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Lipid Rafts and Detergent-Resistant Membranes in Epithelial Keratinocytes

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Book cover Epidermal Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1195))

Abstract

Our understanding of the plasma membrane has markedly increased since Singer and Nicolson proposed the fluid mosaic model in 1972. While their revolutionary theory of the lipid bilayer remains largely valid, it is now known that lipids and proteins are not randomly dispersed throughout the plasma membrane but instead may be organized within membrane microdomains, commonly referred to as lipid rafts. Lipid rafts are highly dynamic, detergent resistant, and enriched with both cholesterol and glycosphingolipids. The two main types are flotillin-rich planar lipid rafts and caveolin-rich caveolae. It is proposed that flotillin and caveolin proteins regulate cell communication by compartmentalizing and interacting with signal transduction proteins within their respective lipid microdomains. Consequently, membrane rafts play an important role in vital cellular functions including migration, invasion, and signaling; thus, alterations in their microenvironment can initiate signaling pathways that affect cellular function and behavior. Therefore, the identification of lipid rafts and their associated proteins is integral to the study of transmembrane signaling. Here, we review the current standard protocols and biochemical approaches used to isolate and define raft proteins from epithelial cells and tissues. Furthermore, in Section 3 of this chapter, detailed protocols are offered for isolating lipid rafts by subjection to detergent and sucrose density centrifugation, as well as an approach for selectively isolating caveolae. Methods to manipulate rafts with treatments such as methyl-β-cyclodextrin and flotillin III are also described.

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Acknowledgments

We thank Dr. Michael DiPersio (Center for Cell Biology & Cancer Research, Albany Medical College), Dr. Mon-Li Chu, Donna Brennan, and Andrew Overmiller for critically reading the paper and for their insightful discussions. We thank Jordan Wesolowski and Dr. Fabienne Paumet for the protocol using the SW60 rotor. This work was supported by grants from the National Institutes of Health (Mahoney, R01AR056067).

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Authors and Affiliations

  1. Department of Dermatology and Cutaneous Biology, Jefferson Medical College, 233 S. 10th Street, Suite 428 BLSB, Philadelphia, PA, 19107, USA

    Kathleen P. McGuinn & Mỹ G. Mahoney Ph.D.

  2. Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA

    Mỹ G. Mahoney Ph.D.

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  1. Kathleen P. McGuinn
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  2. Mỹ G. Mahoney Ph.D.
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Correspondence to Mỹ G. Mahoney Ph.D. .

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Editors and Affiliations

  1. Sprott Centre for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada

    Kursad Turksen

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McGuinn, K.P., Mahoney, M.G. (2014). Lipid Rafts and Detergent-Resistant Membranes in Epithelial Keratinocytes. In: Turksen, K. (eds) Epidermal Cells. Methods in Molecular Biology, vol 1195. Springer, New York, NY. https://doi.org/10.1007/7651_2014_71

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  • DOI: https://doi.org/10.1007/7651_2014_71

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  • Publisher Name: Springer, New York, NY

  • Print ISBN: 978-1-4939-1223-0

  • Online ISBN: 978-1-4939-1224-7

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