Biochemical characterization of membrane fractions in murine sperm: Identification of three distinct sub‐types of membrane rafts
- 12 November 2008
- journal article
- research article
- Published by Wiley in Journal of Cellular Physiology
- Vol. 218 (3), 537-548
- https://doi.org/10.1002/jcp.21623
Abstract
Despite enormous interest in membrane raft micro‐domains, no studies in any cell type have defined the relative compositions of the raft fractions on the basis of their major components—sterols, phospholipids, and proteins—or additional raft‐associating lipids such as the ganglioside, GM1. Our previous localization data in live sperm showed that the plasma membrane overlying the acrosome represents a stabilized platform enriched in GM1 and sterols. These findings, along with the physiological requirement for sterol efflux for sperm to function, prompted us to characterize sperm membrane fractions biochemically. After confirming limitations of commonly used detergent‐based approaches, we utilized a non‐detergent‐based method, separating membrane fractions that were reproducibly distinct based on sterol, GM1, phospholipid, and protein compositions (both mass amounts and molar ratios). Based on fraction buoyancy and biochemical composition, we identified at least three highly reproducible sub‐types of membrane raft. Electron microscopy revealed that raft fractions were free of visible contaminants and were separated by buoyancy rather than morphology. Quantitative proteomic comparisons and fluorescence localization of lipids suggested that different organelles contributed differentially to individual raft sub‐types, but that multiple membrane micro‐domain sub‐types could exist within individual domains. This has important implications for scaffolding functions broadly associated with rafts. Most importantly, we show that the common practice of characterizing membrane domains as either “raft” or “non‐raft” oversimplifies the actual biochemical complexity of cellular membranes. J. Cell. Physiol. 218: 537–548, 2009.This publication has 83 references indexed in Scilit:
- Mechanisms underlying the micron‐scale segregation of sterols and GM1 in live mammalian spermJournal of Cellular Physiology, 2008
- Structural elements of the cholesterol-dependent cytolysins that are responsible for their cholesterol-sensitive membrane interactionsProceedings of the National Academy of Sciences of the United States of America, 2007
- External Ca2+ acts upstream of adenylyl cyclase SACY in the bicarbonate signaled activation of sperm motilityDevelopmental Biology, 2007
- Lipid Rafts, Detergent-Resistant Membranes, and Raft Targeting SignalsPhysiology, 2006
- Segregation of micron‐scale membrane sub‐domains in live murine spermJournal of Cellular Physiology, 2005
- Multiplexed Protein Quantitation in Saccharomyces cerevisiae Using Amine-reactive Isobaric Tagging ReagentsMolecular & Cellular Proteomics, 2004
- Lipid Rafts: Elusive or Illusive?Cell, 2003
- Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factorsProceedings of the National Academy of Sciences of the United States of America, 2003
- Involvement of a Na+/HCO −3Cotransporter in Mouse Sperm CapacitationOnline Journal of Public Health Informatics, 2003
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970