Cryo-electron tomography of homophilic adhesion mediated by the neural cell adhesion molecule L1.

The neural cell adhesion molecule L1 participates in homophilic interactions important for axon guidance and neuronal development. The structural details of homophilic adhesion mediated by L1 and other immunoglobulin superfamily members containing an N-terminal horseshoe arrangement of four immunoglobulin-like domains are unknown. Here we used cryo-electron tomography to study liposomes to which intact or truncated forms of the L1 ectodomain were attached. Tomographic reconstructions revealed an adhesion interface with a regular and repeating pattern consistent with interactions between paired horseshoes contributed by L1 proteins from neighboring liposomes. The characteristics of the pattern changed when N-linked carbohydrates were altered by removing sialic acids or converting from complex to high mannose or oligomannose glycans, suggesting a regulatory role for carbohydrates in L1-mediated homophilic adhesion. Using the results from tomograms and crystal structures of L1-related molecules, we present a structural model for L1-mediated homophilic adhesion that depends on protein-protein, protein-carbohydrate, and carbohydrate-carbohydrate interactions.

Role of neuropsin in formation and maturation of Schaffer-collateral L1cam-immunoreactive synaptic boutons.

We report that neuropsin is involved in the synaptogenesis/maturation of orphan and small synaptic boutons in the Schaffer-collateral pathway. Most non-synaptic orphan boutons and a number of immature small synaptic boutons expressed the cell adhesion molecule L1 in presynaptic Schaffer-collateral terminals, whereas mature large boutons on mushroom spines were devoid of L1. The number of L1-immunoreactive boutons was markedly higher in neuropsin-deficient mice than in wild-type mice, whereas there were far fewer mature large boutons. L1-immunoreactive boutons were hypertrophied in the mutant mice. When a recombinant active neuropsin was microinjected into the mutant hippocampus, the number of immunoreactive synaptic boutons reverted to wild-type levels after one day. These results strongly suggest that enzymatically active neuropsin allows a maturational change of L1-immunoreactive small boutons, both orphan and synaptic, and this step may be important in synaptic plasticity based on activity-dependent structural change.

Mechanical properties, proteolytic degradability and biological modifications affect angiogenic process extension into native and modified fibrin matrices in vitro.

During initial stages of wound healing, fibrin clots provide a three-dimensional scaffold that induces cell infiltration and regeneration. Here, L1Ig6, a ligand for alphavbeta3 integrin was covalently incorporated within fibrin matrices to explore it as a matrix-immobilized angiogenic factor. Incorporation at concentrations greater than 1 microg/ml reduced the fibrin crosslink density, as reflected by measurements of elastic modulus and swelling. The influence of crosslink density on endothelial cell process extension was characterized by modulating factor XIII concentrations in the coagulation mixture. At low incorporated concentrations of L1Ig6, it was possible to compensate gel elastic modulus via increased factor XIII, but not at high concentrations of L1Ig6. Similar findings were found when matrix swelling was analyzed. Fibrin crosslink density strongly influenced endothelial cell process extension, fewer and shorter processes were observed at high crosslink density. Matrix metalloproteinases (MMPs) were required for process extension and zymography and Western blots identified MMP-2 but not MMP-9. The amount of active MMP-2 increased for endothelial cells cultured in native and L1Ig6-modified matrices or when stimulated with VEGF-A165. The data indicate that distinct matrix properties can be tailored such that they become biologically stimulating and respond to cellular proteolytic activities, being a prerequisite for potential use of such matrices in biomedical applications.