SUMMARY The precise role of caveolae, the characteristic plasma membrane invaginations present in many cells, still remains debated. The high density of caveolae in cells experiencing mechanical stress led us to investigate their role in membrane-mediated mechanical response. Acute mechanical stress induced by cell osmotic swelling or by uniaxial stretching results in the immediate disappearance of caveolae, which is associated with a reduced caveolin/Cavin1 interaction, and an increase of free caveolins at the plasma membrane. Tether pulling force measurements in live cells and in plasma membrane spheres demonstrate that caveola flattening and disassembly is the primary actin and ATP-independent cell response which buffers membrane tension surges during mechanical stress. Conversely, stress release leads to complete caveola reassembly in an actin and ATP-dependent process. The absence of a functional caveola reservoir in myotubes from muscular dystrophic patients enhanced membrane fragility under mechanical stress. Our findings support a new role for caveolae as a physiological membrane reservoir that allows cells to quickly accommodate sudden and acute mechanical stresses.

Clathrin seems to be dispensable for some endocytic processes and, in several instances, no cytosolic coat protein complexes could be detected at sites of membrane invagination. Hence, new principles must in these cases be invoked to account for the mechanical force driving membrane shape changes. Here we show that the Gb3 (glycolipid)-binding B-subunit of bacterial Shiga toxin induces narrow tubular membrane invaginations in human and mouse cells and model membranes. In cells, tubule occurrence increases on energy depletion and inhibition of dynamin or actin functions. Our data thus demonstrate that active cellular processes are needed for tubule scission rather than tubule formation. We conclude that the B-subunit induces lipid reorganization that favours negative membrane curvature, which drives the formation of inward membrane tubules. Our findings support a model in which the lateral growth of B-subunit-Gb3 microdomains is limited by the invagination process, which itself is regulated by membrane tension. The physical principles underlying this basic cargo-induced membrane uptake may also be relevant to other internalization processes, creating a rationale for conceptualizing the perplexing diversity of endocytic routes.

The molecular mechanisms underlying early/recycling endosomes-to-TGN transport are still not understood. We identified interactions between the TGN-localized putative t-SNAREs syntaxin 6, syntaxin 16, and Vti1a, and two early/recycling endosomal v-SNAREs, VAMP3/cellubrevin, and VAMP4. Using a novel permeabilized cell system, these proteins were functionally implicated in the post-Golgi retrograde transport step. The function of Rab6a' was also required, whereas its closely related isoform, Rab6a, has previously been implicated in Golgi-to-endoplasmic reticulum transport. Thus, our study shows that membrane exchange between the early endocytic and the biosynthetic/secretory pathways involves specific components of the Rab and SNARE machinery, and suggests that retrograde transport between early/recycling endosomes and the endoplasmic reticulum is critically dependent on the sequential action of two members of the Rab6 subfamily.

Shiga toxin and other toxins of this family can escape the endocytic pathway and reach the Golgi apparatus. To synchronize endosome to Golgi transport, Shiga toxin B-fragment was internalized into HeLa cells at low temperatures. Under these conditions, the protein partitioned away from markers destined for the late endocytic pathway and colocalized extensively with cointernalized transferrin. Upon subsequent incubation at 37°C, ultrastructural studies on cryosections failed to detect B-fragment–specific label in multivesicular or multilamellar late endosomes, suggesting that the protein bypassed the late endocytic pathway on its way to the Golgi apparatus. This hypothesis was further supported by the rapid kinetics of B-fragment transport, as determined by quantitative confocal microscopy on living cells and by B-fragment sulfation analysis, and by the observation that actin- depolymerizing and pH-neutralizing drugs that modulate vesicular transport in the late endocytic pathway had no effect on B-fragment accumulation in the Golgi apparatus. B-fragment sorting at the level of early/recycling endosomes seemed to involve vesicular coats, since brefeldin A treatment led to B-fragment accumulation in transferrin receptor–containing membrane tubules, and since B-fragment colocalized with adaptor protein type 1 clathrin coat components on early/recycling endosomes. Thus, we hypothesize that Shiga toxin B-fragment is transported directly from early/recycling endosomes to the Golgi apparatus. This pathway may also be used by cellular proteins, as deduced from our finding that TGN38 colocalized with the B-fragment on its transport from the plasma membrane to the TGN.

The efficacy of the anti-cancer immunomodulatory agent cyclophosphamide (CTX) relies on intestinal bacteria. How and which relevant bacterial species are involved in tumor immunosurveillance, and their mechanism of action are unclear. Here, we identified two bacterial species, Enterococcus hirae and Barnesiella intestinihominis that are involved during CTX therapy. Whereas E. hirae translocated from the small intestine to secondary lymphoid organs and increased the intratumoral CD8/Treg ratio, B. intestinihominis accumulated in the colon and promoted the infiltration of IFN-γ-producing γδT cells in cancer lesions. The immune sensor, NOD2, limited CTX-induced cancer immunosurveillance and the bioactivity of these microbes. Finally, E. hirae and B. intestinihominis specific-memory Th1 cell immune responses selectively predicted longer progression-free survival in advanced lung and ovarian cancer patients treated with chemo-immunotherapy. Altogether, E. hirae and B. intestinihominis represent valuable "oncomicrobiotics" ameliorating the efficacy of the most common alkylating immunomodulatory compound.

Head and neck cancers positive for human papillomavirus (HPV) have a more favorable clinical outcome than HPV-negative cancers, but it is unknown why this is the case. We hypothesized that prognosis was affected by intrinsic features of HPV-infected tumor cells or differences in host immune response. In this study, we focused on a comparison of regulatory

Shiga toxin-producing Escherichia coli is an emergent pathogen that can induce haemolytic uraemic syndrome. The toxin has received considerable attention not only from microbiologists but also in the field of cell biology, where it has become a powerful tool to study intracellular trafficking. In this Review, we summarize the Shiga toxin family members and their structures, receptors, trafficking pathways and cellular targets. We discuss how Shiga toxin affects cells not only by inhibiting protein biosynthesis but also through the induction of signalling cascades that lead to apoptosis. Finally, we discuss how Shiga toxins might be exploited in cancer therapy and immunotherapy.

Incoming simian virus 40 (SV40) particles enter tight-fitting plasma membrane invaginations after binding to the carbohydrate moiety of GM1 gangliosides in the host cell plasma membrane through pentameric VP1 capsid proteins. This is followed by activation of cellular signalling pathways, endocytic internalization and transport of the virus via the endoplasmic reticulum to the nucleus. Here we show that the association of SV40 (as well as isolated pentameric VP1) with GM1 is itself sufficient to induce dramatic membrane curvature that leads to the formation of deep invaginations and tubules not only in the plasma membrane of cells, but also in giant unilamellar vesicles (GUVs). Unlike native GM1 molecules with long acyl chains, GM1 molecular species with short hydrocarbon chains failed to support such invagination, and endocytosis and infection did not occur. To conceptualize the experimental data, a physical model was derived based on energetic considerations. Taken together, our analysis indicates that SV40, other polyoma viruses and some bacterial toxins (Shiga and cholera) use glycosphingolipids and a common pentameric protein scaffold to induce plasma membrane curvature, thus directly promoting their endocytic uptake into cells.