Dynamic Partitioning of a Glycosyl-Phosphatidylinositol-Anchored Protein in Glycosphingolipid-Rich Microdomains Imaged by Single-Quantum Dot Tracking

Authors

  • Fabien Pinaud,

    Corresponding author
    1. Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, CA, USA
    2. Current address: Ecole Normale Supérieure Département de Biologie, 46 rue d’ Ulm, 75005-Paris, France
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  • Xavier Michalet,

    Corresponding author
    1. Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, CA, USA
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  • Gopal Iyer,

    1. Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, CA, USA
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  • Emmanuel Margeat,

    1. Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, CA, USA
    2. Current address: Centre de Biochimie Structurale, 29 Rue de Navacelles 34090, Montpellier, France
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  • Hsiao-Ping Moore,

    1. Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA, USA
    2. Current address: Lawrence Technological University, 21000 West Ten Mile Road Southfield, MI 48075, USA
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  • Shimon Weiss

    Corresponding author
    1. Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, CA, USA
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Fabien Pinaud, fabien.pinaud@lkb.ens.fr;

Xavier Michalet, michalet@chem.ucla.eduand

Shimon Weiss, sweiss@chem.ucla.edu

Abstract

Recent experimental developments have led to a revision of the classical fluid mosaic model proposed by Singer and Nicholson more than 35 years ago. In particular, it is now well established that lipids and proteins diffuse heterogeneously in cell plasma membranes. Their complex motion patterns reflect the dynamic structure and composition of the membrane itself, as well as the presence of the underlying cytoskeleton scaffold and that of the extracellular matrix. How the structural organization of plasma membranes influences the diffusion of individual proteins remains a challenging, yet central, question for cell signaling and its regulation. Here we have developed a raft-associated glycosyl-phosphatidyl-inositol-anchored avidin test probe (Av-GPI), whose diffusion patterns indirectly report on the structure and dynamics of putative raft microdomains in the membrane of HeLa cells. Labeling with quantum dots (qdots) allowed high-resolution and long-term tracking of individual Av-GPI and the classification of their various diffusive behaviors. Using dual-color total internal reflection fluorescence (TIRF) microscopy, we studied the correlation between the diffusion of individual Av-GPI and the location of glycosphingolipid GM1-rich microdomains and caveolae. We show that Av-GPI exhibit a fast and a slow diffusion regime in different membrane regions, and that slowing down of their diffusion is correlated with entry in GM1-rich microdomains located in close proximity to, but distinct, from caveolae. We further show that Av-GPI dynamically partition in and out of these microdomains in a cholesterol-dependent manner. Our results provide direct evidence that cholesterol-/sphingolipid-rich microdomains can compartmentalize the diffusion of GPI-anchored proteins in living cells and that the dynamic partitioning raft model appropriately describes the diffusive behavior of some raft-associated proteins across the plasma membrane.

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