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To investigate the morphology of these RF puncta, we carried out scanning electron microscopy (SEM) analysis. On the far end of RFs, large numbers of round, small extracellular vesicles are left behind; in many cases these vesicles are linked together, like a string of beads (Fig. 1b). To check whether these vesicles have a continuous membrane, we carried out transmission electron microscopy (TEM). TEM revealed that these vesicles indeed have a continuous membrane (Fig. 1c), and thus they are true vesicles instead of broken membrane fragments. Cryo-TEM analysis, which can better preserve the morphology of delicate membrane structures, further confirmed that the RF puncta are small extracellular vesicles (Fig. 1d). These vesicles are much smaller than migrasomes, with size varing from 50 nm to 250 nm (Fig. 1e). These data suggest that when RFs break up, they form a type of previously unreported extracellular vesicle. Similar to migrasomes, these small extracellular vesicles are migration-dependent: treating cells with blebbistatin blocks their formation (Supplementary information, Fig. S1a–c). Thus, we named these vesicles “retractosomes”, to reflect their RF and plasma membrane origin. The beads-on-a-string structure is frequently observed under cryo-TEM (Fig. 1f). This indicates that, before breaking, the RF goes through a stage of morphological transformation, with sections of the RF bulging into small vesicles, which likely form retractosomes. Wheat germ agglutinin (WGA) effectively labels RFs and migrasomes.3 WGA staining revealed that retractosomes are formed by all the migratory cells we tested, none of which ecotopically expressed Tspan4 (Supplementary information, Fig. S1d). Moreover, we observed formation of retractosomes in various in vivo settings, including circulating neutrophils in mice, and embryonic cells during zebrafish gastrulation (Supplementary information, Fig. S2a, b).

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