期刊
MOLECULAR BIOLOGY OF THE CELL
卷 18, 期 8, 页码 3131-3143出版社
AMER SOC CELL BIOLOGY
DOI: 10.1091/mbc.E06-12-1101
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We investigated the interplay between surface trafficking and binding dynamics of the immunoglobulin cell adhesion molecule Ll at neuronal growth cones. Primary neurons were transfected with Ll constructs bearing thrombin-cleavable green fluorescent protein (GFP), allowing visualization of newly exocytosed Ll or labeling of membrane Ll molecules by Quantum dots. Intracellular L1-GFP vesicles showed preferential centrifugal motion, whereas surface L1-GFP diffused randomly, revealing two pathways to address Ll to adhesive sites. We triggered Ll adhesions using microspheres coated with L1-Fc protein or anti-L1 antibodies, manipulated by optical tweezers. Microspheres coupled to the actin retrograde flow at the growth cone periphery while recruiting L1-GFP molecules, of which 50% relied on exocytosis. Fluorescence recovery after photobleaching experiments revealed a rapid recycling of L1-GFP molecules at L1-Fc (but not anti-L1) bead contacts, attributed to a high]ability of L1-L1 bonds at equilibrium. L1-GFP molecules truncated in the intracellular tail as well as neuronal cell adhesion molecules (NrCAMs) missing the clathrin adaptor binding sequence showed both little internalization and reduced turnover rates, indicating a role of endocytosis in the recycling of mature Ll contacts at the base of the growth cone. Thus, unlike for other molecules such as NrCAM or N-cadherin, diffusion/trapping and exo/endocytosis events cooperate to allow the fast renewal of Ll adhesions.
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