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Published online
doi:10.1083/jcb.200702151
The Journal of Cell Biology, Vol. 179, No. 5, 1067-1082
The Rockefeller University Press, 0021-9525 $30.00
© Caiolfa et al.
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Article

Monomer–dimer dynamics and distribution of GPI-anchored uPAR are determined by cell surface protein assemblies



Valeria R. Caiolfa1,2, Moreno Zamai1,2, Gabriele Malengo1,3, Annapaola Andolfo4, Chris D. Madsen4, Jason Sutin5, Michelle A. Digman5, Enrico Gratton5, Francesco Blasi1,3,4, and Nicolai Sidenius1,4

1 Department of Molecular Biology and Functional Genomics and 2 Italian Institute of Technology Network Research, Unit of Molecular Neuroscience, San Raffaele Scientific Institute, 20132 Milano, Italy
3 Università Vita-Salute San Raffaele, 20132 Milano, Italy
4 Fondazione Italiana per la Ricerca sul Cancro Institute of Molecular Oncology, 20139 Milano, Italy
5 Laboratory for Fluorescence Dynamics, University of California, Irvine, Irvine 92697, CA

Correspondence to Valeria R. Caiolfa: valeria.caiolfa{at}hsr.it; or Francesco Blasi: francesco.blasi{at}hsr.it

To search for functional links between glycosylphosphatidylinositol (GPI) protein monomer–oligomer exchange and membrane dynamics and confinement, we studied urokinase plasminogen activator (uPA) receptor (uPAR), a GPI receptor involved in the regulation of cell adhesion, migration, and proliferation. Using a functionally active fluorescent protein–uPAR in live cells, we analyzed the effect that extracellular matrix proteins and uPAR ligands have on uPAR dynamics and dimerization at the cell membrane. Vitronectin directs the recruitment of dimers and slows down the diffusion of the receptors at the basal membrane. The commitment to uPA–plasminogen activator inhibitor type 1–mediated endocytosis and recycling modifies uPAR diffusion and induces an exchange between uPAR monomers and dimers. This exchange is fully reversible. The data demonstrate that cell surface protein assemblies are important in regulating the dynamics and localization of uPAR at the cell membrane and the exchange of monomers and dimers. These results also provide a strong rationale for dynamic studies of GPI-anchored molecules in live cells at steady state and in the absence of cross-linker/clustering agents.

Abbreviations used in this paper: ACF, autocorrelation function; cpsm, counts per second per molecule; FCS, fluorescence correlation spectroscopy; FLIM, fluorescence lifetime imaging microscopy; Fn, fibronectin; FRET, Förster resonance energy transfer; GPI, glycosylphosphatidylinositol; mRFP, monomeric RFP; PAI1, plasminogen activator inhibitor type 1; PCH, photon-counting histogram; TCSPC, time-correlated single-photon counting; uPA, urokinase plasminogen activator; uPAR, uPA receptor; Vn, vitronectin; wt, wild type.


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