In colorectal cancer patients, a high density of cytotoxic CD8 T cells in tumors is associated with better prognosis. Using a Stat3 loss-of-function approach in two wnt/β-catenin-dependent autochthonous models of sporadic intestinal tumorigenesis, we unravel a complex intracellular process in intestinal epithelial cells (IECs) that controls the induction of a CD8 T cell based adaptive immune response. Elevated mitophagy in IECs causes iron(II)-accumulation in epithelial lysosomes, in turn, triggering lysosomal membrane permeabilization. Subsequent release of proteases into the cytoplasm augments MHC class I presentation and activation of CD8 T cells via cross-dressing of dendritic cells. Thus, our findings highlight a so-far-unrecognized link between mitochondrial function, lysosomal integrity, and MHC class I presentation in IECs and suggest that therapies triggering mitophagy or inducing LMP in IECs may prove successful in shifting the balance toward anti-tumor immunity in colorectal cancer.

We investigate the microtubule polymerization dynamics with catastrophe and rescue events for three different confinement scenarios, which mimic typical cellular environments: (i) The microtubule is confined by rigid and fixed walls, (ii) it grows under constant force, and (iii) it grows against an elastic obstacle with a linearly increasing force. We use realistic catastrophe models and analyze the microtubule dynamics, the resulting microtubule length distributions, and force generation by stochastic and mean field calculations; in addition, we perform stochastic simulations. Freely growing microtubules exhibit a phase of bounded growth with finite microtubule length and a phase of unbounded growth. The main results for the three confinement scenarios are as follows: (i) In confinement by fixed rigid walls, we find exponentially decreasing or increasing stationary microtubule length distributions instead of bounded or unbounded phases, respectively. We introduce a realistic model for wall-induced catastrophes and investigate the behavior of the average length as a function of microtubule growth parameters. (ii) Under a constant force, the boundary between bounded and unbounded growth is shifted to higher tubulin concentrations and rescue rates. The critical force f(c) for the transition from unbounded to bounded growth increases logarithmically with tubulin concentration and the rescue rate, and it is smaller than the stall force. (iii) For microtubule growth against an elastic obstacle, the microtubule length and polymerization force can be regulated by microtubule growth parameters. For zero rescue rate, we find that the average polymerization force depends logarithmically on the tubulin concentration and is always smaller than the stall force in the absence of catastrophes and rescues. For a nonzero rescue rate, we find a sharply peaked steady-state length distribution, which is tightly controlled by microtubule growth parameters. The corresponding average microtubule length self-organizes such that the average polymerization force equals the critical force f(c) for the transition from unbounded to bounded growth. We also investigate the force dynamics if growth parameters are perturbed in dilution experiments. Finally, we show the robustness of our results against changes of catastrophe models and load distribution factors.

SummaryChaperone-assisted sorting of post-translationally imported proteins is a general mechanism among all eukaryotic organisms. Interaction of some preproteins with the organellar membranes is mediated by chaperones, which are recognised by membrane-bound tetratricopeptide repeat (TPR) domain containing proteins. We have characterised AtTPR7 as an endoplasmic reticulum protein in plants and propose a potential function for AtTPR7 in post-translational protein import. Our data demonstrate that AtTPR7 interacts with the heat shock proteins HSP90 and HSP70 via a cytosol-exposed TPR domain. We further show by in vitro and in vivo experiments that AtTPR7 is associated with the Arabidopsis Sec63 homologue, AtERdj2. Interestingly, AtTPR7 can functionally complement a Dsec71 yeast mutant that is impaired in post-translational protein transport. These data strongly suggest that AtTPR7 not only has a role in chaperone binding but also in post-translational protein import into the endoplasmic reticulum, pointing to a general mechanism of chaperone-mediated post-translational sorting between the endoplasmic reticulum, mitochondria and chloroplasts in plant cells.

A/B toxins such as cholera toxin, Pseudomonas exotoxin and killer toxin K28 contain a KDEL-like amino acid motif at one of their subunits which ensures retrograde toxin transport through the secretory pathway of a target cell. As key step in host cell invasion, each toxin binds to distinct plasma membrane receptors that are utilized for cell entry. Despite intensive efforts, some of these receptors are still unknown. Here we identify the yeast H/KDEL receptor Erd2p as membrane receptor of K28, a viral A/B toxin carrying an HDEL motif at its cell binding β-subunit. While initial toxin binding to the yeast cell wall is unaffected in cells lacking Erd2p, binding to spheroplasts and in vivo toxicity strongly depend on the presence of Erd2p. Consistently, Erd2p is not restricted to membranes of the early secretory pathway but extends to the plasma membrane where it binds and internalizes HDEL-cargo such as K28 toxin, GFPHDEL and Kar2p. Since human KDEL receptors are fully functional in yeast and restore toxin sensitivity in the absence of endogenous Erd2p, toxin uptake by H/KDEL receptors at the cell surface might likewise contribute to the intoxication efficiency of A/B toxins carrying a KDEL-motif at their cytotoxic A-subunit(s).

Abstract. We introduce a model for microtubule mechanics containing lateral bonds between dimers in neighboring protofilaments, bending rigidity of dimers, and repulsive interactions between protofilaments modeling steric constraints to investigate the influence of mechanical forces on hydrolysis and catastrophes. We use the allosteric dimer model, where tubulin dimers are characterized by an equilibrium bending angle, which changes from 0• to 22• by hydrolysis of a dimer. This also affects the lateral interaction and bending energies and, thus, the mechanical equilibrium state of the microtubule. As hydrolysis gives rise to conformational changes in dimers, mechanical forces also influence the hydrolysis rates by mechanical energy changes modulating the hydrolysis rate. The interaction via the microtubule mechanics then gives rise to correlation effects in the hydrolysis dynamics, which have not been taken into account before. Assuming a dominant influence of mechanical energies on hydrolysis rates, we investigate the most probable hydrolysis pathways both for vectorial and random hydrolysis. Investigating the stability with respect to lateral bond rupture, we identify initiation configurations for catastrophes along the hydrolysis pathways and values for a lateral bond rupture force. If we allow for rupturing of lateral bonds between dimers in neighboring protofilaments above this threshold force, our model exhibits avalanche-like catastrophe events.

No abstract

BackgroundNatural killer group 2D (NKG2D) is an activating receptor of natural killer (NK) cells and other lymphocytes that mediates lysis of malignant cells through recognition of stress-induced ligands such as MICA and MICB. Such ligands are broadly expressed by cancer cells of various origins and serve as targets for adoptive immunotherapy with effector cells endogenously expressing NKG2D or carrying an NKG2D-based chimeric antigen receptor (CAR). However, shedding or downregulation of NKG2D ligands (NKG2DL) can prevent NKG2D activation, resulting in escape of cancer cells from NKG2D-dependent immune surveillance.MethodsTo enable tumor-specific targeting of NKG2D-expressing effector cells independent of membrane-anchored NKG2DLs, we generated a homodimeric recombinant antibody which harbors an N-terminal single-chain fragment variable (scFv) antibody domain for binding to NKG2D, linked via a human IgG4 Fc region to a second C-terminal scFv antibody domain for recognition of the tumor-associated antigen ErbB2 (HER2). The ability of this molecule, termed NKAB-ErbB2, to redirect NKG2D-expressing effector cells to ErbB2-positive tumor cells of different origins was investigated using peripheral blood mononuclear cells, ex vivo expanded NK cells, and NK and T cells engineered with an NKG2D-based chimeric receptor.ResultsOn its own, bispecific NKAB-ErbB2 increased lysis of ErbB2-positive breast carcinoma cells by peripheral blood-derived NK cells endogenously expressing NKG2D more effectively than an ErbB2-specific IgG1 mini-antibody able to induce antibody-dependent cell-mediated cytotoxicity via activation of CD16. Furthermore, NKAB-ErbB2 synergized with NK-92 cells or primary T cells engineered to express an NKG2D-CD3ζ chimeric antigen receptor (NKAR), leading to targeted cell killing and greatly enhanced antitumor activity, which remained unaffected by soluble MICA known as an inhibitor of NKG2D-mediated natural cytotoxicity. In an immunocompetent mouse glioblastoma model mimicking low or absent NKG2DL expression, the combination of NKAR-NK-92 cells and NKAB-ErbB2 effectively suppressed outgrowth of ErbB2-positive tumors, resulting in treatment-induced endogenous antitumor immunity and cures in the majority of animals.ConclusionsOur results demonstrate that combining an NKAB antibody with effector cells expressing an activating NKAR receptor represents a powerful and versatile approach to simultaneously enhance tumor antigen-specific as well as NKG2D-CAR and natural NKG2D-mediated cytotoxicity, which may be particularly useful to target tumors with heterogeneous target antigen expression.

No abstract