Phase Separation of MAGI2-Mediated Complex Underlies Formation of Slit Diaphragm Complex in Glomerular Filtration Barrier

Significance Statement Slit diaphragms between podocytes play a critical role in maintaining the filtration function in kidney. At each slit diaphragm there is an electron-dense junctional plaque crucial for slit diaphragm integrity and podocyte signal transduction. However, the molecular basis underlying slit diaphragm assembly is not well understood. Here, we demonstrate that MAGI2, a unique MAGUK family scaffold protein at slit diaphragm, can autonomously undergo liquid-liquid phase separation. Multivalent interactions among the MAGI2-Dendrin-CD2AP complex drive the formation of the slit diaphragm condensates at physiologic conditions. The reconstituted slit diaphragm condensates can effectively enrich Nephrin. A nephrotic syndrome–associated mutation of MAGI2 interferes with slit diaphragm condensate formation, leading to impaired recruitment of Nephrin. Therefore, the electron-dense slit diaphragm assembly might form via phase separation of the slit diaphragm complex. Background Slit diaphragm is a specialized adhesion junction between the opposing podocytes, establishing the final filtration barrier to urinary protein loss. At the cytoplasmic insertion site of each slit diaphragm there is an electron-dense and protein-rich cellular compartment that is essential for slit diaphragm integrity and signal transduction. Mutations in genes that encode components of this membrane-less compartment have been associated with glomerular diseases. However, the molecular mechanism governing formation of compartmentalized slit diaphragm assembly remains elusive. Methods We systematically investigated the interactions between key components at slit diaphragm, such as MAGI2, Dendrin, and CD2AP, through a combination of biochemical, biophysical, and cell biologic approaches. Results We demonstrated that MAGI2, a unique MAGUK family scaffold protein at slit diaphragm, can autonomously undergo liquid-liquid phase separation. Multivalent interactions among the MAGI2-Dendrin-CD2AP complex drive the formation of the highly dense slit diaphragm condensates at physiologic conditions. The reconstituted slit diaphragm condensates can effectively recruit Nephrin. A nephrotic syndrome–associated mutation of MAGI2 interfered with formation of the slit diaphragm condensates, thus leading to impaired enrichment of Nephrin. Conclusions Key components at slit diaphragm ( e.g. , MAGI2 and its complex) can spontaneously undergo phase separation. The reconstituted slit diaphragm condensates can be enriched in adhesion molecules and cytoskeletal adaptor proteins. Therefore, the electron-dense slit diaphragm assembly might form via phase separation of core components of the slit diaphragm in podocytes.