The slit diaphragm in Drosophila features a bi-layered, fishnet-like architecture

The kidney filters large volumes of blood plasma, relying on the glomerulus for the filtration. The slit diaphragm, a critical component of the glomerulus, is formed between podocytes by the immunoglobulin domain proteins nephrin and Neph1. The molecular architecture of the slit diaphragm has remained elusive for decades. Using cryo-electron tomography on focused ion beam-milled Drosophila nephrocytes, an invertebrate podocyte model, we show that the slit diaphragm adopts a fishnet-like pattern. Comparison of hundreds of slit diaphragm segments reveals that it is bi-layered and highly periodic. Based on the cryo-electron tomography map, we propose four possible models for the arrangement of the Drosophila nephrin ortholog (Sns), and the Drosophila Neph1 ortholog (Kirre), the main components of the slit diaphragm in Drosophila. In each model, precise and consistent homo- and heterophilic interactions between crossing immunoglobulin domains of Sns and Kirre become apparent, with immediate implications for the stability and the assembly of the slit diaphragm. Cryo-electron tomography shows that sns silencing disrupts this fishnet pattern, linking this directly to Drosophila nephrin. After Rab5 silencing, causing Sns mistrafficking and ectopic SD formation, the fishnet pattern appears ectopically as well. Our findings align with observations applying cryo-electron tomography to podocytes in mice, indicating that the molecular architecture is evolutionarily conserved across animals. This highlights the value of the nephrocyte as a podocyte model and establishes a crucial link between the architecture of the slit diaphragm and its function.