LAUSR

Dear Editor, Migrasomes are newly discovered cellular organelles, first described in 2015. Migrasomes are vesicles with diameters of 0.5–3 μm which are generated during cell migration. Cellular contents such as cytosolic components are actively transported to migrasomes and eventually released extracellularly. Thus, migrasomes are proposed as a mechanism for cell–cell communications. Migrasomes are essential for organ morphogenesis during zebrafish embryonic development. Moreover, it has been shown that migrasomes are detected in human serum. Assembly of tetraspaninand cholesterol-enriched membrane microdomains into micron-scale macrodomains are necessary and sufficient for migrasome formation. In addition, integrins provide the adhesion force for retraction fiber tethering, which are pivotal in migrasome biogenesis process. Pairing of integrins with specific ECM partners for proper adhesion is a determinant for migrasome formation. So far, the systematic studies on detailed regulatory mechanisms of migrasome biogenesis are still lacking. We designed a chemical genetic screening to identify chemical compounds and their protein targets which interfered with migrasome formation. We used NRK cells stably expressing TSPAN4-GFP to generate migrasomes in 96-well plates and treated with compounds. A diagram of the workflow used for screening is shown in Fig. 1a. Image acquisition was achieved automatically. To assay migrasome generation, the number of cells and migrasomes was quantified and the average migrasome number per cell was calculated. It has been reported that fibronectin (FN) promotes migrasome formation. Using our assay, we tested the effect of increasing the concentration of fibronectin. The average migrasome number per cell increased as the fibronectin concentration increased (Supplementary Fig. S1a). GLPG0187 is the inhibitor of integrin α5β1, which is essential for migrasome biogenesis. GLPG0187 inhibited migrasome biogenesis in a concentration-dependent manner without cytotoxicity (Supplementary Fig. S1b). Based on these results, we concluded that the assay was sufficiently robust and we proceeded with high-throughput screening. We performed the assay with 2240 compounds at a concentration of 10 μM in a 96-well plate format. We identified 507 compounds which had significant inhibitory effect on migrasome generation (Fig. 1b). Indeed, we found that 463 out of the 507 hits showed no or less retraction fibers indicating defect of cell migration (Fig. 1b, Supplementary Fig. S1c). This is a confirmation of the notion that migrasome formation is migration dependent. We focused on the 12 candidates which show significant decreased migrasome number with relatively normal retraction fiber (Fig. 1b, Supplementary Fig. S1c). We performed secondary screening of the 12 candidates. SAR407899 showed stable inhibition of migrasome formation without cytotoxicity or impaired cell proliferation (Fig. 1c, d). The number of migrasomes/100 μm was also significantly reduced compared to DMSO-treated cells (Fig. 1e), which excluded the effect of retraction fiber and cell migration on migrasome formation. In zebrafish embryos, generation of migrasomes has been observed during gastrulation. Migrasomes were shown to be essential for organ morphogenesis during embryonic development. We thus tested the inhibitory