Abstract Migrasomes are recently discovered signalling organelles, enriched with tetraspanin proteins (TSPAN) 1 . They form by local swelling of retraction fibers, the cylindrical protrusions of cell membranes that form as a result of cell migration along external substrates. Migrasomes can grow up to several micrometers in diameter 2 , and allow cells to release contents such as chemokines at specific locations, hence, transmitting signals to surrounding cells through the relevant chemokine receptors. Recently, evidence emerged showing that migrasomes play essential roles in fundamental cellular processes such transfer of mRNA and proteins 3 , organ morphogenesis 4 , and mitochondria quality control 5 . Thus, understanding the mechanism of migrasome biogenesis is of outstanding importance. Previously, it was established that the molecules crucial for migrasome formation are tetraspanin proteins and cholesterol forming macrodomains in the migrasome membrane, while the physical forces driving local swelling of the retraction fibers originate from membrane tension and bending rigidity 1 . Yet, it remained unknown how and in which time sequence these factors are involved in migrasome nucleation, growth, and stabilization, and what are the possible intermediate stages of migrasome biogenesis.

Tetraspanin 4 mediates migrasome formation via a two-stage mechanism