FAT1, which encodes a protocadherin, is one of the most frequently mutated genes in human cancers
. However, the role and the molecular mechanisms by which FAT1 mutations control tumour initiation ...and progression are poorly understood. Here, using mouse models of skin squamous cell carcinoma and lung tumours, we found that deletion of Fat1 accelerates tumour initiation and malignant progression and promotes a hybrid epithelial-to-mesenchymal transition (EMT) phenotype. We also found this hybrid EMT state in FAT1-mutated human squamous cell carcinomas. Skin squamous cell carcinomas in which Fat1 was deleted presented increased tumour stemness and spontaneous metastasis. We performed transcriptional and chromatin profiling combined with proteomic analyses and mechanistic studies, which revealed that loss of function of FAT1 activates a CAMK2-CD44-SRC axis that promotes YAP1 nuclear translocation and ZEB1 expression that stimulates the mesenchymal state. This loss of function also inactivates EZH2, promoting SOX2 expression, which sustains the epithelial state. Our comprehensive analysis identified drug resistance and vulnerabilities in FAT1-deficient tumours, which have important implications for cancer therapy. Our studies reveal that, in mouse and human squamous cell carcinoma, loss of function of FAT1 promotes tumour initiation, progression, invasiveness, stemness and metastasis through the induction of a hybrid EMT state.
Humans can survive bloodstream infection by African trypanosomes, owing to the activity of serum complexes that have efficient trypanosome-killing ability. The two trypanosome subspecies that are ...responsible for human sleeping sickness--Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense--can evade this defence mechanism by expressing distinct resistance proteins. In turn, sequence variation in the gene that encodes the trypanosome-killing component in human serum has enabled populations in western Africa to restore resistance to T. b. rhodesiense, at the expense of the high probability of developing kidney sclerosis. These findings highlight the importance of resistance to trypanosomes in human evolution.
Tsetse flies are the sole vectors of Trypanosoma brucei parasites that cause sleeping sickness. Our knowledge on the early interface between the infective metacyclic forms and the mammalian host skin ...is currently highly limited. Glossina morsitans flies infected with fluorescently tagged T. brucei parasites were used in this study to initiate natural infections in mice. Metacyclic trypanosomes were found to be highly infectious through the intradermal route in sharp contrast with blood stream form trypanosomes. Parasite emigration from the dermal inoculation site resulted in detectable parasite levels in the draining lymph nodes within 18 hours and in the peripheral blood within 42 h. A subset of parasites remained and actively proliferated in the dermis. By initiating mixed infections with differentially labeled parasites, dermal parasites were unequivocally shown to arise from the initial inoculum and not from a re-invasion from the blood circulation. Scanning electron microscopy demonstrated intricate interactions of these skin-residing parasites with adipocytes in the connective tissue, entanglement by reticular fibers of the periadipocytic baskets and embedment between collagen bundles. Experimental transmission experiments combined with molecular parasite detection in blood fed flies provided evidence that dermal trypanosomes can be acquired from the inoculation site immediately after the initial transmission. High resolution thermographic imaging also revealed that intradermal parasite expansion induces elevated skin surface temperatures. Collectively, the dermis represents a delivery site of the highly infective metacyclic trypanosomes from which the host is systemically colonized and where a proliferative subpopulation remains that is physically constrained by intricate interactions with adipocytes and collagen fibrous structures.
Immortal spheroids were generated from fetal mouse intestine using the culture system initially developed to culture organoids from adult intestinal epithelium. Spheroid proportion progressively ...decreases from fetal to postnatal period, with a corresponding increase in production of organoids. Like organoids, spheroids show Wnt-dependent indefinite self-renewing properties but display a poorly differentiated phenotype reminiscent of incompletely caudalized progenitors. The spheroid transcriptome is strikingly different from that of adult intestinal stem cells, with minimal overlap of Wnt target gene expression. The receptor LGR4, but not LGR5, is essential for their growth. Trop2/Tacstd2 and Cnx43/Gja1, two markers highly enriched in spheroids, are expressed throughout the embryonic-day-14 intestinal epithelium. Comparison of in utero and neonatal lineage tracing using Cnx43-CreER and Lgr5-CreERT2 mice identified spheroid-generating cells as developmental progenitors involved in generation of the prenatal intestinal epithelium. Ex vivo, spheroid cells have the potential to differentiate into organoids, qualifying as a fetal type of intestinal stem cell.
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•Fetal and adult intestinal epithelia are generated from two kinds of progenitor cells•Trop2 and Cnx43 are markers of fetal intestinal progenitors•Ex vivo, fetal intestinal progenitors generate immortal undifferentiated spheroids•Spheroids have the ability to convert to differentiated Lgr5-positive organoids
Vassart, Garcia, and colleagues now use lineage-tracing experiments to demonstrate that definitive mouse intestinal epithelium is generated in two waves depending on different sets of progenitors. The authors show that immortal, undifferentiated spheroids can be obtained from culturing fetal intestinal epithelium under conditions yielding differentiated organoids from adult intestine. Spheroid cells display a transcriptome strikingly different from that of adult intestinal stem cells. The cells correspond to progenitors “frozen” in a state preceding differentiation into fetal intestinal epithelium and generation of adult stem cells.
Approximately 20% of sleeping sickness patients exhibit respiratory complications, however, with a largely unknown role of the parasite. Here we show that tsetse fly-transmitted Trypanosoma brucei ...parasites rapidly and permanently colonize the lungs and occupy the extravascular spaces surrounding the blood vessels of the alveoli and bronchi. They are present as nests of multiplying parasites exhibiting close interactions with collagen and active secretion of extracellular vesicles. The local immune response shows a substantial increase of monocytes, macrophages, dendritic cells and γδ and activated αβ T cells and a later influx of neutrophils. Interestingly, parasite presence results in a significant reduction of B cells, eosinophils and natural killer cells. T. brucei infected mice show no infection-associated pulmonary dysfunction, mirroring the limited pulmonary clinical complications during sleeping sickness. However, the substantial reduction of the various immune cells may render individuals more susceptible to opportunistic infections, as evident by a co-infection experiment with respiratory syncytial virus. Collectively, these observations provide insights into a largely overlooked target organ, and may trigger new diagnostic and supportive therapeutic approaches for sleeping sickness.
Humans resist infection by the African parasite Trypanosoma brucei owing to the trypanolytic activity of the serum apolipoprotein L1 (APOL1). Following uptake by endocytosis in the parasite, APOL1 ...forms pores in endolysosomal membranes and triggers lysosome swelling. Here we show that APOL1 induces both lysosomal and mitochondrial membrane permeabilization (LMP and MMP). Trypanolysis coincides with MMP and consecutive release of the mitochondrial TbEndoG endonuclease to the nucleus. APOL1 is associated with the kinesin TbKIFC1, of which both the motor and vesicular trafficking VHS domains are required for MMP, but not for LMP. The presence of APOL1 in the mitochondrion is accompanied by mitochondrial membrane fenestration, which can be mimicked by knockdown of a mitochondrial mitofusin-like protein (TbMFNL). The BH3-like peptide of APOL1 is required for LMP, MMP and trypanolysis. Thus, trypanolysis by APOL1 is linked to apoptosis-like MMP occurring together with TbKIFC1-mediated transport of APOL1 from endolysosomal membranes to the mitochondrion.
Mesoderm arises at gastrulation and contributes to both the mouse embryo proper and its extra‐embryonic membranes. Two‐photon live imaging of embryos bearing a keratin reporter allowed recording ...filament nucleation and elongation in the extra‐embryonic region. Upon separation of amniotic and exocoelomic cavities, keratin 8 formed apical cables co‐aligned across multiple cells in the amnion, allantois, and blood islands. An influence of substrate rigidity and composition on cell behavior and keratin content was observed in mesoderm explants. Embryos lacking all keratin filaments displayed a deflated extra‐embryonic cavity, a narrow thick amnion, and a short allantois. Single‐cell RNA sequencing of sorted mesoderm cells and micro‐dissected amnion, chorion, and allantois, provided an atlas of transcriptomes with germ layer and regional information. It defined the cytoskeleton and adhesion expression profile of mesoderm‐derived keratin 8‐enriched cells lining the exocoelomic cavity. Those findings indicate a novel role for keratin filaments in the expansion of extra‐embryonic structures and suggest mechanisms of mesoderm adaptation to the environment.
Synopsis
Keratin intermediate filaments form apical cables continuous across multiple cells in mesoderm‐derived extra‐embryonic tissues lining the exocoelomic cavity of the mouse embryo. Loss of keratin results in defective growth of extra‐embryonic membranes.
At late gastrulation, keratin filaments nucleate, elongate, and co‐align across multiple cells.
In explanted mesoderm cells, cell speed and keratin content vary according to the composition and rigidity of the substrate.
Embryos devoid of keratin filaments have a smaller cavity, amnion, and allantois.
A single cell transcriptomic atlas of extra‐embryonic membranes defines early differentiation of clusters in extra‐embryonic mesoderm, amnion, chorion, and allantois.
Cells lining the exocoelomic cavity in mouse embryos assemble keratin cables that are continuous intercellularly, and whose loss affects the growth of extra‐embryonic membranes.
Apolipoproteins L (ApoLs) are Bcl‐2‐like proteins expressed under inflammatory conditions in myeloid and endothelial cells. We found that Toll‐like receptor (TLR) stimuli, particularly the viral ...mimetic polyinosinic:polycytidylic acid (poly(I:C)), specifically induce ApoLs7/11 subfamilies in murine CD8α+ dendritic cells (DCs). This induction requires the TLR3/TRIF (where TRIF is TIR domain containing adapter‐inducing interferon β) signaling pathway and is dependent on IFN‐β in all ApoLs subfamilies except for ApoL7c. Poly(I:C) treatment of DCs is also associated with induction of both cell death and autophagy. ApoLs expression is related to promotion of DC death by poly(I:C), as ApoLs7/11 knockdown increases DC survival and ApoLs7 are associated with the anti‐apoptotic protein Bcl‐xL (where Bcl‐xL is B‐cell lymphoma extra large). Similarly, in human monocyte‐derived DCs poly(I:C) induces both cell death and the expression of ApoLs, principally ApoL3. Finally, the BH3‐like peptide of ApoLs appears to be involved in the DC death‐promoting activity. We would like to propose that ApoLs are involved in cell death linked to activation of DCs by viral stimuli.
In dendritic cells (DCs), stimulation of the TLR3/TRIF/IRF3/IFN‐β signaling pathway by the viral mimetic poly(I:C) triggers the synthesis of Apolipoproteins L (ApoLs). ApoLs are involved in DC death induced by poly(I:C). ApoLs interact with the anti‐apoptotic Bcl‐xL protein, presumably through interactions mediated by BH3 peptides.
Apolipoproteins L1 and L3 (APOLs) are associated at the Golgi with the membrane fission factors phosphatidylinositol 4-kinase-IIIB (PI4KB) and non-muscular myosin 2A. Either APOL1 C-terminal ...truncation (APOL1Δ) or APOL3 deletion (APOL3-KO knockout) reduces PI4KB activity and triggers actomyosin reorganization. We report that APOL3, but not APOL1, controls PI4KB activity through interaction with PI4KB and neuronal calcium sensor-1 or calneuron-1. Both APOLs are present in Golgi-derived autophagy-related protein 9A vesicles, which are involved in PI4KB trafficking. Like APOL3-KO, APOL1Δ induces PI4KB dissociation from APOL3, linked to reduction of mitophagy flux and production of mitochondrial reactive oxygen species. APOL1 and APOL3, respectively, can interact with the mitophagy receptor prohibitin-2 and the mitophagosome membrane fusion factor vesicle-associated membrane protein-8 (VAMP8). While APOL1 conditions PI4KB and APOL3 involvement in mitochondrion fission and mitophagy, APOL3-VAMP8 interaction promotes fusion between mitophagosomal and endolysosomal membranes. We propose that APOL3 controls mitochondrial membrane dynamics through interactions with the fission factor PI4KB and the fusion factor VAMP8.
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•APOL3 controls the activity of the Golgi and mitochondrion membrane fission factor PI4KB•APOL1 allows APOL3 and PI4KB traffic to MERCSs, linked to mitochondrion fission and mitophagy•Through interaction with VAMP8, APOL3 promotes mitophagosome-endolysosome fusion•C-terminal APOL1 variants reduce the mitophagy flux through APOL3 and PI4KB inactivation
Lecordier et al. report that APOL1 and APOL3 are involved in mitophagy. Whereas APOL1 mediates APOL3 and PI4KB traffic from the Golgi to MERCSs, APOL3 controls mitochondrial membrane fission and fusion through interactions with PI4KB and VAMP8, respectively. APOL1 C-terminal variants inhibit mitophagy through interference with APOL3 and PI4KB activities.
The C-terminal variants G1 and G2 of apolipoprotein L1 (APOL1) confer human resistance to the sleeping sickness parasite Trypanosoma rhodesiense, but they also increase the risk of kidney disease. ...APOL1 and APOL3 are death-promoting proteins that are partially associated with the endoplasmic reticulum and Golgi membranes. We report that in podocytes, either APOL1 C-terminal helix truncation (APOL1Δ) or APOL3 deletion (APOL3KO) induces similar actomyosin reorganization linked to the inhibition of phosphatidylinositol-4-phosphate PI(4)P synthesis by the Golgi PI(4)-kinase IIIB (PI4KB). Both APOL1 and APOL3 can form K+ channels, but only APOL3 exhibits Ca2+-dependent binding of high affinity to neuronal calcium sensor-1 (NCS-1), promoting NCS-1-PI4KB interaction and stimulating PI4KB activity. Alteration of the APOL1 C-terminal helix triggers APOL1 unfolding and increased binding to APOL3, affecting APOL3-NCS-1 interaction. Since the podocytes of G1 and G2 patients exhibit an APOL1Δ or APOL3KO-like phenotype, APOL1 C-terminal variants may induce kidney disease by preventing APOL3 from activating PI4KB, with consecutive actomyosin reorganization of podocytes.
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•C-terminal helix alteration unfolds APOL1, increasing APOL1 interaction with APOL3•APOL3 binds to NCS-1, promoting NCS-1-PI4KB interaction and PI4KB activation•C-terminal APOL1 variants interfere with APOL3-NCS-1 interaction, inactivating PI4KB•PI4KB inactivation occurs in podocytes from kidney disease patients with APOL1 variants
Uzureau et al. propose a molecular explanation for the linkage between resistance to sleeping sickness and high risk of kidney disease in African individuals expressing APOL1 variants. These variants resist neutralization by T. rhodesiense SRA and kill the parasite, but they also indirectly trigger podocyte actomyosin reorganization through interaction with APOL3, inactivating the NCS-1-PI4KB complex.
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