Dufrêne,et al.  Detection and localization of single molecular recognition events using atomic force microscopy , 2006, Nature Methods.

[15]  D. Müller AFM: a nanotool in membrane biology. , 2008 .

[16]  Daniel J Müller,et al.  Atomic force microscopy as a multifunctional molecular toolbox in nanobiotechnology. , 2008, Nature nanotechnology.

[17]  S. Singer,et al.  The Fluid Mosaic Model of the Structure of Cell Membranes , 1972, Science.

[18]  L. Foster,et al.  Contributions of quantitative proteomics to understanding membrane microdomains , 2009, Journal of Lipid Research.

[19]  Ken Jacobson,et al.  Partitioning of Thy-1, GM1, and cross-linked phospholipid analogs into lipid rafts reconstituted in supported model membrane monolayers , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[20]  Xin Shang,et al.  Localization of Na+-K+ ATPases in quasi-native cell membranes. , 2009, Nano letters.

[21]  L. Rajendran,et al.  Lipid rafts and membrane dynamics , 2005, Journal of Cell Science.

[22]  Kai Simons,et al.  Lipid Rafts As a Membrane-Organizing Principle , 2010, Science.

[23]  E Gratton,et al.  Lipid rafts reconstituted in model membranes. , 2001, Biophysical journal.

[24]  C. le Grimellec,et al.  In situ imaging of detergent-resistant membranes by atomic force microscopy. , 2000, Journal of structural biology.

[25]  E. Ikonen,et al.  Functional rafts in cell membranes , 1997, Nature.

[26]  Guillaume Andre,et al.  Imaging the nanoscale organization of peptidoglycan in living Lactococcus lactis cells , 2010, Nature communications.

[27]  K. Motoyama,et al.  Involvement of lipid rafts of rabbit red blood cells in morphological changes induced by methylated beta-cyclodextrins. , 2009, Biological & pharmaceutical bulletin.

[28]  Mingjun Cai,et al.  Preparation of cell membranes for high resolution imaging by AFM. , 2010, Ultramicroscopy.

[29]  D. Engelman,et al.  Protein area occupancy at the center of the red blood cell membrane , 2008, Proceedings of the National Academy of Sciences.

[30]  M. Tanner,et al.  The structure and function of band 3 (AE1): recent developments (review). , 1997, Molecular membrane biology.

[31]  D. Lingwood,et al.  Detergent resistance as a tool in membrane research , 2007, Nature Protocols.

[32]  R C Macdonald,et al.  Atomic force microscopy of the erythrocyte membrane skeleton , 2001, Journal of microscopy.

[33]  N. Hooper,et al.  The prion protein and lipid rafts (Review) , 2006 .

[34]  K. El Kirat,et al.  Cholesterol modulation of membrane resistance to Triton X-100 explored by atomic force microscopy. , 2007, Biochimica et biophysica acta.

[35]  Richard G. W. Anderson,et al.  Lipid rafts: at a crossroad between cell biology and physics , 2007, Nature Cell Biology.

[36]  W. Rodgers,et al.  Cytoskeleton–membrane interactions in membrane raft structure , 2009, Cellular and Molecular Life Sciences.

[37]  Deborah A. Brown,et al.  Lipid rafts, detergent-resistant membranes, and raft targeting signals. , 2006, Physiology.

[38]  G. Meer,et al.  Membrane lipids: where they are and how they behave , 2008, Nature Reviews Molecular Cell Biology.

[39]  Jennifer Lippincott-Schwartz,et al.  Dynamics of putative raft-associated proteins at the cell surface , 2004, The Journal of cell biology.

[40]  E. Sackmann,et al.  Chemically induced phase separation in mixed vesicles containing phosphatidic acid. An optical study. , 1975, Journal of the American Chemical Society.

[41]  Xin Shang,et al.  Locating the Band III protein in quasi-native cell membranes , 2010 .

[42]  P. Gane,et al.  Flow cytometric analysis of the association between blood group‐related proteins and the detergent‐insoluble material of K562 cells and erythroid precursors , 2001, British journal of haematology.

[43]  A. Shevchenko,et al.  Resistance of cell membranes to different detergents , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[44]  P. Low,et al.  Erythrocyte detergent-resistant membrane proteins: their characterization and selective uptake during malarial infection. , 2004, Blood.

[45]  Deborah A. Brown,et al.  Structure and Function of Sphingolipid- and Cholesterol-rich Membrane Rafts* , 2000, The Journal of Biological Chemistry.

[46]  Chengyu Jiang,et al.  SARS coronavirus entry into host cells through a novel clathrin- and caveolae-independent endocytic pathway , 2008, Cell Research.

[47]  L. Pike Lipid rafts: heterogeneity on the high seas. , 2004, The Biochemical journal.