Highlights d Intestine-Chip reveals that Shigella infects enterocytes efficiently from the gut lumen d The crypt-like structure of the intestine is critical for Shigella adhesion d Peristaltic motion (mechanical forces) enhances Shigella invasion d Shigella exploits intestine architecture and mechanical forces to maximize infectivity

SummaryReceptor-mediated endocytosis is an essential process used by eukaryotic cells to internalise many molecules. Several clathrinindependent endocytic routes exist, but the molecular mechanism of each pathway remains to be uncovered. The present study focuses on a clathrin-independent dynamin-dependent pathway used by interleukin 2 receptors (IL-2R), essential players of the immune response. Ras-related C3 botulinum toxin substrate (Rac1) and its targets, the p21-activated kinases (Pak), are specific regulators of this pathway, acting on cortactin and actin polymerization. The present study reveals a dual and specific role of phosphatidylinositol 3-kinase (PI3K) in IL-2R endocytosis. Inhibition of the catalytic activity of PI3K strongly affects IL-2R endocytosis, in contrast to transferrin (Tf) uptake, a marker of the clathrin-mediated pathway. Moreover, Vav2, a GTPase exchange factor (GEF) induced upon PI3K activation, is specifically involved in IL-2R entry. The second action of PI3K is through its regulatory subunit, p85a, which binds to and recruits Rac1 during IL-2R internalisation. Indeed, the overexpression of a p85a mutant missing the Rac1 binding motif leads to the specific inhibition of IL-2R endocytosis. The inhibitory effect of this p85a mutant could be rescued by the overexpression of either Rac1 or the active form of Pak, indicating that p85a acts upstream of the Rac1-Pak cascade. Finally, biochemical and fluorescent microscopy techniques reveal an interaction between p85a, Rac1 and IL-2R that is enhanced by IL-2. In summary, our results indicate a key role of class I PI3K in IL-2R endocytosis that creates a link with IL-2 signalling.

Endocytosis controls many functions including nutrient uptake, cell division, migration and signal transduction. A clathrin-and caveolin-independent endocytosis pathway is used by important physiological cargos, including interleukin-2 receptors (IL-2R). However, this process lacks morphological and dynamic data. Our electron microscopy (EM) and tomography studies reveal that IL-2R-pits and vesicles are initiated at the base of protrusions. We identify the WAVE complex as a specific endocytic actor. The WAVE complex interacts with IL-2R, via a WAVE-interacting receptor sequence (WIRS) present in the receptor polypeptide, and allows for receptor clustering close to membrane protrusions. In addition, using total internal reflection fluorescent microscopy (TIRF) and automated analysis we demonstrate that two timely distinct bursts of actin polymerization are required during IL-2R uptake, promoted first by the WAVE complex and then by N-WASP. Finally, our data reveal that dynamin acts as a transition controller for the recruitment of Arp2/3 activators required for IL-2R endocytosis. Altogether, our work identifies the spatiotemporal specific role of factors initiating clathrin-independent endocytosis by a unique mechanism that does not depend on the deformation of a flat membrane, but rather on that of membrane protrusions.