COPI-coated vesicles mediate trafficking within the Golgi apparatus and from the Golgi to the endoplasmic reticulum. The structures of membrane protein coats, including COPI, have been extensively studied with in vitro reconstitution systems using purified components. In a previous paper (Dodonova et al., 2017), we determined a complete structural model of the in vitro reconstituted COPI coat. Here, we applied cryo-focused ion beam milling, cryo-electron tomography and subtomogram averaging to determine the native structure of the COPI coat within vitrified Chlamydomonas reinhardtii cells. The native algal structure resembles the in vitro mammalian structure, but additionally reveals cargo bound beneath β'-COP. We find that all coat components disassemble simultaneously and relatively rapidly after budding. Structural analysis in situ, maintaining Golgi topology, shows that vesicles change their size, membrane thickness, and cargo content as they progress from cis to trans, but the structure of the coat machinery remains constant.
Chlamydia trachomatis is obligate intracellular bacterial pathogen that remains a significant public health burden worldwide. A critical early event during infection is chlamydial entry into non-phagocytic host epithelial cells. Like other Gram-negative bacteria, C.trachomatis uses a type III secretion system (T3SS) to deliver virulence effector proteins into host cells. These effectors trigger bacterial uptake and promote bacterial survival and replication within the host cell. In this review, we highlight recent cryo-electron tomography that has provided striking insights into the initial interactions between Chlamydia and its host. We describe the polarised structure of extracellular C.trachomatis elementary bodies (EBs), and the supramolecular organisation of T3SS complexes on the EB surface, in addition to the changes in host and pathogen architecture that accompany bacterial internalisation and EB encapsulation into early intracellular vacuoles. Finally, we consider the implications for further understanding the mechanism of C.trachomatis entry and how this might relate to those of other bacteria and viruses.
Tumor-infiltrating regulatory T lymphocytes (Treg) can suppress effector T cells specific for tumor antigens. Deeper molecular definitions of tumor-infiltrating-lymphocytes could thus offer therapeutic opportunities. Transcriptomes of T helper 1 (Th1), Th17, and Treg cells infiltrating colorectal or non-small-cell lung cancers were compared to transcriptomes of the same subsets from normal tissues and validated at the single-cell level. We found that tumor-infiltrating Treg cells were highly suppressive, upregulated several immune-checkpoints, and expressed on the cell surfaces specific signature molecules such as interleukin-1 receptor 2 (IL1R2), programmed death (PD)-1 Ligand1, PD-1 Ligand2, and CCR8 chemokine, which were not previously described on Treg cells. Remarkably, high expression in whole-tumor samples of Treg cell signature genes, such as LAYN, MAGEH1, or CCR8, correlated with poor prognosis. Our findings provide insights into the molecular identity and functions of human tumor-infiltrating Treg cells and define potential targets for tumor immunotherapy.
COPI mediates retrograde trafficking from the Golgi to the endoplasmic reticulum (ER) and within the Golgi stack, sorting transmembrane proteins bearing C-terminal KKxx or KxKxx motifs. The structure of KxKxx motifs bound to the N-terminal WD-repeat domain of β'-COP identifies electrostatic contacts between the motif and complementary patches at the center of the β'-COP propeller. An absolute requirement of a two-residue spacing between the terminal carboxylate group and first lysine residue results from interactions of carbonyl groups in the motif backbone with basic side chains of β'-COP. Similar interactions are proposed to mediate binding of KKxx motifs by the homologous α-COP domain. Mutation of key interacting residues in either domain or in their cognate motifs abolishes in vitro binding and results in mistrafficking of dilysine-containing cargo in yeast without compromising cell viability. Flexibility between β'-COP WD-repeat domains and the location of cargo binding have implications for COPI coat assembly.
Bacteria utilize complex type IV secretion systems (T4SSs) to translocate diverse effector proteins or DNA into target cells. Despite the importance of T4SSs in bacterial pathogenesis, the mechanism by which these translocation machineries deliver cargo across the bacterial envelope remains poorly understood, and very few studies have investigated the use of synthetic molecules to disrupt T4SS-mediated transport. Here, we describe two synthetic small molecules (C10 and KSK85) that disrupt T4SS-dependent processes in multiple bacterial pathogens. Helicobacter pylori exploits a pilus appendage associated with the cag T4SS to inject an oncogenic effector protein (CagA) and peptidoglycan into gastric epithelial cells. In H. pylori, KSK85 impedes biogenesis of the pilus appendage associated with the cag T4SS, while C10 disrupts cag T4SS activity without perturbing pilus assembly. In addition to the effects in H. pylori, we demonstrate that these compounds disrupt interbacterial DNA transfer by conjugative T4SSs in Escherichia coli and impede vir T4SS-mediated DNA delivery by Agrobacterium tumefaciens in a plant model of infection. Of note, C10 effectively disarmed dissemination of a derepressed IncF plasmid into a recipient bacterial population, thus demonstrating the potential of these compounds in mitigating the spread of antibiotic resistance determinants driven by conjugation. To our knowledge, this study is the first report of synthetic small molecules that impair delivery of both effector protein and DNA cargos by diverse T4SSs.
Delineating the crosstalk between distinct signaling pathways is key to understanding the diverse and dynamic responses of adult stem cells during tissue regeneration. Here, we demonstrate that the Edn/EdnrB signaling pathway can interact with other signaling pathways to elicit distinct stem cell functions during tissue regeneration. EdnrB signaling promotes proliferation and differentiation of melanocyte stem cells (McSCs), dramatically enhancing the regeneration of hair and epidermal melanocytes. This effect is dependent upon active Wnt signaling that is initiated by Wnt ligand secretion from the hair follicle epithelial niche. Further, this Wnt-dependent EdnrB signaling can rescue the defects in melanocyte regeneration caused by Mc1R loss. This suggests that targeting Edn/EdnrB signaling in McSCs can be a therapeutic approach to promote photoprotective-melanocyte regeneration, which may be useful for those with increased risk of skin cancers due to Mc1R variants.
An important feature of the mammary gland is its ability to undergo repeated morphological changes during each reproductive cycle with profound tissue expansion in pregnancy and regression in involution. However, the mechanisms that determine the tissue’s cyclic regenerative capacity remain elusive. We have now discovered that Cre-Lox ablation of Rac1 in mammary epithelia causes gross enlargement of the epithelial tree and defective alveolar regeneration in a second pregnancy. Architectural defects arise because loss of Rac1 disrupts clearance in involution following the first lactation. We show that Rac1 is crucial for mammary alveolar epithelia to switch from secretion to a phagocytic mode and rapidly remove dying neighbors. Moreover, Rac1 restricts the extrusion of dying cells into the lumen, thus promoting their eradication by live phagocytic neighbors while within the epithelium. Without Rac1, residual milk and cell corpses flood the ductal network, causing gross dilation, chronic inflammation, and defective future regeneration.
Stromal vascular fraction (SVF) can easily be obtained from a mini-lipoaspirate procedure of fat tissue. The SVF contains a mixture of cells including ADSCs and growth factors and has been depleted of the adipocyte (fat cell) population. We evaluated the safety and efficacy of administering SVF intra-myocardially into patients with chronic ischemic cardiomyopathy.
Diabetes and its concurrent complications impact a significant proportion of the population of the US and create a large financial burden on the American health care system. FDA-approved maggot debridement therapy (MDT), the application of sterile laboratory-reared Lucilia sericata (green bottle fly) larvae to wounds, is a cost-effective and successful treatment for diabetic foot ulcers and other medical conditions. Human platelet derived growth factor-BB (PDGF-BB) is a secreted dimeric peptide growth factor that binds the PDGF receptor. PDGF-BB stimulates cell proliferation and survival, promotes wound healing, and has been investigated as a possible topical treatment for non-healing wounds. Genetic engineering has allowed for expression and secretion of human growth factors and other proteins in transgenic insects. Here, we present a novel concept in MDT technology that combines the established benefits of MDT with the power of genetic engineering to promote healing. The focus of this study is to create and characterize strains of transgenic L. sericata that express and secrete PDGF-BB at detectable levels in adult hemolymph, whole larval lysate, and maggot excretions/ secretions (ES), with potential for clinical utility in wound healing.
The generation of insulin-producing pancreatic β cells from stem cells in vitro would provide an unprecedented cell source for drug discovery and cell transplantation therapy in diabetes. However, insulin-producing cells previously generated from human pluripotent stem cells (hPSC) lack many functional characteristics of bona fide β cells. Here, we report a scalable differentiation protocol that can generate hundreds of millions of glucose-responsive β cells from hPSC in vitro. These stem-cell-derived β cells (SC-β) express markers found in mature β cells, flux Ca(2+) in response to glucose, package insulin into secretory granules, and secrete quantities of insulin comparable to adult β cells in response to multiple sequential glucose challenges in vitro. Furthermore, these cells secrete human insulin into the serum of mice shortly after transplantation in a glucose-regulated manner, and transplantation of these cells ameliorates hyperglycemia in diabetic mice.