Abstract
L1 is a neural cell adhesion molecule critical for neural development. Full-length L1 (L1(FL)) contains an alternatively spliced cytoplasmic sequence, RSLE, which is absent in L1 expressed in nonneuronal cells. The RSLE sequence follows a tyrosine, creating an endocytic motif that allows rapid internalization via clathrin-mediated endocytosis. We hypothesized that L1(FL) would internalize more rapidly than L1 lacking the RSLE sequence (L1(Delta)(RSLE)) and that internalization might regulate L1-mediated adhesion. L1 internalization was measured by immunofluorescence microscopy and by uptake of (125)I-anti-rat-L1 antibody, demonstrating that L1(FL) is internalized 2-3 times faster than L1(Delta)(RSLE). Inhibition of clathrin-mediated endocytosis slowed internalization of L1(FL) but did not affect initial uptake of L1(Delta)(RSLE). To test whether L1 endocytosis regulates L1 adhesion, cell aggregation rates were tested. L1(Delta)(RSLE) cells aggregated two times faster than L1(FL) cells. Inhibition of clathrin-mediated endocytosis increases the aggregation rate of the L1(FL) cells to that of L1(Delta)(RSLE) cells. Our results demonstrate that rapid internalization of L1 dramatically affects L1 adhesion.
Highlights
Development of the nervous system is a complex process that requires coordination of many cellular events including cell migration, axon outgrowth, and synapse formation
Immunocytochemistry Demonstrates That L1FL Internalizes Faster Than L1 lacking the RSLE sequence (L1⌬RSLE)—To study the time course of internalization of different L1 isoforms, L1FL cells or L1⌬RSLE cells were incubated in the presence of rabbit anti-L1 antibody for 10 or 60 min at 37 or at 4 °C for 30 min
The current results indicate that differences in L1 internalization rate between L1FL and L1⌬RSLE explains the slower aggregation of L1FL versus L1⌬RSLE cells
Summary
Development of the nervous system is a complex process that requires coordination of many cellular events including cell migration, axon outgrowth, and synapse formation. Many cell adhesion molecules (CAMs) participate in these events. An important question in developmental neurobiology is how expression of CAMs is regulated to allow for the precise adhesive events necessary for proper neural development. CAM-CAM binding can produce intracellular signals that regulate adhesion and trigger other events such as migration, proliferation, and synapse formation (6 –11). L1FL, but not L1⌬RSLE, is internalized via clathrin-mediated endocytosis [25]. L1FL cells aggregated more slowly than L1⌬RSLE cells, but when clathrin-mediated endocytosis was inhibited, the L1FL cells aggregated more rapidly, at a rate indistinguishable from L1⌬RSLE cells. This demonstrates that alterations in L1 internalization can regulate L1-mediated adhesion
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