CD44 restricts EGFR mobility to polarize cytoskeletal signalling modules driving bleb-based migration.

Cells under high confinement migrate efficiently in low-adhesion environments by forming stable, polarized, hydrostatic pressure-driven leader blebs. Here we investigated the basis of polarized bleb morphology in metastatic melanoma cells migrating under low-adhesive and highly confined microenvironments. Using high-resolution live imaging, molecular perturbations and biosensors, we show that EGF signalling through PI3K stabilizes and maintains polarized leader blebs. EGFR and PI3K activities form a gradient within leader blebs that decreases from rear to front, promoting phosphatidylinositol 3,4,5-trisphosphate and Rac1-GTP accumulation at the bleb rear, whereas phosphatidylinositol 4,5-bisphosphate and RhoA-GTP concentrate at the bleb tip, the inverse of the organization observed in integrin-dependent mesenchymal migration. Optogenetic disruption of this gradient triggers bleb retraction, underscoring its functional importance. Mathematical modelling and experiments identified a mechanism whereby during bleb initiation, CD44 and ERM proteins restrict EGFR mobility within a membrane-apposed cortical actin meshwork at the bleb rear, establishing the EGFR-PI3K-Rac gradient. Together, these findings define the biophysical and molecular mechanisms that underlie polarity in bleb-based migration and highlight how alternative spatial organization of signalling modules supports distinct migration modes in different microenvironments.