Palmitoylation of Tspan4 is essential for migrasome formation

Migrasomes are key organelles in cell-cell communication, playing a role in embryonic morphogenesis, angiogenesis, coagulation, and mitochondrial homeostasis. Migrasome formation involves the assembly of tetraspanin-enriched microdomains (TEMs) into larger macrodomains (TEMAs), but the underlying mechanisms are unclear. Here, we demonstrate that tetraspanin 4 (Tspan4) is highly palmitoylated at six juxtamembrane cysteines. DHHC6 and PPT1 are identified as the main enzymes regulating this modification. Palmitoylation of Tspan4 is critical for Tspan4 clustering and cholesterol recruitment, enabling the TEM to TEMA assembly required for migrasome formation and stabilization. Notably, the palmitoylation-deficient Tspan4 mutant acts in a dominant-negative manner, suppressing migrasome formation not only in cultured cells but also in zebrafish embryos, where it disrupts left-right asymmetry and organ morphogenesis. Collectively, our study establishes protein palmitoylation as a conserved and essential regulator of migrasome assembly, delineating a mechanism whereby Tspan4 palmitoylation drives cholesterol-dependent membrane macrodomain organization to enable migrasome formation and function.