The study of host-parasite interactions has increased considerably in the last decades, with many studies focusing on the identification of parasite molecules (i.e. surface or excretory/secretory proteins (ESP)) as potential targets for new specific treatments and/or diagnostic tools. In parallel, in the last few years there have been significant advances in the field of extracellular vesicles research. Among these vesicles, exosomes of endocytic origin, with a characteristic size ranging from 30-100 nm, carry several atypical secreted proteins in different organisms, including parasitic protozoa. Here, we present experimental evidence for the existence of exosome-like vesicles in parasitic helminths, specifically the trematodes Echinostoma caproni and Fasciola hepatica. These microvesicles are actively released by the parasites and are taken up by host cells. Trematode extracellular vesicles contain most of the proteins previously identified as components of ESP, as confirmed by proteomic, immunogold labeling and electron microscopy studies. In addition to parasitic proteins, we also identify host proteins in these structures. The existence of extracellular vesicles explains the secretion of atypical proteins in trematodes, and the demonstration of their uptake by host cells suggests an important role for these structures in host-parasite communication, as described for other infectious agents.
Ionizing radiation is a universal tool in tumor therapy but may also cause secondary cancers or cell invasiveness. These negative side effects could be causally related to the human-intermediate-conductance Ca(2+)-activated-K(+)-channel (hIK), which is activated by X-ray irradiation and affects cell proliferation and migration. To analyze the signaling cascade downstream of ionizing radiation we use genetically encoded reporters for H2O2 (HyPer) and for the dominant redox-buffer glutathione (Grx1-roGFP2) to monitor with high spatial and temporal resolution, radiation-triggered excursions of H2O2 in A549 and HEK293 cells. The data show that challenging cells with ≥1 Gy X-rays or with UV-A laser micro-irradiation causes a rapid rise of H2O2 in the nucleus and in the cytosol. This rise, which is determined by the rate of H2O2 production and glutathione-buffering, is sufficient for triggering a signaling cascade that involves an elevation of cytosolic Ca(2+) and eventually an activation of hIK channels.
B7-H4, one of the costimulatory molecules of B7 family, has been found to be widely expressed in many kinds of tumor tissues and to play an important part in tumor progression and poor prognosis. However, the role of B7-H4 in esophageal squamous cell carcinoma (ESCC) cells has not been elucidated. In this study, we found that, compared with normal esophageal tissue, B7-H4 was highly expressed in three ESCC cell lines, Eca109, TE1 and TE13. Besides, B7-H4 silence suppressed cells proliferation and colony formation. Additionally, compared with control cells, B7-H4 silence cells showed higher apoptosis rate, Bcl-2 and Survivin upregulation as well as BAX downregulation. Further study demonstrated that B7-H4 silence cells also exhibited reduction of IL-6 secretion, STAT3 activation and p-STAT3 translocation from cytoplasm to nucleus. Moreover, B7-H4 depletion inhibited the IL-6 secretion of control cells but not JAK2/STAT3 inhibitor FLLL32 treated cells. IL-6 receptor antagonist Tocilizumab didn’t block the p-JAK2 and p-STAT3 downregulation induced by B7-H4 silence. It was suggested that B7-H4 silence suppressed IL-6 secretion through JAK2/STAT3 inactivation. Furthermore, cells proliferation and colony formation were downregulated by Tocilizumab in control cells but not B7-H4 silence cells demonstrating that IL-6 upregulation induced by B7-H4 was necessary for cells growth. On the other hand, B7-H4 expression was downregulated by Tocilizumab. In all, our study provided the first evidence that B7-H4 facilitated ESCC cells proliferation through promoting IL-6/STAT3 positive loopback pathway activation. This article is protected by copyright. All rights reserved.
Nonreplicating type I uracil auxotrophic mutants of Toxoplasma gondii possess a potent ability to activate therapeutic immunity to established solid tumors by reversing immune suppression in the tumor microenvironment. Here we engineered targeted deletions of parasite secreted effector proteins using a genetically tractable Δku80 vaccine strain to show that the secretion of specific rhoptry (ROP) and dense granule (GRA) proteins by uracil auxotrophic mutants of T. gondii in conjunction with host cell invasion activates antitumor immunity through host responses involving CD8α+ dendritic cells, the IL-12/interferon-gamma (IFN-γ) TH1 axis, as well as CD4+ and CD8+ T cells. Deletion of parasitophorous vacuole membrane (PVM) associated proteins ROP5, ROP17, ROP18, ROP35 or ROP38, intravacuolar network associated dense granule proteins GRA2 or GRA12, and GRA24 which traffics past the PVM to the host cell nucleus severely abrogated the antitumor response. In contrast, deletion of other secreted effector molecules such as GRA15, GRA16, or ROP16 that manipulate host cell signaling and transcriptional pathways, or deletion of PVM associated ROP21 or GRA3 molecules did not affect the antitumor activity. Association of ROP18 with the PVM was found to be essential for the development of the antitumor responses. Surprisingly, the ROP18 kinase activity required for resistance to IFN-γ activated host innate immunity related GTPases and virulence was not essential for the antitumor response. These data show that PVM functions of parasite secreted effector molecules, including ROP18, manipulate host cell responses through ROP18 kinase virulence independent mechanisms to activate potent antitumor responses. Our results demonstrate that PVM associated rhoptry effector proteins secreted prior to host cell invasion and dense granule effector proteins localized to the intravacuolar network and host nucleus that are secreted after host cell invasion coordinately control the development of host immune responses that provide effective antitumor immunity against established ovarian cancer.
The bacterial Type VI secretion system (T6SS) delivers proteins into target cells using fast contraction of a long sheath anchored to the cell envelope and wrapped around an inner Hcp tube associated with the secreted proteins. Mechanisms of sheath assembly and length regulation are unclear. Here we study these processes using spheroplasts formed from ampicillin-treated Vibrio cholerae. We show that spheroplasts secrete Hcp and deliver T6SS substrates into neighbouring cells. Imaging of sheath dynamics shows that the sheath length correlates with the diameter of spheroplasts and may reach up to several micrometres. Analysis of sheath assembly after partial photobleaching shows that subunits are exclusively added to the sheath at the end that is distal from the baseplate and cell envelope attachment. We suggest that this mode of assembly is likely common for all phage-like contractile nanomachines, because of the conservation of the structures and connectivity of sheath subunits.
The uptake of cytoplasmic calcium into mitochondria is critical for a variety of physiological processes, including calcium buffering, metabolism, and cell survival. Here, we demonstrate that inhibiting the mitochondrial calcium uniporter in the Drosophila mushroom body neurons (MBn)-a brain region critical for olfactory memory formation-causes memory impairment without altering the capacity to learn. Inhibiting uniporter activity only during pupation impaired adult memory, whereas the same inhibition during adulthood was without effect. The behavioral impairment was associated with structural defects in MBn, including a decrease in synaptic vesicles and an increased length in the axons of the αβ MBn. Our results reveal an in vivo developmental role for the mitochondrial uniporter complex in establishing the necessary structural and functional neuronal substrates for normal memory formation in the adult organism.
We employed electron cryo-tomography to visualize cytosolic ribosomes on the surface of mitochondria. Translation-arrested ribosomes reveal the clustered organization of the TOM complex, corroborating earlier reports of localized translation. Ribosomes are shown to interact specifically with the TOM complex, and nascent chain binding is crucial for ribosome recruitment and stabilization. Ribosomes are bound to the membrane in discrete clusters, often in the vicinity of the crista junctions. This interaction highlights how protein synthesis may be coupled with transport. Our work provides unique insights into the spatial organization of cytosolic ribosomes on mitochondria.
There has been tremendous interest in constructing in vitro cardiac tissue for a range of fundamental studies of cardiac development and disease and as a commercial system to evaluate therapeutic drug discovery prioritization and toxicity. Although there has been progress towards studying 2-dimensional cardiac function in vitro, there remain challenging obstacles to generate rapid and efficient scaffold-free 3-dimensional multiple cell type co-culture cardiac tissue models. Herein, we develop a programmed rapid self-assembly strategy to induce specific and stable cell-cell contacts among multiple cell types found in heart tissue to generate 3D tissues through cell-surface engineering based on liposome delivery and fusion to display bio-orthogonal functional groups from cell membranes. We generate, for the first time, a scaffold free and stable self assembled 3 cell line co-culture 3D cardiac tissue model by assembling cardiomyocytes, endothelial cells and cardiac fibroblast cells via a rapid inter-cell click ligation process. We compare and analyze the function of the 3D cardiac tissue chips with 2D co-culture monolayers by assessing cardiac specific markers, electromechanical cell coupling, beating rates and evaluating drug toxicity.
Tight regulation of signalling activity is crucial for proper tissue patterning and growth. Here we investigate the function of Pentagone (Pent), a secreted protein that acts in a regulatory feedback during establishment and maintenance of BMP/Dpp morphogen signalling during Drosophila wing development. We show that Pent internalises the Dpp co-receptors, the glypicans Dally and Dally-like protein (Dlp), and propose that this internalisation is important in the establishment of a long range Dpp gradient. Pent-induced endocytosis and degradation of glypicans requires dynamin- and Rab5, but not clathrin or active BMP signalling. Thus, Pent modifies the ability of cells to trap and transduce BMP by fine-tuning the levels of the BMP reception system at the plasma membrane. In addition, and in accordance with the role of glypicans in multiple signalling pathways, we establish a requirement of Pent for Wg signalling. Our data propose a novel mechanism by which morphogen signalling is regulated.
By dividing asymmetrically, stem cells can generate two daughter cells with distinct fates. However, evidence is limited in mammalian systems for the selective apportioning of subcellular contents between daughters. We followed the fates of old and young organelles during the division of human mammary stemlike cells and found that such cells apportion aged mitochondria asymmetrically between daughter cells. Daughter cells that received fewer old mitochondria maintained stem cell traits. Inhibition of mitochondrial fission disrupted both the age-dependent subcellular localization and segregation of mitochondria and caused loss of stem cell properties in the progeny cells. Hence, mechanisms exist for mammalian stemlike cells to asymmetrically sort aged and young mitochondria, and these are important for maintaining stemness properties.