Paper BI-MoM9
Probing the Determinants of Sphingolipid Distribution in the Plasma Membrane with SIMS
Monday, December 8, 2014, 11:20 am, Room Milo
| Session: |
Nanobio Imaging |
| Presenter: |
Mary Kraft, University of Illinois at Urbana-Champaign |
| Authors: |
M. Kraft, University of Illinois at Urbana-Champaign
J. Frisz, University of Illinois at Urbana-Champaign
P. Weber, Lawrence Livermore National Laboratory
R. Wilson, University of Illinois at Urbana-Champaign
J. Zimmerberg, National Institutes of Health
H. Klitzing, University of Illinois at Urbana-Champaign
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| Correspondent: |
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The plasma membrane is a selectively permeable lipid bilayer that separates cells from their surroundings. Numerous different lipid species, cholesterol, and a variety of different proteins form the plasma membranes of mammalian cells. One class of lipids, the sphingolipids, and their metabolites serve both as structural components in the plasma membranes of mammalian cells, and as bioactive signaling molecules that modulate fundamental cellular processes. Though segregation of the sphingolipids into distinct membrane domains is likely essential for cellular function, the sphingolipid distribution within the plasma membrane and the mechanisms that regulate it are poorly understood. To address this issue, we have combined metabolic labeling with stable isotopes and SIMS performed on a Cameca NanoSIMS 50 to image the distributions of stable isotope-labeled sphingolipids in the plasma membranes of fixed cells with ~100 nm lateral resolution. Using this approach, we previously discovered that the 15N-sphingolipids were enriched within distinct domains in the plasma membranes of fibroblast cells [1]. Here we report how we have used this approach to probe the mechanisms responsible for this sphingolipid organization. To determine whether the sphingolipid domains are dependent on molecular interactions with cholesterol or protein-based barriers that are established by the cytoskeleton and its associated membrane proteins, we used SIMS to image the 15N-sphingolipid distribution in the plasma membrane following cholesterol depletion and actin depolymerization. We also assessed whether these 15N-sphingolipid domains were co-localized with hemagglutinin, a specific membrane protein that is thought to have an affinity for sphingolipid-enriched membrane domains. Our results indicate that the sphingolipid organizations in the plasma membrane are dependent on the cytoskeleton, but not on favorable interactions with cholesterol or hemagglutinin.[1] J. F. Frisz, K. Lou, H. A. Klitzing, W. P. Hanafin, V. Lizunov, R. L. Wilson, K. J. Carpenter, R. Kim, I. D. Hutcheon, J. Zimmerberg, P. K. Weber, M. L. Kraft, Proc. Natl. Acad. Sci. U.S.A., 2013, 110 (8), E613-E622.