Cytokine receptor cluster size impacts its endocytosis and signaling.

The interleukin-2 receptor (IL-2R) is a cytokine receptor essential for immunity that transduces proliferative signals regulated by its uptake and degradation. IL-2R is a well-known marker of clathrin-independent endocytosis (CIE), a process devoid of any coat protein, raising the question of how the CIE vesicle is generated. Here, we investigated the impact of IL-2Rγ clustering in its endocytosis. Combining total internal reflection fluorescence (TIRF) live imaging of a CRISPR-edited T cell line endogenously expressing IL-2Rγ tagged with green fluorescent protein (GFP), with multichannel imaging, single-molecule tracking, and quantitative analysis, we were able to decipher IL-2Rγ stoichiometry at the plasma membrane in real time. We identified three distinct IL-2Rγ cluster populations. IL-2Rγ is secreted to the cell surface as a preassembled small cluster of three molecules maximum, rapidly diffusing at the plasma membrane. A medium-sized cluster composed of four to six molecules is key for IL-2R internalization and is promoted by interleukin 2 (IL-2) binding, while larger clusters (more than six molecules) are static and inefficiently internalized. Moreover, we identified membrane cholesterol and the branched actin cytoskeleton as key regulators of IL-2Rγ clustering and IL-2-induced signaling. Both cholesterol depletion and Arp2/3 inhibition lead to the assembly of large IL-2Rγ clusters, arising from the stochastic interaction of receptor molecules in close correlation with their enhanced lateral diffusion at the membrane, thus resulting in a default in IL-2R endocytosis. Despite similar clustering outcomes, while cholesterol depletion leads to a sustained IL-2-dependent signaling, Arp2/3 inhibition prevents signal initiation. Taken together, our results reveal the importance of cytokine receptor clustering for CIE initiation and signal transduction.

Bioengineered Human Organ-on-Chip Reveals Intestinal Microenvironment and Mechanical Forces Impacting Shigella Infection.

Intestinal epithelial cells are constantly exposed to pathogens and mechanical forces. However, the impact of mechanical forces on infections leading to diarrheal diseases remains largely unknown. Here, we addressed whether flow and peristalsis impact the infectivity of the human pathogen Shigella within a 3D colonic epithelium using Intestine-Chip technology. Strikingly, infection is significantly increased and minimal bacterial loads are sufficient to invade enterocytes from the apical side and trigger loss of barrier integrity, thereby shifting the paradigm about early stage Shigella invasion. Shigella quickly colonizes epithelial crypt-like invaginations and demonstrates the essential role of the microenvironment. Furthermore, by modulating the mechanical forces of the microenvironment, we find that peristalsis impacts Shigella invasion. Collectively, our results reveal that Shigella leverages the intestinal microenvironment by taking advantage of the microarchitecture and mechanical forces to efficiently invade the intestine. This approach will enable molecular and mechanistic interrogation of human-restricted enteric pathogens.

Shigella promotes major alteration of gut epithelial physiology and tissue invasion by shutting off host intracellular transport.

Intracellular trafficking pathways in eukaryotic cells are essential to maintain organelle identity and structure, and to regulate cell communication with its environment. Shigella flexneri invades and subverts the human colonic epithelium by the injection of virulence factors through a type 3 secretion system (T3SS). In this work, we report the multiple effects of two S. flexneri effectors, IpaJ and VirA, which target small GTPases of the Arf and Rab families, consequently inhibiting several intracellular trafficking pathways. IpaJ and VirA induce large-scale impairment of host protein secretion and block the recycling of surface receptors. Moreover, these two effectors decrease clathrin-dependent and -independent endocytosis. Therefore, S. flexneri infection induces a global blockage of host cell intracellular transport, affecting the exchange between cells and their external environment. The combined action of these effectors disorganizes the epithelial cell polarity, disturbs epithelial barrier integrity, promotes multiple invasion events, and enhances the pathogen capacity to penetrate into the colonic tissue in vivo.

Membrane protrusion powers clathrin-independent endocytosis of interleukin-2 receptor.

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 spatio-temporal 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.

The signalling factor PI3K is a specific regulator of the clathrin-independent dynamin-dependent endocytosis of IL-2 receptors.

Receptor-mediated endocytosis is an essential process used by eukaryotic cells to internalise many molecules. Several clathrin-independent 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, p85α, which binds to and recruits Rac1 during IL-2R internalisation. Indeed, the overexpression of a p85α mutant missing the Rac1 binding motif leads to the specific inhibition of IL-2R endocytosis. The inhibitory effect of this p85α mutant could be rescued by the overexpression of either Rac1 or the active form of Pak, indicating that p85α acts upstream of the Rac1-Pak cascade. Finally, biochemical and fluorescent microscopy techniques reveal an interaction between p85α, 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.

Ectodomain shedding of interleukin-2 receptor beta and generation of an intracellular functional fragment.

Interleukin-2 (IL-2) regulates different functions of various lymphoid cell subsets. These are mediated by its binding to the IL-2 receptor (IL-2R) composed of three subunits (IL2-Ralpha, -beta, and -gamma(c)). IL-2Rbeta is responsible for the activation of several signaling pathways. Ectodomain shedding of membrane receptors is thought to be an important mechanism for down-regulation of cell surface receptor abundance but is also emerging as a mechanism that cell membrane-associated molecules require for proper action in vivo. Here, we demonstrate that IL-2Rbeta is cleaved in cell lines of different origin, including T cells, generating an intracellular 37-kDa fragment (37beta ic) that comprises the full intracellular C-terminal and transmembrane domains. Ectodomain shedding of IL-2Rbeta decreases in a mutant deleted of the juxtamembrane region, where cleavage is predicted to occur, and is inhibited by tissue inhibitor of metalloproteases-3. 37Beta ic is tyrosine-phosphorylated and associates with STAT-5, a canonic signal transducer of IL-2R. Finally, lymphoid cell transfection with a truncated form of IL-2Rbeta mimicking 37beta ic increases their proliferation. These data indicate that IL-2Rbeta is subject to ectodomain shedding generating an intracellular fragment biologically functional, because (i) it is phosphorylated, (ii) it associates with STAT5A, and (iii) it increases cell proliferation.

NEDD4-2 associates with gamma(c) and regulates its degradation rate.

Interleukin-2 (IL-2) is a cytokine that regulates proliferation, differentiation and survival of various lymphoid cell subsets. Its actions are mediated through its binding to the IL-2 receptor which is composed of three subunits (IL-2Ralpha, IL-2Rbeta and gamma(c)). Only beta and gamma(c) have been shown to transduce intra cellular signals. The gamma(c) chain is shared by the interleukin-2, 4, 7, 9, 15 and 21 receptors, and is essential for lymphocyte functions. The regulation of gamma(c) expression level is therefore critical for the ability of cells to respond to these cytokines. In the present work, we show that the IL-2R constitutively associates with the ubiquitin ligase NEDD4-2, and to a lesser extent NEDD4-1. We identified the specific binding site on gamma(c). And we show that the loss of NEDD4 association on gamma(c) is accompanied by a dramatic increase of the half-life of the receptor subunit.

Ubiquitination of the common cytokine receptor gammac and regulation of expression by an ubiquitination/deubiquitination machinery.

The common cytokine receptor gamma(c) is shared by the interleukin-2, -4, -7, -9, -15, and -21 receptors, and is essential for lymphocyte proliferation and survival. The regulation of gamma(c) receptor expression level is therefore critical for the ability of cells to respond to these cytokines. We previously reported that gamma(c) is efficiently constitutively internalized and addressed towards a degradation endocytic compartment. We show that gamma(c) is ubiquitinated and also associated to ubiquitinated proteins. We report that the ubiquitin-ligase c-Cbl induces gamma(c) down-regulation. In addition, the ubiquitin-hydrolase, DUB-2, counteracts the effect of c-Cbl on gamma(c) expression. We show that an increase in DUB-2 expression correlates with an increased gamma(c) half-life, resulting in the up-regulation of the receptor. Altogether, we show that gamma(c) is the target of an ubiquitination mechanism and its expression level can be regulated through the activities of a couple of ubiquitin-ligase/ubiquitin-hydrolase enzymes, namely c-Cbl/DUB-2.