Supporting data for "Discovery of a Basement Membrane Sensing Mechanism and Pursuit of Its Structural Basis"

Basement membranes are thin sheets of extracellular matrices that support epithelial and endothelial tissues while delivering instructive signals to cells through specific receptor-ligand pairings. Nidogen-1, a ubiquitous basement membrane glycoprotein, is essential for assembling these membranes by linking laminin and collagen IV networks, yet the mechanisms by which cells sense and respond to nidogen-1 remain not fully understood. Here we identify basal cell adhesion molecule (BCAM), a well-known laminin receptor, as a novel, direct, and functional receptor for nidogen-1. Through an unbiased CRISPR activation receptor screen, BCAM emerged as a principal “sensor” of nidogen-1, conferring robust nidogen-1 binding at the cell surface. We confirmed their direct physical interaction in vitro albeit with far weaker affinity than the high-affinity laminin-521-BCAM interaction, but demonstrated a clear functional relevance of their direct engagement to nidogen-1-guided haptotactic migration. This stark contrast indicates cellular context could potentiate the nidogen-1-BCAM interaction, suggesting regulatory mechanisms at play. Parallel efforts to produce recombinant full-length human nidogen-1 for structural studies yielded only trace monomeric material due to severe aggregation and secretion toxicity upon overexpression. Negative-stain electron microscopy of this fraction, nevertheless, confirmed the conserved three-globule dumbbell shape of the intact human molecule seen in previous mouse studies. Our findings uncover a new receptor-ligand axis in basement membrane sensing and reveal how weak interactions can drive potent cellular responses, advancing our understanding of basement membrane biology. Such cues may also influence ECM-driven development and diseases where basement membrane integrity is compromised.

基底膜 (basement membranes) 是支撑上皮与内皮组织的薄层细胞外基质 (extracellular matrices)，可通过特定受体-配体配对向细胞传递指导性信号。巢蛋白-1 (nidogen-1) 是一种广泛分布的基底膜糖蛋白，通过连接层粘连蛋白 (laminin) 与IV型胶原 (collagen IV) 网络参与基底膜组装，但其被细胞感知并产生应答的完整机制尚未完全阐明。本研究鉴定出基底细胞黏附分子 (basal cell adhesion molecule, BCAM)——一种公认的层粘连蛋白受体——作为巢蛋白-1的新型、直接且具备功能活性的受体。通过无偏倚CRISPR激活受体筛选，BCAM被确定为巢蛋白-1的核心“传感器”，可在细胞表面介导强效的巢蛋白-1结合。我们在体外证实了二者的直接物理相互作用，尽管其亲和力远低于高亲和力的层粘连蛋白-521-BCAM配对，但实验证明二者的直接结合与巢蛋白-1引导的趋触性迁移具有明确的功能相关性。这种显著差异提示细胞微环境可增强巢蛋白-1与BCAM的相互作用，暗示存在相关调控机制。为开展结构研究制备重组全长人源巢蛋白-1的平行尝试，因过表达时发生严重聚集并产生分泌毒性，仅获得微量单体产物。不过，对该组分进行负染电子显微镜 (negative-stain electron microscopy) 分析，证实了完整人源分子保守的三球状哑铃结构，该结构此前在小鼠研究中已被观测到。本研究发现揭示了基底膜感知通路中的全新受体-配体轴，阐明了弱相互作用如何驱动强效细胞应答，增进了我们对基底膜生物学的认知。这类信号或可影响细胞外基质 (ECM) 驱动的发育过程，以及基底膜完整性受损相关疾病的发生发展。