1 May 2008 Fluorescence correlation spectroscopy and photon counting histogram on membrane proteins: functional dynamics of the glycosylphosphatidylinositol-anchored urokinase plasminogen activator receptor
Author Affiliations +
J. of Biomedical Optics, 13(3), 031215 (2008). doi:10.1117/1.2940570
Abstract
The oligomerization of glycosylphosphatidylinositol-anchored proteins is thought to regulate their association with membrane microdomains, subcellular sorting, and activity. However, these mechanisms need to be comprehensively explored in living, unperturbed cells, without artificial clustering agents, and using fluorescent protein-tagged chimeras that are fully biologically active. We expressed in human embryo kidnay 293 (HEK293) cells a biologically active chimera of the urokinase plasminogen activator receptor (uPAR), the uPAR-mEGFP-GPI. We also produced HEK293/D2D3-mEGFP-GPI cells expressing the truncated form of the receptor, lacking biological activity. We studied the dynamics and oligomerization of the two proteins, combining fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analyses, and using subclones with homogenously low expression levels. Overall, the mobile fractions of the two proteins, constituted by monomers and dimers, had comparable diffusion coefficients. However, the diffusion coefficient decreased in monomer-enriched fractions only for the active receptor, suggesting that uPAR monomers might be preferentially engaged in multiprotein transmembrane signaling complexes. Our approach helps in limiting the alteration of the data due to out-of-focus effects and in minimizing the overestimation of the molecular brightness. In addition to a careful design of the cellular model, it gives reliable estimates of diffusion coefficients and oligomerization of GPI-anchored proteins, in steady-state conditions, at low expression levels, and in live, unperturbed cells.
Gabriele Malengo, Annapaola Andolfo, Nicolai Sidenius, Enrico Gratton, Moreno Zamai, Valeria R. Caiolfa, "Fluorescence correlation spectroscopy and photon counting histogram on membrane proteins: functional dynamics of the glycosylphosphatidylinositol-anchored urokinase plasminogen activator receptor," Journal of Biomedical Optics 13(3), 031215 (1 May 2008). http://dx.doi.org/10.1117/1.2940570
JOURNAL ARTICLE
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KEYWORDS
Proteins

Diffusion

Fluorescence correlation spectroscopy

Receptors

Photon counting

Luminescence

Molecules

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