Polysialylation Increases Lateral Diffusion of Neural Cell Adhesion Molecule in the Cell Membrane

Fabien Conchonaud, Stéphane Nicolas, Marie-Claude Amoureux, Céline Ménager, Didier Marguet, Pierre-François Lenne, Geneviève Rougon, Valéry Matarazzo
2007 Journal of Biological Chemistry  
Polysialic acid (PSA) is a polymer of N-acetylneuraminic acid residues added post-translationally to the membrane-bound neural cell adhesion molecule (NCAM). The large excluded volume created by PSA polymer is thought to facilitate cell migration by decreasing cell adhesion. Here we used live cell imaging (spot fluorescence recovery after photobleaching and fluorescence correlation spectroscopy) combined with biochemical approaches in an attempt to uncover a link between cell motility and the
more » ... pact of polysialylation on NCAM dynamics. We show that PSA regulates specifically NCAM lateral diffusion and this is dependent on the integrity of the cytoskeleton. However, whereas the glial-derivative neurotrophic factor chemotactic effect is dependent on PSA, the molecular dynamics of PSA-NCAM is not directly affected by glial-derivative neurotrophic factor. These findings reveal a new intrinsic mechanism by which polysialylation regulates NCAM dynamics and thereby a biological function like cell migration. The spatial and temporal expression pattern of cell adhesion molecules is a prerequisite for the correct development and functioning of the nervous system. These molecules fulfill their role by regulating cell-cell and cell-substrate adhesion as well as intracellular signaling pathways (1, 2). NCAM 5 (neural cell adhesion molecule), the prototype member of the immunoglobulin superfamily proteins, is widely expressed in the nervous system. NCAM mediates a large number of biological functions both through homophilic interactions and heterophilic interactions with other membrane receptors such as fibroblast growth factor and glial-derivative neurotrophic factor (GDNF) receptors (3, 4). For the latter, Paratcha et al. (4) and Iwase et al. (5) showed that the NCAM140 isoform can function as a co-receptor of the GDNF family receptor ␣1 (GFR␣1) independently of Ret tyrosine kinase, a known GDNF-signaling receptor. The functional outcome of this cross-talk is an increase of axonal growth in neurons and cell migration in Schwann cells. In vertebrates, NCAM is the only carrier of polysialic acid (PSA), a long carbohydrate composed of ␣ 2,8-linked N-acetylneuraminic acid (Neu5Ac) residues (6). The polysialylated form of NCAM (PSA-NCAM) plays a critical and unique role during brain development and in some brain tumors, modulating adhesion between cells, stimulating cell migration and neurite outgrowth (7, 8) . Observations based upon enzymatic digestion of PSA by endoneuraminidase (EndoN) (9), knock out of the polysialyltransferase coding genes responsible for addition of PSA to NCAM (10), or the use of mimotope peptide of PSA (11) indicate that the carbohydrate, more than the core protein, is critical to account for PSA-NCAM biological functions. At the molecular level, previous studies have demonstrated that PSA doubles the hydrodynamic radius of the extracellular domain of NCAM (12, 13) and thereby increases the range and magnitude of intermembrane repulsion (14). In this vein, it is accepted that the repulsion conferred by PSA is a mechanism by which cellcell interactions are decreased. However, there is no direct evidence that PSA volume affects NCAM dynamics at the cell membrane. Therefore, exploring whether PSA would regulate NCAM spatial distribution, endocytosis, mobile fraction, lateral diffusion, or NCAM confinement in a living cell could help in understanding PSA-NCAM biological functions. In this study, to investigate whether these parameters were affected by PSA removal we used a cellular model in which PSA potentiates cell migration and conducts a chemotactic effect. Biochemical and molecular imaging techniques performed on living cells allowed us to show that polysialylation conveys to NCAM an intrinsic capacity to modify its lateral diffusion at the cell surface even when molecules are engaged in mediating cell-cell contact or activated by an extracellular factor such as GDNF. In
doi:10.1074/jbc.m608590200 pmid:17623676 fatcat:qzyls3ccjbcj5iwrvql5mtq7xm