Imaging of mobile long-lived nanoplatforms in the live cell plasma membrane

Mario Brameshuber, Julian Weghuber, Verena Ruprecht, Imre Gombos, Ibolya Horváth, László Vigh, Paul Eckerstorfer, Endre Kiss, Hannes Stockinger, Gerhard J. Schütz

    Research output: Contribution to journalArticlepeer-review

    82 Citations (Scopus)

    Abstract

    The plasma membrane has been hypothesized to contain nanoscopic lipid platforms, which are discussed in the context of "lipid rafts" or "membrane rafts." Based on biochemical and cell biological studies, rafts are believed to play a crucial role in many signaling processes. However, there is currently not much information on their size, shape, stability, surface density, composition, and heterogeneity. We present here a method that allows for the first time the direct imaging of nanoscopic long-lived platforms with raft-like properties diffusing in the live cell plasma membrane. Our method senses these platforms by their property to assemble a characteristic set of fluorescent marker proteins or lipids on a time scale of seconds. A special photobleaching protocol was used to reduce the surface density of labeled mobile platforms down to the level of well isolated diffraction-limited spots without altering the single spot brightness. The statistical distribution of probe molecules per platform was determined by single molecule brightness analysis. For demonstration, we used the consensus raft marker glycosylphosphatidylinositol-anchored monomeric GFP and the fluorescent lipid analog BODIPY-GM1, which preferentially partitions into liquid-ordered phases. For both markers, we found cholesterol-dependent homo-association in the plasma membrane of living CHO and Jurkat T cells in the resting state, thereby demonstrating the existence of small, mobile, long-lived platforms containing these probes. We further applied the technology to address structural changes in the plasma membrane during fever-type heat shock: at elevated temperatures, the glycosylphosphatidylinositol- anchored monomeric GFP homo-association disappeared, accompanied by an increase in the expression of the small heat shock protein Hsp27.

    Original languageEnglish
    Pages (from-to)41765-41771
    Number of pages7
    JournalJournal of Biological Chemistry
    Volume285
    Issue number53
    DOIs
    Publication statusPublished - 31 Dec 2010

    Keywords

    • Animals
    • Cell Membrane/metabolism
    • Cholesterol/chemistry
    • Cricetinae
    • Cricetulus
    • Diffusion
    • Glycosylphosphatidylinositols/chemistry
    • Green Fluorescent Proteins/chemistry
    • Humans
    • Jurkat Cells
    • Membrane Microdomains/chemistry
    • Microscopy/methods
    • Nanostructures/chemistry
    • Nanotechnology/methods
    • Surface Properties

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