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Papers In Press, published online ahead of print May 1, 2005
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Centre for Vascular Research, University of New South Wales, Sydney, NSW 2052
Corresponding Author: k.gaus{at}unsw.edu.au
Lipid rafts, defined as cholesterol- and sphingolipid- rich domains, provide specialized lipid environments understood to regulate the organization and function of many plasma membrane proteins. Growing evidence of their existence, protein cargo and regulation is based largely on study of isolated lipid rafts, however, the consistency and validity of common isolation methods is controversial. Here, we provide a detailed and direct comparison of the lipid and protein composition of plasma membrane ‘rafts’ prepared from human macrophages by different methods, including several detergent-based isolations and a detergent-free method. We find that detergent-based and detergent-free methods can generate ‘raft’ fractions with similar lipid contents and a biophysical structure close to that previously found on living cells, even in cells not expressing caveolin 1 such as primary human macrophages. However, important differences between isolation methods are demonstrated. Triton X100-resistant ‘rafts’ are less sensitive to cholesterol or sphingomyelin depletion than those prepared by detergent-free methods. Moreover, we show that detergent-based methods can scramble membrane lipids during the isolation process, reorganizing lipids previously in sonciation-derived non-raft domains to generate new detergent-resistant ‘rafts’. The role of rafts in regulating biological activities of macrophage plasma membrane proteins may require careful re-evaluation using multiple isolation procedures, analyses of lipids, and microscopic techniques.
Revised on April 20, 2005
Accepted on April 20, 2005
Domain-specific lipid distribution in macrophage plasma membranes
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