GPI‑anchored protein nanoclusters license migrasome expansion and serve as exocytic platforms for migrasome

Migrasomes are dynamic organelles that form on migrating cells and mediate intercellular communication through secretory cargo release. Expansion of migrasomes has classically been attributed to tetraspanin-enriched microdomains (TEMs). Here we show that nanoclusters of glycosylphosphatidylinositol-anchored proteins (GPI-APs) act upstream to license expansion, while TEMs provide the stabilizing scaffold. We find that GPI-AP biosynthesis is important for migrasome formation, and that insertion of the GPI anchor alone is sufficient to drive precursor expansion, producing unstable migrasomes that retract. Stimulated emission depletion (STED) microscopy resolves a meshwork of GPI-AP nanoclusters interlocked with, yet largely segregated from, tetraspanin domains in stable migrasomes. Exocytic machinery localizes to GPI-AP regions, where secretion occurs. In cells that naturally generate unstable migrasomes, elevated tetraspanin levels convert them into stable vesicles. We propose a two-module architecture generating unstable, secretion-specialized migrasomes in some cells and stable migrasomes as extracellular extensions of the secretory pathway in others. This study reveals GPI-anchored protein nanoclusters license migrasome expansion and serve as exocytic platforms, enabling local secretion; while tetraspanin-enriched microdomains stabilize them for long-range communication in migrating cells.