A hippocampal mossy fiber synapse has a complex structure in which presynaptic boutons attach to the dendritic trunk by puncta adherentia junctions (PAJs) and wrap multiply-branched spines, forming synaptic junctions. It was previously shown that afadin regulates the formation of the PAJs cooperatively with nectin-1, nectin-3, and N-cadherin. Afadin is a nectin-binding protein with two splice variants, l-afadin and s-afadin: l-afadin has an actin filament-binding domain, whereas s-afadin lacks it. It remains unknown which variant is involved in the formation of the PAJs or how afadin regulates it. We showed here that re-expression of l-afadin, but not s-afadin, in the afadin-deficient cultured hippocampal neurons in which the PAJ-like structure was disrupted, restored this structure as estimated by the accumulation of N-cadherin and αΝ-catenin. The l-afadin mutant, in which the actin filament-binding domain was deleted, or the l-afadin mutant, in which the αΝ-catenin-binding domain was deleted, did not restore the PAJ-like structure. These results indicate that l-afadin, but not s-afadin, regulates the formation of the hippocampal synapse PAJ-like structure through the binding to actin filaments and αN-catenin. We further found here that l-afadin bound αN-catenin, but not γ-catenin, whereas s-afadin bound γ-catenin, but hardly αN-catenin. These results suggest that the inability of s-afadin to form the hippocampal synapse PAJ-like structure is due to its inability to efficiently bind αN-catenin.

海马长满苔藓的纤维突触具有复杂的结构，其中突触前的by突通过点状粘附连接（PAJ）连接到树突状主干，并包裹多支棘，形成突触连接。先前显示，阿法丁与nectin-1，nectin-3和N-cadherin协同调节PAJ的形成。阿法丁是一种具有两个剪接变体的lecta结合蛋白和s-afadin结合蛋白，它具有一个肌动蛋白丝结合域，而s-afadin则缺乏肌动蛋白丝结合域。尚不清楚PAJ的形成涉及哪种变体或阿法丁如何调节它。在这里，我们发现，重新表达-1- afadin，但不支持S-afadin，在afadin缺乏培养的海马神经元（其中PAJ样结构被破坏）恢复了该结构，这是通过N-钙粘着蛋白和αN-连环蛋白的积累来估计的。其中肌动蛋白丝结合结构域被删除的l-afadin突变体，或其中αN-catenin结合结构域被删除的l-afadin突变体没有恢复PAJ样结构。这些结果表明，l-阿法丁而非s-阿法丁通过与肌动蛋白丝和αN-连环蛋白的结合来调节海马突触PAJ样结构的形成。我们在这里进一步发现，l-afadin结合αN-catenin，但不结合γ-catenin，而s-afadin结合γ-catenin，但几乎不结合αN-catenin。这些结果表明，s-阿法丁不能形成海马突触PAJ样结构是由于其不能有效结合αN-catenin。