NrCAM coupling to the cytoskeleton depends on multiple protein domains and partitioning into lipid rafts.
NrCAM is a cell adhesion molecule of the L1 family that is implicated in the control of axonal growth. Adhesive contacts may promote advance of the growth cone by triggering the coupling of membrane receptors with the F-actin retrograde flow. We sought to understand the mechanisms leading to clutching the F-actin at the site of ligand-mediated clustering of NrCAM. Using optical tweezers and single particle tracking of beads coated with the ligand TAG-1, we analyzed the mobility of NrCAM-deletion mutants transfected in a neuroblastoma cell line. Deletion of the cytoplasmic tail did not prevent the coupling of NrCAM to the actin flow. An additional deletion of the FNIII domains to remove cis-interactions, was necessary to abolish the rearward movement of TAG-1 beads, which instead switched to a stationary behavior. Next, we showed that the actin-dependent retrograde movement of NrCAM required partitioning into lipid rafts as indicated by cholesterol depletion experiments using methyl-beta-cyclodextrin. Recruitment of the raft component caveolin-1 was induced at the adhesive contact between the cell surface and TAG-1 beads, indicating that enlarged rafts were generated. Photobleaching experiments showed that the lateral mobility of NrCAM increased with raft dispersion in these contact areas, further suggesting that TAG-1-coated beads induced the coalescence of lipid rafts. In conclusion, we propose that anchoring of NrCAM with the retrograde actin flow can be triggered by adhesive contacts via cooperative processes including interactions with the cytoplasmic tail, formation of cis-complex via the FNIII repeats, and lipid raft aggregation.
Falk
Julien
J
Neurogenèse et Morphogenèse du Développement à l'Adulte, UMR 6156 CNRS, Institut de Biologie du Développement de Marseille, Marseille, France.
Thoumine
Olivier
O
Dequidt
Caroline
C
Choquet
Daniel
D
Faivre-Sarrailh
Catherine
C
D000199:Actins / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D015816:Cell Adhesion Molecules, Neuronal / Q000378:metabolism; D045744:Cell Line, Tumor; D058969:Contactin 2; D003599:Cytoskeleton / Q000378:metabolism*; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D000199:Actins; D015815:Cell Adhesion Molecules; D015816:Cell Adhesion Molecules, Neuronal; C548676:Cntn2 protein, rat; D058969:Contactin 2; C504754:NrCAM protein, rat; D011993:Recombinant Fusion Proteins