Tetraspanin 4 stabilizes membrane swellings and facilitates their maturation into migrasomes

Migrasomes are newly discovered cell organelles forming by local swelling of retraction fibers. The migrasome formation critically depends on tetraspanin proteins present in the retraction fiber membranes and is modulated by the membrane tension and bending rigidity. 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. Here using live cell imaging and a biomimetic system for migrasomes and retraction fibers, we reveal that migrasome formation is a two-stage process. At the first stage, which in biomimetic system is mediated by membrane tension, local swellings largely devoid of tetraspanin 4 form on the retraction fibers. At the second stage, tetraspanin 4 molecules migrate toward and onto these swellings, which grow up to several microns in size and transform into migrasomes. This tetraspanin 4 recruitment to the swellings is essential for migrasome growth and stabilization. Based on these findings we propose that the major role of tetraspanin proteins is in stabilizing the migrasome structure, while the migrasome nucleation and initial growth stages can be driven by membrane mechanical stresses.