Ludger Johannes

Shiga toxin induces tubular membrane invaginations for its uptake into cells

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.

Cells Respond to Mechanical Stress by Rapid Disassembly of Caveolae

The functions of caveolae, the characteristic plasma membrane invaginations, remain debated. Their abundance in cells experiencing mechanical stress led us to investigate their role in membrane-mediated mechanical response. Acute mechanical stress induced by osmotic swelling or by uniaxial stretching results in a rapid disappearance of caveolae, in a reduced caveolin/Cavin1 interaction, and in an increase of free caveolins at the plasma membrane. Tether-pulling force measurements in cells and in plasma membrane spheres demonstrate that caveola flattening and disassembly is the primary actin- and ATP-independent cell response that 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 quickly accommodates sudden and acute mechanical stresses.

Evidence for a COP-I-independent transport route from the Golgi complex to the endoplasmic reticulum.

The cytosolic coat-protein complex COP-I interacts with cytoplasmic ‘retrieval’ signals present in membrane proteins that cycle between the endoplasmic reticulum (ER) and the Golgi complex, and is required for both anterograde and retrograde transport in the secretory pathway. Here we study the role of COP-I in Golgi-to-ER transport of several distinct marker molecules. Microinjection of anti-COP-I antibodies inhibits retrieval of the lectin-like molecule ERGIC-53 and of the KDEL receptor from the Golgi to the ER. Transport to the ER of protein toxins, which contain a sequence that is recognized by the KDEL receptor, is also inhibited. In contrast, microinjection of anti-COP-I antibodies or expression of a GTP-restricted Arf-1 mutant does not interfere with Golgi-to-ER transport of Shiga toxin/Shiga-like toxin-1 or with the apparent recycling to the ER of Golgi-resident glycosylation enzymes. Overexpression of a GDP-restricted mutant of Rab6 blocks transport to the ER of Shiga toxin/Shiga-like toxin-1 and glycosylation enzymes, but not of ERGIC-53, the KDEL receptor or KDEL-containing toxins. These data indicate the existence of at least two distinct pathways for Golgi-to-ER transport, one COP-I dependent and the other COP-I independent. The COP-I-independent pathway is specifically regulated by Rab6 and is used by Golgi glycosylation enzymes and Shiga toxin/Shiga-like toxin-1.

Shiga toxins — from cell biology to biomedical applications

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.

PD-1–Expressing Tumor-Infiltrating T Cells Are a Favorable Prognostic Biomarker in HPV-Associated Head and Neck Cancer

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 Foxp3(+) T cells and programmed death-1 (PD-1)(+) T cells in the microenvironment of tumors that were positive or negative for HPV, in two groups that were matched for various clinical and biologic parameters. HPV-positive head and neck cancers were more heavily infiltrated by regulatory T cells and PD-1(+) T cells and the levels of PD-1(+) cells were positively correlated with a favorable clinical outcome. In explaining this paradoxical result, we showed that these PD-1(+) T cells expressed activation markers and were functional after blockade of the PD-1-PD-L1 axis in vitro. Approximately 50% of PD-1(+) tumor-infiltrating T cells lacked Tim-3 expression and may indeed represent activated T cells. In mice, administration of a cancer vaccine increased PD-1 on T cells with concomitant tumor regression. In this setting, PD-1 blockade synergized with vaccine in eliciting antitumor efficacy. Our findings prompt a need to revisit the significance of PD-1-infiltrating T cells in cancer, where we suggest that PD-1 detection may reflect a previous immune response against tumors that might be reactivated by PD-1/PD-L1 blockade.

GM1 structure determines SV40-induced membrane invagination and infection

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.

Tracing the Retrograde Route in Protein Trafficking

Retrograde transport, in which proteins and lipids are shuttled between endosomes and biosynthetic/secretory compartments such as the Golgi apparatus, is crucial for a diverse range of cellular functions. Mechanistic studies that explore the molecular machinery involved in this retrograde trafficking route are shedding light on the functions of transport proteins and are providing fresh insights into possible new therapeutic directions.

Clathrin‐Dependent or Not: Is It Still the Question?

Whether the endocytic uptake of a given molecule is mediated through clathrin‐coated pits or not is a classical criterion used to characterize its endocytic pathway(s). Hence, clathrin‐dependent endocytosis has been associated with highly selective and efficient uptake, whereas clathrin‐independent endocytosis appeared to be confined to bulk uptake of fluid‐phase markers. This scholastic view has recently been challenged using newly developed molecular tools that allow for the first time a functional and mechanistic analysis of these less well‐characterized clathrin‐independent pathways, including caveolar uptake and macropinocytosis. Furthermore, several studies point to a critical role of lateral lipid asymmetry – lipid rafts/microdomains – in membrane sorting. We will discuss the potential role of these structures in endocytosis and the possibility that differential sorting at the plasma membrane predisposes the ensuing intracellular fate of a given molecule as well as its physiological function.

The GTPase Rab3a negatively controls calcium-dependent exocytosis in neuroendocrine cells.

There is accumulating evidence that small GTPases of the rab family regulate intracellular vesicle traffic along biosynthetic and endocytotic pathways in eukaryotic cells. It has been suggested that Rab3a, which is associated with synaptic vesicles in neurons and with secretory granules in adrenal chromaffin cells, might regulate exocytosis. We report here that overexpression in PC12 cells of Rab3a mutant proteins defective in either GTP hydrolysis or in guanine nucleotide binding inhibited exocytosis, as measured by a double indirect immunofluorescence assay. Moreover, injection of the purified mutant proteins into bovine adrenal chromaffin cells also inhibited exocytosis, as monitored by membrane capacitance measurements. Finally, the electrophysiological approach showed that bovine chromaffin cells which were intracellularly injected with antisense oligonucleotides targeted to the rab3a messenger exhibited an increasing potential to respond to repetitive stimulations. In contrast, control cells showed a phenomenon of desensitization. These results provide clear evidence that Rab3a is involved in regulated exocytosis and suggest that Rab3a is a regulatory factor that prevents exocytosis from occurring unless secretion is triggered. Furthermore, it is proposed that Rab3a is involved in adaptive processes such as response habituation.

Retrograde Transport of KDEL-bearing B-fragment of Shiga Toxin

To investigate retrograde transport along the biosynthetic/secretory pathway, we have constructed a recombinant Shiga toxin B-fragment carrying an N-glycosylation site and a KDEL retrieval motif at its carboxyl terminus (B-Glyc-KDEL). After incubation with HeLa cells, B-Glyc-KDEL was progressively glycosylated in the endoplasmic reticulum (ER) and remained stably associated with this compartment. B-fragment with a nonfunctional KDEL sequence (B-Glyc-KDELGL) was glycosylated with about the same kinetics as B-Glyc-KDEL but localized at steady state to the Golgi apparatus. Morphological studies showed that B-Glyc-KDEL was delivered from the plasma membrane, via endosomes and the cisternae of the Golgi apparatus, to the ER. Moreover, the addition of a sulfation site allowed us to show that B-Glyc-KDEL on transit to the ER entered the Golgi apparatus through the trans-Golgi network. Transport of B-Glyc-KDEL to the ER was slowed down by nocodazole, indicating that microtubules are important for the retrograde pathway. Our results document the existence of a continuous pathway from the plasma membrane to the endoplasmic reticulum via the Golgi apparatus and show that a fully folded exogenous protein arriving in the endoplasmic reticulum via this pathway can undergo N-glycosylation.