RESUMO
Homozygous Apolipoprotein L1 (APOL1) variants G1 and G2 cause APOL1-mediated kidney disease, purportedly acting as surface cation channels in podocytes. APOL1-G0 exhibits various single nucleotide polymorphisms, most commonly haplotype E150K, M228I and R255K ("KIK"; the Reference Sequence is "EMR"), whereas variants G1 and G2 are mostly found in a single "African" haplotype background ("EIK"). Several labs reported cytotoxicity with risk variants G1 and G2 in KIK or EIK background haplotypes, but used HEK-293 cells and did not verify equal surface expression. To see if haplotype matters in a more relevant cell type, we induced APOL1-G0, G1 and G2 EIK, KIK and EMR at comparable surface levels in immortalized podocytes. G1 and G2 risk variants (but not G0) caused dose-dependent podocyte death within 48h only in their native African EIK haplotype and correlated with K+ conductance (thallium FLIPR). We ruled out differences in localization and trafficking, except for possibly greater surface clustering of cytotoxic haplotypes. APOL1 surface expression was required, since Brefeldin A rescued cytotoxicity; and cytoplasmic isoforms vB3 and vC were not cytotoxic. Thus, APOL1-EIK risk variants kill podocytes in a dose and haplotype-dependent manner (as in HEK-293 cells), whereas unlike in HEK-293 cells the KIK risk variants did not.
Assuntos
Podócitos , Humanos , Podócitos/metabolismo , Haplótipos , Apolipoproteína L1/genética , Apolipoproteína L1/metabolismo , Células HEK293 , Variação GenéticaRESUMO
Disruption of epithelial barriers is a common disease manifestation in chronic degenerative diseases of the airways, lung, and intestine. Extensive human genetic studies have identified risk loci in such diseases, including in chronic obstructive pulmonary disease (COPD) and inflammatory bowel diseases. The genes associated with these loci have not fully been determined, and functional characterization of such genes requires extensive studies in model organisms. Here, we report the results of a screen in Drosophila melanogaster that allowed for rapid identification, validation, and prioritization of COPD risk genes that were selected based on risk loci identified in human genome-wide association studies (GWAS). Using intestinal barrier dysfunction in flies as a readout, our results validate the impact of candidate gene perturbations on epithelial barrier function in 56% of the cases, resulting in a prioritized target gene list. We further report the functional characterization in flies of one family of these genes, encoding for nicotinic acetylcholine receptor (nAchR) subunits. We find that nAchR signaling in enterocytes of the fly gut promotes epithelial barrier function and epithelial homeostasis by regulating the production of the peritrophic matrix. Our findings identify COPD-associated genes critical for epithelial barrier maintenance, and provide insight into the role of epithelial nAchR signaling for homeostasis.
Assuntos
Doença Pulmonar Obstrutiva Crônica , Receptores Nicotínicos , Animais , Humanos , Receptores Nicotínicos/genética , Estudo de Associação Genômica Ampla , Drosophila melanogaster/genética , PulmãoRESUMO
MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) is widely used as marker of autophagic compartments at different stages of maturation. Electron microscopy (EM) combined with immunolabeling is the only technique that can reveal the ultrastructural identity of LC3-labeled compartments. However, immuno-EM of endogenous LC3 proteins has proven difficult. Here, we test a panel of commercially available antibodies and apply different labeling conditions to present an optimized procedure for LC3 immuno-EM. Using ultrathin cryosections and protein A-colloidal gold or gold enhancement labeling, we localize endogenous LC3 in starved cells or tissues in the presence or absence of the proton pump inhibitor bafilomycin A1. We localize LC3 to early and late stage autophagic compartments that can be classified by their morphology. By on-section correlative light-electron microscopy (CLEM) we show that comparable fluorescent LC3-positive puncta can represent different autophagic intermediates. We also show that our approach is sufficiently robust to label endogenous LC3 simultaneously with other lysosomal and autophagy markers, LAMP1 or SQSTM1/p62, and can be used for quantitative approaches. Thus, we demonstrate that bafilomycin A1 treatment from 2.5 up to 24 h does not inhibit fusion between autophagosomes and lysosomes, but leads to the accumulation of LC3-positive material inside autolysosomes. Together, this is the first study presenting an extensive overview of endogenous LC3 localization at ultrastructural resolution without the need for cell permeabilization and using a commercially available antibody. This provides researchers with a tool to study canonical and non-canonical roles of LC3 in native conditions.Abbreviations: BafA1: bafilomycin A1; BSA: bovine serum albumin; BSA-c: acetylated BSA; BSA5: BSA conjugated to 5-nm gold particles; CLEM: correlative light-electron microscopy; EGFP: enhanced green fluorescent protein; EM: electron microscopy; FBS: fetal bovine serum; FSG: fish skin gelatin; GA: glutaraldehyde; IF: immunofluorescence; LAMP1: lysosomal associated membrane protein 1; LC3s: LC3 proteins; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; ON: overnight; PAG: protein A-conjugated gold particles; PAG1-3: PAG5, PAG10, PAG15, protein A conjugated to 1-3-, 5-, 10-, or 15-nm gold particles; PB: Sorensen's phosphate buffer; PBS: phosphate-buffered saline; PFA: paraformaldehyde; RT: room temperature.
Assuntos
Autofagia , Lisossomos , Animais , Microscopia Imunoeletrônica , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfatos/metabolismoRESUMO
Microglia and complement can mediate neurodegeneration in Alzheimer's disease (AD). By integrative multi-omics analysis, here we show that astrocytic and microglial proteins are increased in TauP301S synapse fractions with age and in a C1q-dependent manner. In addition to microglia, we identified that astrocytes contribute substantially to synapse elimination in TauP301S hippocampi. Notably, we found relatively more excitatory synapse marker proteins in astrocytic lysosomes, whereas microglial lysosomes contained more inhibitory synapse material. C1q deletion reduced astrocyte-synapse association and decreased astrocytic and microglial synapses engulfment in TauP301S mice and rescued synapse density. Finally, in an AD mouse model that combines ß-amyloid and Tau pathologies, deletion of the AD risk gene Trem2 impaired microglial phagocytosis of synapses, whereas astrocytes engulfed more inhibitory synapses around plaques. Together, our data reveal that astrocytes contact and eliminate synapses in a C1q-dependent manner and thereby contribute to pathological synapse loss and that astrocytic phagocytosis can compensate for microglial dysfunction.
Assuntos
Doença de Alzheimer , Camundongos , Animais , Doença de Alzheimer/genética , Complemento C1q/genética , Microglia/metabolismo , Astrócitos/metabolismo , Sinapses/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/metabolismoRESUMO
The signaling network of the unfolded protein response (UPR) adjusts the protein-folding capacity of the endoplasmic reticulum (ER) according to need. The most conserved UPR sensor, IRE1α, spans the ER membrane and activates through oligomerization. IRE1α oligomers accumulate in dynamic foci. We determined the in situ structure of IRE1α foci by cryogenic correlated light and electron microscopy combined with electron cryo-tomography and complementary immunoelectron microscopy in mammalian cell lines. IRE1α foci localized to a network of narrow anastomosing ER tubes (diameter, ~28 nm) with complex branching. The lumen of the tubes contained protein filaments, which were likely composed of arrays of IRE1α lumenal domain dimers that were arranged in two intertwined, left-handed helices. This specialized ER subdomain may play a role in modulating IRE1α signaling.
Assuntos
Estresse do Retículo Endoplasmático , Endorribonucleases/química , Proteínas Serina-Treonina Quinases/química , Resposta a Proteínas não Dobradas , Linhagem Celular Tumoral , Microscopia Crioeletrônica , Humanos , Domínios Proteicos , Multimerização Proteica , Transdução de SinaisRESUMO
Antigen cross presentation, whereby exogenous antigens are presented by MHC class I molecules to CD8+ T cells, is essential for generating adaptive immunity to pathogens and tumor cells. Following endocytosis, it is widely understood that protein antigens must be transferred from endosomes to the cytosol where they are subject to ubiquitination and proteasome degradation prior to being translocated into the endoplasmic reticulum (ER), or possibly endosomes, via the TAP1/TAP2 complex. Revealing how antigens egress from endocytic organelles (endosome-to-cytosol transfer, ECT), however, has proved vexing. Here, we used two independent screens to identify the hydrogen peroxide-transporting channel aquaporin-3 (AQP3) as a regulator of ECT. AQP3 overexpression increased ECT, whereas AQP3 knockout or knockdown decreased ECT. Mechanistically, AQP3 appears to be important for hydrogen peroxide entry into the endosomal lumen where it affects lipid peroxidation and subsequent antigen release. AQP3-mediated regulation of ECT was functionally significant, as AQP3 modulation had a direct impact on the efficiency of antigen cross presentation in vitro. Finally, AQP3-/- mice exhibited a reduced ability to mount an anti-viral response and cross present exogenous extended peptide. Together, these results indicate that the AQP3-mediated transport of hydrogen peroxide can regulate endosomal lipid peroxidation and suggest that compromised membrane integrity and coordinated release of endosomal cargo is a likely mechanism for ECT.
Assuntos
Aquaporina 3/metabolismo , Citosol/metabolismo , Endossomos/metabolismo , Animais , Apresentação de Antígeno , Aquaporina 3/genética , Transporte Biológico , Células Cultivadas , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Peroxidação de Lipídeos , CamundongosRESUMO
BACKGROUND: APOL1 is found in human kidney podocytes and endothelia. Variants G1 and G2 of the APOL1 gene account for the high frequency of nondiabetic CKD among African Americans. Proposed mechanisms of kidney podocyte cytotoxicity resulting from APOL1 variant overexpression implicate different subcellular compartments. It is unclear where endogenous podocyte APOL1 resides, because previous immunolocalization studies utilized overexpressed protein or commercially available antibodies that crossreact with APOL2. This study describes and distinguishes the locations of both APOLs. METHODS: Immunohistochemistry, confocal and immunoelectron microscopy, and podocyte fractionation localized endogenous and transfected APOL1 using a large panel of novel APOL1-specific mouse and rabbit monoclonal antibodies. RESULTS: Both endogenous podocyte and transfected APOL1 isoforms vA and vB1 (and a little of isoform vC) localize to the luminal face of the endoplasmic reticulum (ER) and to the cell surface, but not to mitochondria, endosomes, or lipid droplets. In contrast, APOL2, isoform vB3, and most vC of APOL1 localize to the cytoplasmic face of the ER and are consequently absent from the cell surface. APOL1 knockout podocytes do not stain for APOL1, attesting to the APOL1-specificity of the antibodies. Stable re-transfection of knockout podocytes with inducible APOL1-G0, -G1, and -G2 showed no differences in localization among variants. CONCLUSIONS: APOL1 is found in the ER and plasma membrane, consistent with either the ER stress or surface cation channel models of APOL1-mediated cytotoxicity. The surface localization of APOL1 variants potentially opens new therapeutic targeting avenues.
Assuntos
Apolipoproteína L1/análise , Membrana Celular/química , Retículo Endoplasmático/química , Podócitos/química , Animais , Anticorpos/imunologia , Apolipoproteína L1/imunologia , Apolipoproteínas L/análise , Células COS , Células Cultivadas , Chlorocebus aethiops , Reações Cruzadas , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Podócitos/ultraestruturaRESUMO
Cystine-knot peptides are attractive templates in drug discovery due to a number of features they possess including their 3D conformation, physicochemical stability and synthetic tractability. Yet, their cellular uptake mechanisms remain largely unexplored. Recently, we demonstrated that the cystine-knot peptide EETI-II is internalized into cells and that its cellular uptake could be modulated by using a protein transfection reagent Xfect. However, the mechanism of Xfect-mediated cellular internalization of EETI-II remained unclear. Here, by using high resolution electron microscopy, we observe the formation of EETI-II-positive macropinosomes and clathrin-coated pits at early time points after treatment of cells with EETI-II/Xfect complexes. Internalized EETI-II subsequently accumulates in intracellular Xfect-induced detergent-resistant membrane compartments which appear to lack characteristic endosomal or lysosomal markers. Notably, Xfect enables the uptake of cell impermeable nuclear dyes into similar intracellular compartments that do not seem to deliver the cargo to the cytosol or nucleus. Altogether, our findings reveal mechanistic insights into the cellular uptake route of Xfect, and underscore the need for the development of effective tools to enhance the cytosolic delivery of cystine-knot peptides. Finally, our data illustrate that electron microscopy is a powerful approach for studying endocytic mechanisms of cell-penetrating peptides and their effects on cellular membranes.
Assuntos
Cistina , Microscopia Eletrônica , Peptídeos/química , Peptídeos/metabolismo , Transfecção , Membrana Celular/metabolismo , Clatrina/metabolismo , Endossomos/metabolismo , Células HeLa , Humanos , Lisossomos/metabolismo , Permeabilidade , Transporte ProteicoRESUMO
Synapse loss and Tau pathology are hallmarks of Alzheimer's disease (AD) and other tauopathies, but how Tau pathology causes synapse loss is unclear. We used unbiased proteomic analysis of postsynaptic densities (PSDs) in Tau-P301S transgenic mice to identify Tau-dependent alterations in synapses prior to overt neurodegeneration. Multiple proteins and pathways were altered in Tau-P301S PSDs, including depletion of a set of GTPase-regulatory proteins that leads to actin cytoskeletal defects and loss of dendritic spines. Furthermore, we found striking accumulation of complement C1q in the PSDs of Tau-P301S mice and AD patients. At synapses, C1q decorated perisynaptic membranes, accumulated in correlation with phospho-Tau, and was associated with augmented microglial engulfment of synapses and decline of synapse density. A C1q-blocking antibody inhibited microglial synapse removal in cultured neurons and in Tau-P301S mice, rescuing synapse density. Thus, inhibiting complement-mediated synapse removal by microglia could be a potential therapeutic target for Tau-associated neurodegeneration.
Assuntos
Anticorpos/uso terapêutico , Complemento C1q/imunologia , Sinapses/metabolismo , Tauopatias/tratamento farmacológico , Tauopatias/patologia , Proteínas tau/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Animais Recém-Nascidos , Diferenciação Celular , Células Cultivadas , Complemento C1q/metabolismo , Complemento C1q/ultraestrutura , Embrião de Mamíferos , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Densidade Pós-Sináptica/metabolismo , Densidade Pós-Sináptica/patologia , Densidade Pós-Sináptica/ultraestrutura , Presenilina-2/genética , Presenilina-2/metabolismo , Proteínas Priônicas/genética , Proteínas Priônicas/metabolismo , Proteoma/metabolismo , Ratos , Sinapses/efeitos dos fármacos , Sinapses/ultraestrutura , Tauopatias/diagnóstico por imagem , Tauopatias/genética , Proteínas tau/genéticaRESUMO
The detection of microbial pathogens involves the recognition of conserved microbial components by host cell sensors such as Toll-like receptors (TLRs) and NOD-like receptors (NLRs). TLRs are membrane receptors that survey the extracellular environment for microbial infections, whereas NLRs are cytosolic complexes that detect microbial products that reach the cytosol. Upon detection, both sensor classes trigger innate inflammatory responses and allow the engagement of adaptive immunity. Endo-lysosomes are the entry sites for a variety of pathogens, and therefore the sites at which the immune system first senses their presence. Pathogens internalized by endocytosis are well known to activate TLRs 3 and 7-9 that are localized to endocytic compartments and detect ligands present in the endosomal lumen. Internalized pathogens also activate sensors in the cytosol such as NOD1 and NOD2 (ref. 2), indicating that endosomes also provide for the translocation of bacterial components across the endosomal membrane. Despite the fact that NOD2 is well understood to have a key role in regulating innate immune responses and that mutations at the NOD2 locus are a common risk factor in inflammatory bowel disease and possibly other chronic inflammatory states, little is known about how its ligands escape from endosomes. Here we show that two endo-lysosomal peptide transporters, SLC15A3 and SLC15A4, are preferentially expressed by dendritic cells, especially after TLR stimulation. The transporters mediate the egress of bacterially derived components, such as the NOD2 cognate ligand muramyl dipeptide (MDP), and are selectively required for NOD2 responses to endosomally derived MDP. Enhanced expression of the transporters also generates endosomal membrane tubules characteristic of dendritic cells, which further enhanced the NOD2-dependent response to MDP. Finally, sensing required the recruitment of NOD2 and its effector kinase RIPK2 (refs 8, 9) to the endosomal membrane, possibly by forming a complex with SLC15A3 or SLC15A4. Thus, dendritic cell endosomes are specialized platforms for both the lumenal and cytosolic sensing of pathogens.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Endossomos/imunologia , Endossomos/metabolismo , Proteína Adaptadora de Sinalização NOD2/imunologia , Proteína Adaptadora de Sinalização NOD2/metabolismo , Salmonella typhimurium/imunologia , Acetilmuramil-Alanil-Isoglutamina/imunologia , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Animais , Proteínas de Transporte/metabolismo , Citoplasma/imunologia , Citoplasma/metabolismo , Citoplasma/microbiologia , Células Dendríticas/citologia , Imunidade Inata , Inflamação , Doenças Inflamatórias Intestinais/genética , Ligantes , Lisossomos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Fagossomos/imunologia , Fagossomos/metabolismoRESUMO
Many oncology drugs are administered at their maximally tolerated dose without the knowledge of their optimal efficacious dose range. In this study, we describe a multifaceted approach that integrated preclinical and clinical data to identify the optimal dose for an antiangiogenesis agent, anti-EGFL7. EGFL7 is an extracellular matrix-associated protein expressed in activated endothelium. Recombinant EGFL7 protein supported EC adhesion and protected ECs from stress-induced apoptosis. Anti-EGFL7 antibodies inhibited both of these key processes and augmented anti-VEGF-mediated vascular damage in various murine tumor models. In a genetically engineered mouse model of advanced non-small cell lung cancer, we found that anti-EGFL7 enhanced both the progression-free and overall survival benefits derived from anti-VEGF therapy in a dose-dependent manner. In addition, we identified a circulating progenitor cell type that was regulated by EGFL7 and evaluated the response of these cells to anti-EGFL7 treatment in both tumor-bearing mice and cancer patients from a phase I clinical trial. Importantly, these preclinical efficacy and clinical biomarker results enabled rational selection of the anti-EGFL7 dose currently being tested in phase II clinical trials.
Assuntos
Inibidores da Angiogênese/farmacologia , Anticorpos/farmacologia , Apoptose , Fatores de Crescimento Endotelial/imunologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Bevacizumab , Proteínas de Ligação ao Cálcio , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Ensaios Clínicos Fase I como Assunto , Família de Proteínas EGF , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Insulinoma/irrigação sanguínea , Insulinoma/tratamento farmacológico , Insulinoma/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Camundongos Transgênicos , Células Neoplásicas Circulantes/efeitos dos fármacos , Células Neoplásicas Circulantes/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/irrigação sanguínea , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Carga Tumoral/efeitos dos fármacos , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular/fisiologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Establishment and stabilization of endothelial tubes with patent lumens is vital during vertebrate development. Ras-interacting protein 1 (RASIP1) has been described as an essential regulator of de novo lumenogenesis through modulation of endothelial cell (EC) adhesion to the extracellular matrix (ECM). Here, we show that in mouse and zebrafish embryos, Rasip1-deficient vessels transition from an angioblast cord to a hollow tube, permit circulation of primitive erythrocytes, but ultimately collapse, leading to hemorrhage and embryonic lethality. Knockdown of RASIP1 does not alter EC-ECM adhesion, but causes cell-cell detachment and increases permeability of EC monolayers in vitro. We also found that endogenous RASIP1 in ECs binds Ras-related protein 1 (RAP1), but not Ras homolog gene family member A or cell division control protein 42 homolog. Using an exchange protein directly activated by cyclic adenosine monophosphate 1 (EPAC1)-RAP1-dependent model of nascent junction formation, we demonstrate that a fraction of the RASIP1 protein pool localizes to cell-cell contacts. Loss of RASIP1 phenocopies loss of RAP1 or EPAC1 in ECs by altering junctional actin organization, localization of the actin-bundling protein nonmuscle myosin heavy chain IIB, and junction remodeling. Our data show that RASIP1 regulates the integrity of newly formed blood vessels as an effector of EPAC1-RAP1 signaling.
Assuntos
Proteínas de Transporte/fisiologia , Endotélio Vascular/embriologia , Endotélio Vascular/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Junções Intercelulares/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Knockout , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Neovascularização Fisiológica , Gravidez , Interferência de RNA , Transdução de Sinais , Peixe-Zebra , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/fisiologiaRESUMO
Like several other intracellular pathogens, Mycobacterium marinum (Mm) escapes from phagosomes into the host cytosol where it can polymerize actin, leading to motility that promotes spread to neighboring cells. However, only approximately 25% of internalized Mm form actin tails, and the fate of the remaining bacteria has been unknown. Here we show that cytosolic access results in a new and intricate host pathogen interaction: host macrophages ubiquitinate Mm, while Mm shed their ubiquitinated cell walls. Phagosomal escape and ubiquitination of Mm occurred rapidly, prior to 3.5 hours post infection; at the same time, ubiquitinated Mm cell wall material mixed with host-derived dense membrane networks appeared in close proximity to cytosolic bacteria, suggesting cell wall shedding and association with remnants of the lysed phagosome. At 24 hours post-infection, Mm that polymerized actin were not ubiquitinated, whereas ubiquitinated Mm were found within LAMP-1-positive vacuoles resembling lysosomes. Though double membranes were observed which sequestered Mm away from the cytosol, targeting of Mm to the LAMP-1-positive vacuoles was independent of classical autophagy, as demonstrated by absence of LC3 association and by Atg5-independence of their formation. Further, ubiquitination and LAMP-1 association did not occur with mutant avirulent Mm lacking ESX-1 (type VII) secretion, which fail to escape the primary phagosome; apart from its function in phagosome escape, ESX-1 was not directly required for Mm ubiquitination in macrophages or in vitro. These data suggest that virulent Mm follow two distinct paths in the cytosol of infected host cells: bacterial ubiquitination is followed by sequestration into lysosome-like organelles via an autophagy-independent pathway, while cell wall shedding may allow escape from this fate to permit continued residence in the cytosol and formation of actin tails.
Assuntos
Citosol/microbiologia , Lisossomos/microbiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Mycobacterium marinum/metabolismo , Fagossomos/microbiologia , Actinas/metabolismo , Proteína 5 Relacionada à Autofagia , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Células Cultivadas , Citosol/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Macrófagos/ultraestrutura , Microscopia de Fluorescência , Mycobacterium marinum/ultraestrutura , Fagossomos/metabolismo , UbiquitinaçãoRESUMO
Akt has emerged as an attractive cancer therapeutic target with a central role in cell survival, growth, proliferation and metabolism.A key to the clinical success of Akt inhibitors is the maximal possible antitumor efficacy achievable without intolerable side effects. In our recent work, we show that although Akt inhibition does not always induce a clear apoptotic response, autophagy is a more readily detectable response to pan-Akt knockdown or selective small molecule inhibitors of the PI3K/Akt pathway. Autophagy isa catabolic process of bulk lysosomal degradation and recycling of cytoplasmic material and organelles, which can provide a temporary survival mechanism for cells under stress conditions, but can also make cells vulnerable to several forms of cell death under specific circumstances. We hypothesize that autophagy induced by Akt inhibition may sensitize tumor cells to agents targeting the later steps of this lysosomal degradation process. Indeed, agents that interfere with the lysosomal degradation function could precipitate cell death when combined with Akt inhibition and promote complete tumor remissions in preclinical models. These findings suggest that manipulating the autophagic response may be a promising strategy to increase the therapeutic efficacy of Akt inhibitors.
Assuntos
Autofagia , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Morte Celular , Linhagem Celular Tumoral , Humanos , Lisossomos/metabolismo , Camundongos , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologiaRESUMO
BACKGROUND: Cardiotin expression is observed in adult cardiac tissue. In the present study, we provide evidence for the specific localization of cardiotin in cardiac mitochondria and for its down-regulation during adaptive remodeling (dedifferentiation) of cardiomyocytes. METHODS: Immunocytochemistry was used to study cardiotin localization in adult rabbit papillary muscle, in late-stage embryonic rabbit left ventricular tissue, and in left ventricle samples of rabbits suffering from pressure and volume overload. Western blot analysis of cardiotin was performed in purified pig heart mitochondrial fractions. Cardiotin expression was monitored in vitro in isolated adult rat and rabbit left ventricular cardiomyocytes. RESULTS: Western blot analysis revealed the presence of cardiotin in the mitochondrial fractions of pig heart. Immunoelectron microscopy confirmed the presence of cardiotin in cardiac mitochondria of normal adult rabbits both in vivo and in vitro. Quantification of the localization of immunogold particles suggests an association of cardiotin with the mitochondrial inner membrane. Cardiotin expression is initiated in late-stage embryonic rabbit heart, whereas in adult ventricular tissue cardiotin clearly stained longitudinal arrays of mitochondria. Pressure- and volume-overloaded myocardium showed a reduction in cardiotin expression in dispersed local myocardial areas. Cell cultures of adult cardiomyocytes showed a gradual loss in cardiotin expression in parallel with a sarcomeric remodeling. CONCLUSIONS: Our results demonstrate the specific localization of cardiotin in adult cardiomyocyte mitochondria and propose its use as an early marker for cardiomyocyte adaptive remodeling and dedifferentiation.
Assuntos
Actinina/metabolismo , Insuficiência da Valva Aórtica/metabolismo , Desdiferenciação Celular , Regulação para Baixo , Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Insuficiência da Valva Aórtica/patologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Coração/embriologia , Ventrículos do Coração/química , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Imuno-Histoquímica , Masculino , Microscopia Imunoeletrônica , Mitocôndrias/ultraestrutura , Miócitos Cardíacos/patologia , Miócitos Cardíacos/ultraestrutura , Especificidade de Órgãos , Músculos Papilares/metabolismo , Músculos Papilares/patologia , Músculos Papilares/ultraestrutura , Coelhos , Ratos , SuínosRESUMO
Although Akt is known as a survival kinase, inhibitors of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway do not always induce substantial apoptosis. We show that silencing Akt1 alone, or any combination of Akt isoforms, can suppress the growth of tumors established from phosphatase and tensin homologue-null human cancer cells. Although these findings indicate that Akt is essential for tumor maintenance, most tumors eventually rebound. Akt knockdown or inactivation with small molecule inhibitors did not induce significant apoptosis but rather markedly increased autophagy. Further treatment with the lysosomotropic agent chloroquine caused accumulation of abnormal autophagolysosomes and reactive oxygen species, leading to accelerated cell death in vitro and complete tumor remission in vivo. Cell death was also promoted when Akt inhibition was combined with the vacuolar H(+)-adenosine triphosphatase inhibitor bafilomycin A1 or with cathepsin inhibition. These results suggest that blocking lysosomal degradation can be detrimental to cancer cell survival when autophagy is activated, providing rationale for a new therapeutic approach to enhancing the anticancer efficacy of PI3K-Akt pathway inhibition.
Assuntos
Autofagia/fisiologia , Neoplasias/tratamento farmacológico , PTEN Fosfo-Hidrolase/genética , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/fisiologia , Autofagia/efeitos dos fármacos , Proteína 7 Relacionada à Autofagia , Benzilaminas/farmacologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Cloroquina/farmacologia , Interações Medicamentosas , Feminino , Furanos/farmacologia , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Macrolídeos/farmacologia , Camundongos , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mutação , Neoplasias/genética , Neoplasias/patologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , ATPases Translocadoras de Prótons/antagonistas & inibidores , Piridinas/farmacologia , Pirimidinas/farmacologia , Quinoxalinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Enzimas Ativadoras de Ubiquitina/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Yeast Saccharomyces cerevisiae has been a crucial model system for the study of a multitude of cellular processes because of its amenability to genetics, molecular biology and biochemical procedures. By contrast, the morphological analysis of this organism by immunoelectron microscopy (IEM) has remained in a primordial phase preventing researchers to routinely incorporate this technique into their investigations. Here, in addition to simple but detailed protocols to perform conventional electron microscopy (EM) on plastic embedded sections, we present a new IEM procedure adapted from the Tokuyasu method to prepare cryosections from mildly fixed cells. This novel approach allows an excellent cell preservation and the negatively stained membranes create superb contrast that leads to a unique resolution of the yeast morphology. This, plus the optimal preservation of the epitopes, permits combined localization studies with a fine resolution of protein complexes, vesicular carriers and organelles at an ultrastructural level. Importantly, we also show that this cryo-immunogold protocol can be combined with high-pressure freezing and therefore cryofixation can be employed if difficulties are encountered to immobilize a particular structure with chemical fixation. This new IEM technique will be a valuable tool for the large community of scientists using yeast as a model system, in particular for those studying membrane transport and dynamics.
Assuntos
Crioultramicrotomia/métodos , Imuno-Histoquímica/métodos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Biologia Celular , Membrana Celular/ultraestrutura , Núcleo Celular/ultraestrutura , Criopreservação , Epitopos/química , Processamento de Imagem Assistida por Computador , Microscopia Imunoeletrônica , Organelas , Ácido Periódico/farmacologia , Tomografia/métodosRESUMO
Melanosomes are lysosome-related organelles that coexist with lysosomes in mammalian pigment cells. Melanosomal and lysosomal membrane proteins share similar sorting signals in their cytoplasmic tail, raising the question how they are segregated. We show that in control melanocytes, the melanosomal enzymes tyrosinase-related protein 1 (Tyrp1) and tyrosinase follow an intracellular Golgi to melanosome pathway, whereas in the absence of glycosphingolipids, they are observed to pass over the cell surface. Unexpectedly, the lysosome-associated membrane protein 1 (LAMP-1) and 2 behaved exactly opposite: they were found to travel through the cell surface in control melanocytes but followed an intracellular pathway in the absence of glycosphingolipids. Chimeric proteins having the cytoplasmic tail of Tyrp1 or tyrosinase were transported like lysosomal proteins, whereas a LAMP-1 construct containing the lumenal domain of Tyrp1 localized to melanosomes. In conclusion, the lumenal domain contains sorting information that guides Tyrp1 and probably tyrosinase to melanosomes by an intracellular route that excludes lysosomal proteins and requires glucosylceramide.
Assuntos
Proteínas de Membrana Lisossomal/fisiologia , Melanossomas/fisiologia , Animais , Linhagem Celular Tumoral , Proteínas de Membrana Lisossomal/metabolismo , Proteínas de Membrana Lisossomal/ultraestrutura , Melanócitos/enzimologia , Melanoma/ultraestrutura , Melanossomas/metabolismo , Melanossomas/ultraestrutura , Camundongos , Monofenol Mono-Oxigenase/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , TransfecçãoRESUMO
The endothelial cell (EC) -specific secreted protein EGFL7 is important for tubulogenesis in newly forming blood vessels. We studied its role in vascular tube formation by a quantitative ultrastructural analysis of Egfl7-knockdown zebrafish embryos. At 24 hours postfertilization, the endothelia of dorsal aorta (DA) and posterior cardinal vein (PCV) were correctly anchored to the hypochord and endoderm, respectively, but failed to expand into the vascular area. This resulted in vessels with reduced or split lumen and open sheets of ECs. Concomitantly, the organization of hematopoietic cells-identified by the presence of previously undescribed membrane tubules-between DA and PCV, and within the vessels, was severely disturbed. Strikingly, ectopic cell junctions occurred across the obstructed vessel lumen, on the luminal EC surfaces, which in control conditions never display junctions of any kind. These data suggest that Egfl7 provides ECs with a cue for their extension into the vascular area and in establishing EC cell polarity.
Assuntos
Vasos Sanguíneos/embriologia , Células Endoteliais/ultraestrutura , Neovascularização Fisiológica , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Polaridade Celular , Embrião não Mamífero , Células Endoteliais/fisiologia , Endotélio Vascular/ultraestrutura , Eritroblastos/ultraestrutura , Junções Intercelulares/ultraestrutura , Microscopia Eletrônica de Transmissão , Proteínas de Peixe-Zebra/genéticaRESUMO
Glycosphingolipids are controlled by the spatial organization of their metabolism and by transport specificity. Using immunoelectron microscopy, we localize to the Golgi stack the glycosyltransferases that produce glucosylceramide (GlcCer), lactosylceramide (LacCer), and GM3. GlcCer is synthesized on the cytosolic side and must translocate across to the Golgi lumen for LacCer synthesis. However, only very little natural GlcCer translocates across the Golgi in vitro. As GlcCer reaches the cell surface when Golgi vesicular trafficking is inhibited, it must translocate across a post-Golgi membrane. Concanamycin, a vacuolar proton pump inhibitor, blocks translocation independently of multidrug transporters that are known to translocate short-chain GlcCer. Concanamycin did not reduce LacCer and GM3 synthesis. Thus, GlcCer destined for glycolipid synthesis follows a different pathway and transports back into the endoplasmic reticulum (ER) via the late Golgi protein FAPP2. FAPP2 knockdown strongly reduces GM3 synthesis. Overall, we show that newly synthesized GlcCer enters two pathways: one toward the noncytosolic surface of a post-Golgi membrane and one via the ER toward the Golgi lumen LacCer synthase.