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* CNRS 5091, Université Bordeaux 2, Bordeaux, France; CNRS 6184-NICN, Faculté de Médecine Nord, Marseille, France; and Equipe Avenir, UMR 7592, Institut Jacques Monod, Université Paris VI, Paris, France
Correspondence: Address reprint requests and inquiries to O. Thoumine, Tel: 33-5-57-5740-91, E-mail: olivier.thoumine{at}pcs.u-bordeaux2.fr.
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ABSTRACT |
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 TOP ABSTRACT EXPERIMENTAL METHODSACKNOWLEDGEMENTSREFERENCES |
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The formation of adhesive contacts between cells is fundamental in biology. It involves specific adhesion proteins, e.g., IgCAMs, which are implicated in neurite elongation and growth cone guidance (1
). Contacts are initiated when adhesion molecules find counterreceptors on the surface of neighboring cells and make selective protein-protein bonds. Such interactions depend on the abundance of receptors expressed by the cells, but also on the ability of receptors to diffuse in the cell membrane (2). The regulation of receptor mobility by cytoplasmic partners, e.g., between L1/neurofascin and ankyrin (3,4), may then tune the rate at which adhesions form.
To assess if diffusion could affect the kinetics of receptor recruitment at adhesive sites, we used constructs of varying length (25–180 kD), all tagged extracellularly with green fluorescent protein (GFP). These include L1-GFP, several truncated forms of neuronal-related cell adhesion molecule (NrCAM)-GFP (5), and glycosylphosphatidylinositol (GPI)-GFP (Fig. 1 F). We reasoned that size differences should result in contrasting lateral mobilities. To measure the diffusion coefficient of these receptors, we transfected primary culture neurons and labeled individual receptors with quantum dots (QD). Active growth cones were selected for the recordings (Fig. 1 A), since these structures are implicated in IgCAM-based locomotion and cell recognition; 
40% of the receptors were expressed at the plasma membrane (Table 1), allowing QD to bind specifically to transfected cells (Fig. 1 B). QD attached to the cell surface and moved in two dimensions, exploring the entire growth cone surface (Fig. 1 C). QD showed a variety of behaviors, some moving fast, others staying almost immobile. We tracked individual QD and calculated an instantaneous diffusion coefficient for each trajectory.
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). As receptor size diminishes, the distribution shifts to higher mobility values, resulting in a clear inverse relationship between the molecular weight of the receptor and its average diffusion coefficient (Fig. 1 G). Since these receptors interact similarly with lipid microdomains (5), differences in mobility are unlikely to be associated with variations in the lipid environment. Truncations of intracellular regions caused a slight decrease in lateral mobility (7), which may be attributed to trapping of L1 or NrCAM cytoplasmic tail within the membrane scaffold, or to specific interactions with cytoskeletal partners such as ankyrin or SAP102 (3,4). Deletions of extracellular regions (fibronectin type III, immunoglobin (Ig), or both) strongly reduced receptor diffusion (8). This may be due to steric effects linked to the high glycosylation levels of L1 and NrCAM ectodomains. Alternatively, IgCAMs with intact fibronectin type III and/or Ig domains are able to interact in cis with themselves or other receptors (1), thus forming complexes with lower diffusion properties. We then mimicked adhesive contacts using anti-GFP-coated latex microspheres, which selectively bound to transfected cells (Fig. 2, A and B; Table 1) and recruited GFP-tagged membrane receptors (Fig. 2, C and D). We placed microspheres on growth cones using optical tweezers, and followed the accumulation of receptors around them (Fig. 2 E). We quantified the ratio between the fluorescence level on the microsphere and that on adjacent regions. This enrichment factor increased in a few minutes, slightly faster for smaller receptors (Fig. 2 F), and reached a plateau around 3 with minor differences between the constructs (Table 1). That equilibrium value corresponded to the saturation of antibody binding sites on microspheres by GFP-tagged receptors.
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1000/µm2), L the density of GFP binding sites on microspheres (4000/µm2), C the surface density of bonds between antibodies and receptors, and kon and koff the forward and reverse rate constants, respectively. Fluorescence measurements outside bead contacts indicated that there was no receptor depletion, so we took (R – C) = R. Furthermore, antibody-antigen bonds being very stable, we set koff = 0. This left Eq. 1: C(t) = L[1 – exp(–konRt)], which was used to fit the data and gave the two parameters R/L (Table 1) and konR.
The association rate konR increased weakly with the receptor diffusion coefficient (Fig. 2 C), showing that receptor accumulation at microsphere contacts is not diffusion-limited. This agreed with a theoretical model taking into account the long-range diffusion of receptors toward a narrow zone where they can be irreversibly trapped by immobilized ligands (9). Beads coated with lower affinity ligands such as monoclonal antibodies against GFP (not shown), transient adhesion glycoprotein 1 (5), or N-cadherin (10) all induced slower accumulation of counterreceptors, suggesting that the adhesive reaction is the limiting step there. Thus, there appears to be a large enough reservoir of highly diffusive IgCAMs that can be mobilized quickly at adhesive sites, waiting for ligand binding. It is still possible that subtle differences in the diffusion of less mobile receptor complexes, controlled locally by the cytoskeleton or the lipid environment, can modulate the initiation and durability of neuronal interactions.
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TOPABSTRACTEXPERIMENTAL METHODSACKNOWLEDGEMENTSREFERENCES |
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ACKNOWLEDGEMENTS |
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TOPABSTRACTEXPERIMENTAL METHODSACKNOWLEDGEMENTSREFERENCES |
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We acknowledge financial support from CNRS, the French Ministry of Research, and INSERM.
Submitted on July 29, 2005; accepted for publication August 17, 2005.
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REFERENCES |
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2. Lauffenburger, D. A., and J. J. Linderman. 1993. Receptors: Models for Binding, Trafficking, and Signaling. Oxford University Press, New York.
3. Garver, T. D., Q. Ren, S. Tuvia, and V. Bennett. 1997. Tyrosine phosphorylation at a site highly conserved in the L1 family of cell adhesion molecules abolishes ankyrin binding and increases lateral mobility of neurofascin. J. Cell Biol. 137:703–714.
4. Gil, O. D., T. Sakurai, A. E. Bradley, M. Y. Fink, M. R. Cassella, J. A. Kuo, and D. P. Felsenfeld. 2003. Ankyrin binding mediates L1CAM interactions with static components of the cytoskeleton and inhibits retrograde movement of L1CAM on the cell surface. J. Cell Biol. 162:719–730.
5. Falk, J., O. Thoumine, C. Dequidt, D. Choquet, and C. Faivre-Sarrailh. 2004. NrCAM coupling to the cytoskeleton depends on multiple protein domains and partitioning into lipid rafts. Mol. Biol. Cell. 15:4695–4709.
6. Simson, R., B. Yang, S. E. Moore, P. Doherty, F. S. Walsh, and K. A. Jacobson. 1998. Structural mosaicism on the submicron scale in the plasma membrane. Biophys. J. 74:297–308.
7. Edidin, M., M. C. Zuniga, and M. P. Sheetz. 1994. Truncation mutants define and locate cytoplasmic barriers to lateral mobility of membrane glycoproteins. Proc. Natl. Acad. Sci. USA. 91:3378–3382.
8. Zhang, F., B. Crise, B. Su, Y. Hou, J. K. Rose, A. Bothwell, and K. Jacobson. 1992. The lateral mobility of some membrane proteins is determined by their ectodomains. Biophys. J. 62:92–94.
9. Crank, J. 1975. The Mathematics of Diffusion. Oxford University Press, New York.
10. Thoumine, O., M. Lambert, R. Mège, and D. Choquet. 2005. Regulation of N-cadherin dynamics at neuronal contacts by ligand binding and cytoskeletal coupling. Mol. Biol. Cell. In press.
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Cter is deleted of the ankyrin binding motif, and downstream, 