Mechanisms of transmembrane signaling between neuroglian and the spectrin cytoskeleton

Neuroglian, the Drosophila member of the L1 family of neural cell adhesion molecules, selectively recruits ankyrin to sites of cell-cell contact. Ankyrin, in turn, recruits the spectrin cytoskeleton. I propose that clustering of neuroglian at cell contacts is responsible for the polarized recruitment of ankyrin and spectrin. Point mutations in highly conserved tyrosine residues in the ankyrin-binding region impaired ankyrin recruitment and cell-cell adhesion. In order to uncouple ankyrin binding from cell adhesion, I generated a CD2/neuroglian transgene. Incubation with anti-CD2 antibody caused cap formation and the colocalization of both ankyrin and beta-spectrin with CD2/neuroglian. Thus, the clustering of the neuroglian cytoplasmic domain alone was sufficient to drive ankyrin and beta-spectrin assembly. In order to measure the level of concentration of neuroglian at sites of cell-cell contact, I generated a neuroglian/EGFP transgene. I measured EGFP gray-scale intensity at and away from sites of cell-cell contact (n = 14). The average increase of brightness was between 3 and 3.81 times that of either non-contacting membrane. Thus, the concentration of neuroglian at cell-cell contact was significantly greater than the sum of neuroglian concentration in the two plasma membranes at sites away from contact. In order to determine whether or not the increase in contact neuroglian/EGFP was due to increased concentration of neuroglian/EGFP within the membrane or due to increased membrane accumulation, I examined a series of TEM images of neuroglian-mediated cell clusters for evidence of ruffling or folding at sites of cell-cell contact (n = 24). Over 75% of all measured cell-cell contact was characterized by simple two membrane apposition. Less than 0.45% of all measured contact length in neuroglian-expressing cells had significant infolding of filopodia or ruffles. Thus, the degree and distribution of infolded membrane was insufficient to account for concentration of neuroglian/EGFP at sites of neuroglian-mediated cell-cell contact. I propose a model of adhesion-mediated trapping to account for the accumulation of neuroglian at sites of cell-cell contact and the subsequent triggering of ankyrin recruitment in a concentration-dependent manner.