Structure of cell–cell adhesion mediated by the Down syndrome cell adhesion molecule

The Down syndrome cell adhesion molecule (DSCAM) belongs to the immunoglobulin superfamily (IgSF) and plays important roles in neural development. It has a large ectodomain, including 10 Ig-like domains and 6 fibronectin III (FnIII) domains. Previous data have shown that DSCAM can mediate cell adhesion by forming homophilic dimers between cells and contributes to self-avoidance of neurites or neuronal tiling, which is important for neural network formation. However, the organization and assembly of DSCAM at cell adhesion interfaces has not been fully understood. Here we combine electron microscopy and other biophysical methods to characterize the structure of the DSCAM-mediated cell adhesion and generate three-dimensional views of the adhesion interfaces of DSCAM by electron tomography. The results show that mouse DSCAM forms a regular pattern at the adhesion interfaces. The Ig-like domains contribute to both trans homophilic interactions and cis assembly of the pattern, and the FnIII domains are crucial for the cis pattern formation as well as the interaction with the cell membrane. By contrast, no obvious assembly pattern is observed at the adhesion interfaces mediated by mouse DSCAML1 or Drosophila DSCAMs, suggesting the different structural roles and mechanisms of DSCAMs in mediating cell adhesion and neural network formation.

The Down syndrome cell adhesion molecule (DSCAM) was initially identified by isolating genes responsible for the phenotypes of Down syndrome (1), a genetic disease featured with cognitive and learning deficits (2). The DSCAM gene locates at the Down syndrome critical region (DSCR) on human chromosome 21 and is broadly expressed in nervous system (1, 3, 4), and its expression increases in patients with Down syndrome and in mouse models (3, 5, 6). Therefore, DSCAM has been hypothesized as a candidate gene associated with neurodevelopmental disorders and its dysregulation may lead to cognitive impairment and intellectual disability in Down syndrome (7), but the mechanism for the association between DSCAM and Down syndrome is still poorly understood.In invertebrates, Drosophila DSCAM1 (dDSCAM1) undergoes extensive alternative splicing by generating 38,016 isoforms with distinct recognition specificity (8–10), which is crucial for isoneuronal avoidance (11, 12). Loss of function or overexpression of dDSCAM1 in mutant flies causes defects or disorders in dendrite arborization (13, 14), axon guidance (15, 16), axon branching (17, 18), and synaptic targeting (11, 19, 20). Drosophila DSCAM2 (dDSCAM2) and DSCAM4 (dDSCAM4) also function in neural network formation by directing dendritic targeting but without the massive isoform diversity (21), and dDSCAM2 can mediate axonal tiling as well (22). Aplysia DSCAM (aDSCAM) is involved in transsynaptic protein localization (23).In vertebrates, two paralogous DSCAM genes, DSCAM and DSCAML1 (DSCAM-LIKE1) were identified (1, 24) and both of them could promote isoneuronal and homotypic self-avoidance (25, 26). In mouse, neurons expressing DSCAM (mDSCAM) or DSCAML1 (mDSCAML1) mutants may lose their mosaic pattern and neurite arborization (26, 27). Although the mechanism of mDSCAM-mediated self-avoidance remains unclear, it has been suggested that mDSCAM may function by masking the adhesion mediated by certain cadherin superfamily members (28). In addition, mDSCAM may also regulate neurite outgrowth (29, 30), promote cell death (31, 32), and control neuronal delamination (33). Studies have also shown that it could direct lamina-specific synaptic connections in chick (34) and be involved in cell movement in zebrafish (35). In contrast to dDSCAM1, the extensive alternative splicing has not been found for DSCAM in vertebrates, suggesting the different roles in the formation of neuronal circuits.DSCAM belongs to the immunoglobulin superfamily (IgSF) and consists of 10 immunoglobulin-like (Ig-like) domains, 6 type III fibronectin (FnIII) domains, a transmembrane domain, and a cytoplasmic domain (Fig. 1A). The domain arrangements of DSCAMs from invertebrates and vertebrates are quite similar, and the amino acid sequence identities of DSCAM among homologs are 98% between mDSCAM and hDSCAM (human), 59% between mDSCAM and mDSCAML1, and 33% between mDSCAM and dDSCAM1. The crystal structures of the N-terminal Ig-like domains of dDSCAM1 have been solved (36, 37). The eight N-terminal Ig-like domains form a dimer with a double-S–shaped conformation, which is critical for the homophilic cell adhesion (36). However, it is unclear whether the N-terminal Ig-like domains of mDSCAM and mDSCAML1 adopt a similar conformation to dDSCAM1, and the roles of other domains of DSCAM in cell adhesion remain elusive.Open in a separate windowFig. 1.Conformations of the ectodomains of mDSCAM, mDSCAML1, and dDSCAM1. (A) Diagrams of mDSCAM, mDSCAML1, and dDSCAM1 (ovals, Ig-like domains; rounded rectangles, FnIII domains; vertical rectangles, transmembrane domains; rectangles, cytoplasmic domains). (B–D) Negative staining EM images show the particles of mDSCAM-D_1–8, mDSCAM-D_9–16, and mDSCAM-D_1–16, respectively (Top, red arrows). (Scale bar, 50 nm.) The selected particles (Middle; the particles are picked from different images) and their contours (Bottom) are also listed. (Scale bar, 10 nm.) The schematic models of mDSCAM-D_1–8, mDSCAM-D_9–16, and mDSCAM-D_1–16 are shown in the Top Left Insets, respectively. (E–G) Negative staining EM images show the particles of mDSCAML1-D_1–8, mDSCAML1-D_9–16, and mDSCAML1-D_1–16, respectively (Top, red arrows). (Scale bar, 50 nm.) The selected particles (Middle) and their contours (Bottom) are also listed. (Scale bar, 10 nm.) The schematic models of mDSCAML1-D_1–8, mDSCAML1-D_9–16, and mDSCAML1-D_1–16 are shown in the Top Left Insets, respectively. (H–J) Negative staining EM images show the particles of dDSCAM1-D_1–8, dDSCAM1-D_9–16, and dDSCAM1-D_1–16, respectively (Top, red arrows). (Scale bar, 50 nm.) The selected particles (Middle) and their contours (Bottom) are also listed. (Scale bar, 10 nm.) The schematic models of dDSCAM1-D_1–8, dDSCAM1-D_9–16, and dDSCAM1-D_1–16 are shown in the Top Left Insets, respectively.Recently, electron tomography (ET) has become a powerful tool to provide three-dimensional (3D) views of biological samples (38, 39). By combining correlative light and electron microscopy (CLEM), high-pressure freezing and freeze substitution (HPF-FS), ultrathin sectioning and ET, the 3D structure of cellular or tissue samples can be reconstructed at nanometer resolution, revealing the molecular architecture of macromolecules in situ (40–43). Here we characterize the structures of mDSCAM, mDSCAML1, and dDSCAMs by electron microscopy (EM) as well as other biochemical and biophysical methods and reconstruct the 3D views of the mDSCAM-mediated adhesion interface by electron tomography, thereby unveiling the in situ structural model and the potential mechanism of cell adhesion by DSCAM.