RESUMO
An unbalanced karyotype, a condition known as aneuploidy, has a profound impact on cellular physiology and is a hallmark of cancer. Aneuploid cells experience a number of stresses that are caused by aneuploidy-induced proteomic changes. How the aneuploidy-associated stresses affect cells and whether cells respond to them are only beginning to be understood. Here we show that autophagosomal cargo such as protein aggregates accumulate within lysosomes in aneuploid cells. This causes a lysosomal stress response. Aneuploid cells activate the transcription factor TFEB, a master regulator of autophagic and lysosomal gene expression, thereby increasing the expression of genes needed for autophagy-mediated protein degradation. Accumulation of autophagic cargo within the lysosome and activation of TFEB-responsive genes are also observed in cells in which proteasome function is inhibited, suggesting that proteotoxic stress causes TFEB activation. Our results reveal a TFEB-mediated lysosomal stress response as a universal feature of the aneuploid state.
Assuntos
Aneuploidia , Autofagia/genética , Lisossomos/patologia , Estresse Fisiológico/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Complexo de Endopeptidases do Proteassoma , Agregados Proteicos/fisiologia , Dobramento de Proteína , ProteóliseRESUMO
The HDL receptor scavenger receptor, class B type I (SR-BI) controls the structure and fate of plasma HDL. Female SR-BI KO mice are infertile, apparently because of their abnormal cholesterol-enriched HDL particles. We examined the growth and meiotic progression of SR-BI KO oocytes and found that they underwent normal germinal vesicle breakdown; however, SR-BI KO eggs, which had accumulated excess cholesterol in vivo, spontaneously activated, and they escaped metaphase II (MII) arrest and progressed to pronuclear, MIII, and anaphase/telophase III stages. Eggs from fertile WT mice were activated when loaded in vitro with excess cholesterol by a cholesterol/methyl-ß-cyclodextrin complex, phenocopying SR-BI KO oocytes. In vitro cholesterol loading of eggs induced reduction in maturation promoting factor and MAPK activities, elevation of intracellular calcium, extrusion of a second polar body, and progression to meiotic stages beyond MII. These results suggest that the infertility of SR-BI KO females is caused, at least in part, by excess cholesterol in eggs inducing premature activation and that cholesterol can activate WT mouse eggs to escape from MII arrest. Analysis of SR-BI KO female infertility raises the possibility that abnormalities in cholesterol metabolism might underlie some cases of human female infertility of unknown etiology.
Assuntos
HDL-Colesterol/metabolismo , Colesterol/toxicidade , Infertilidade Feminina/etiologia , Meiose/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Receptores Depuradores Classe B/deficiência , Animais , Sobrevivência Celular , Ácido Egtázico/farmacologia , Feminino , Sistema de Sinalização das MAP Quinases , Meiose/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oócitos/citologia , Corpos Polares , Receptores Depuradores Classe B/fisiologia , Estrôncio/farmacologia , beta-Ciclodextrinas/farmacologiaRESUMO
Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF, VEGF-A) is a multifunctional cytokine with important roles in pathological angiogenesis. Using an adenoviral vector engineered to express murine VEGF-A(164), we previously investigated the steps and mechanisms by which this cytokine induced the formation of new blood vessels in adult immunodeficient mice and demonstrated that the newly formed blood vessels closely resembled those found in VEGF-A-expressing tumors. We now report that, in addition to inducing angiogenesis, VEGF-A(164) also induces a strong lymphangiogenic response. This finding was unanticipated because lymphangiogenesis has been thought to be mediated by other members of the VPF/VEGF family, namely, VEGF-C and VEGF-D. The new "giant" lymphatics generated by VEGF-A(164) were structurally and functionally abnormal: greatly enlarged with incompetent valves, sluggish flow, and delayed lymph clearance. They closely resembled the large lymphatics found in lymphangiomas/lymphatic malformations, perhaps implicating VEGF-A in the pathogenesis of these lesions. Whereas the angiogenic response was maintained only as long as VEGF-A was expressed, giant lymphatics, once formed, became VEGF-A independent and persisted indefinitely, long after VEGF-A expression ceased. These findings raise the possibility that similar, abnormal lymphatics develop in other pathologies in which VEGF-A is overexpressed, e.g., malignant tumors and chronic inflammation.
Assuntos
Indutores da Angiogênese/fisiologia , Fatores de Crescimento Endotelial/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Sistema Linfático/patologia , Linfocinas/fisiologia , Neovascularização Patológica/etiologia , Adenoviridae/genética , Animais , Feminino , Metástase Linfática , Sistema Linfático/fisiologia , Linfoma/etiologia , Camundongos , Timidina/metabolismo , Fator A de Crescimento do Endotélio Vascular , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Fatores de Crescimento do Endotélio VascularRESUMO
Multiprotein complexes are key determinants of Golgi apparatus structure and its capacity for intracellular transport and glycoprotein modification. Three complexes that have previously been partially characterized include (a) the Golgi transport complex (GTC), identified in an in vitro membrane transport assay, (b) the ldlCp complex, identified in analyses of CHO cell mutants with defects in Golgi-associated glycosylation reactions, and (c) the mammalian Sec34 complex, identified by homology to yeast Sec34p, implicated in vesicular transport. We show that these three complexes are identical and rename them the conserved oligomeric Golgi (COG) complex. The COG complex comprises four previously characterized proteins (Cog1/ldlBp, Cog2/ldlCp, Cog3/Sec34, and Cog5/GTC-90), three homologues of yeast Sec34/35 complex subunits (Cog4, -6, and -8), and a previously unidentified Golgi-associated protein (Cog7). EM of ldlB and ldlC mutants established that COG is required for normal Golgi morphology. "Deep etch" EM of purified COG revealed an approximately 37-nm-long structure comprised of two similarly sized globular domains connected by smaller extensions. Consideration of biochemical and genetic data for mammalian COG and its yeast homologue suggests a model for the subunit distribution within this complex, which plays critical roles in Golgi structure and function.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular , Complexo de Golgi/fisiologia , Proteínas/fisiologia , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Linhagem Celular , Clonagem Molecular , Sequência Conservada , Humanos , Substâncias Macromoleculares , Proteínas de Membrana/fisiologia , Complexos Multiproteicos , Mutação , Conformação Proteica , Subunidades Proteicas , Proteínas/química , Proteínas/genética , Proteínas/isolamento & purificação , Ratos , Proteínas de Saccharomyces cerevisiaeRESUMO
OBJECTIVE: High-density lipoprotein (HDL) receptor, scavenger receptor class B, type I (SR-BI), mediated cellular uptake of lipoprotein cholesterol controls HDL structure and plasma HDL and biliary cholesterol levels. In SR-BI knockout (KO) mice, an unusually high plasma unesterified-to-total cholesterol ratio (UC:TC) and abnormally large HDL particles apparently contribute to pathology, including female infertility, susceptibility to atherosclerosis and coronary heart disease, and anemia. Here we examined the influence of SR-BI deficiency on platelets. METHODS AND RESULTS: The high plasma UC:TC ratio in SR-BI KO mice was correlated with platelet abnormalities, including high cholesterol content, abnormal morphologies, high clearance rates, and thrombocytopenia. One day after platelets from wild-type mice were infused into SR-BI KO mice, they exhibited abnormally high cholesterol content and clearance rates similar to those of endogenous platelets. Platelets from SR-BI KO mice exhibited in vitro a blunted aggregation response to the agonist ADP but a normal response to PAR4. CONCLUSIONS: In SR-BI KO mice abnormal circulating lipoproteins, particularly their high UC:TC ratio-rather than the absence of SR-BI in platelets themselves-induce defects in platelet structure and clearance, together with a mild defect in function.
Assuntos
Plaquetas/patologia , Lipoproteínas HDL/genética , Receptores de Lipoproteínas/genética , Receptores Depuradores Classe B/genética , Trombocitopenia/genética , Difosfato de Adenosina/farmacologia , Animais , Aterosclerose/genética , Plaquetas/fisiologia , Agregação Celular/efeitos dos fármacos , Colesterol/metabolismo , Modelos Animais de Doenças , Feminino , Lipoproteínas HDL/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Lipoproteínas/metabolismo , Receptores Depuradores Classe B/metabolismo , Trombocitopenia/metabolismo , Trombocitopenia/patologiaRESUMO
Super-enhancers (SEs) are clusters of enhancers that cooperatively assemble a high density of the transcriptional apparatus to drive robust expression of genes with prominent roles in cell identity. Here we demonstrate that the SE-enriched transcriptional coactivators BRD4 and MED1 form nuclear puncta at SEs that exhibit properties of liquid-like condensates and are disrupted by chemicals that perturb condensates. The intrinsically disordered regions (IDRs) of BRD4 and MED1 can form phase-separated droplets, and MED1-IDR droplets can compartmentalize and concentrate the transcription apparatus from nuclear extracts. These results support the idea that coactivators form phase-separated condensates at SEs that compartmentalize and concentrate the transcription apparatus, suggest a role for coactivator IDRs in this process, and offer insights into mechanisms involved in the control of key cell-identity genes.
Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Proteínas Intrinsicamente Desordenadas/metabolismo , Subunidade 1 do Complexo Mediador/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sequência Conservada , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos/efeitos dos fármacos , Recuperação de Fluorescência Após Fotodegradação , Regulação da Expressão Gênica/efeitos dos fármacos , Glicóis/farmacologia , Células HEK293 , Humanos , Imunoprecipitação , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Subunidade 1 do Complexo Mediador/química , Subunidade 1 do Complexo Mediador/genética , Camundongos , Imagem Molecular , Células NIH 3T3 , Proteínas Nucleares/química , Proteínas Nucleares/genética , Serina/química , Serina/genética , Transativadores/química , Transativadores/genética , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Normal chow (low fat)-fed mice deficient in both the HDL receptor SR-BI and apolipoprotein E (SR-BI/apoE dKO) provide a distinctive model of coronary heart disease (CHD). They exhibit early-onset hypercholesterolemia characterized by unesterified cholesterol-rich abnormal lipoproteins (lamellar/vesicular and stacked discoidal particles), occlusive coronary atherosclerosis, spontaneous myocardial infarction, cardiac dysfunction, and premature death ( approximately 6 weeks of age). Mice in which similar features of CHD could be induced with a lipid-rich diet would represent a powerful tool to study CHD. METHODS AND RESULTS: To generate a diet-inducible model of CHD, we bred SR-BI-deficient (SR-BI KO) mice with hypomorphic apolipoprotein E mice (ApoeR61(h/h)) that express reduced levels of an apoE4-like murine apoE isoform and exhibit diet-induced hypercholesterolemia. When fed a normal chow diet, SR-BI KO/ApoeR61(h/h) mice did not exhibit early-onset atherosclerosis or CHD; the low expression level of the apoE4-like murine apoE was atheroprotective and cardioprotective. However, when fed an atherogenic diet rich in fat, cholesterol, and cholate, they rapidly developed hypercholesterolemia, atherosclerosis, and CHD, a response strikingly similar to that of SR-BI/apoE dKO mice fed a chow diet, and they died 32+/-6 days (50% mortality) after initiation of the high-fat feeding. CONCLUSIONS: The SR-BI KO/ApoeR61(h/h) mouse is a new model of diet-induced occlusive coronary atherosclerosis and CHD (myocardial infarction, cardiac dysfunction and premature death), allowing control of the age of onset, duration, severity, and possibly regression of disease. Thus, SR-BI KO/ApoeR61(h/h) mice have the potential to contribute to our understanding of CHD and its prevention and treatment.
Assuntos
Apolipoproteínas E/fisiologia , Dieta Aterogênica , Modelos Animais de Doenças , Cardiopatias/etiologia , Receptores Depuradores Classe B/deficiência , Animais , Cardiomegalia/patologia , Doença da Artéria Coronariana/etiologia , Eletrocardiografia , Cardiopatias/mortalidade , Cardiopatias/patologia , Cardiopatias/fisiopatologia , Insuficiência Cardíaca/etiologia , Hipercolesterolemia/etiologia , Lipídeos/sangue , Camundongos , Camundongos Knockout , Mortalidade , Infarto do Miocárdio/etiologiaRESUMO
During tumor progression, alternative splicing gives rise to different Mena protein isoforms. We analyzed how Mena11a, an isoform enriched in epithelia and epithelial-like cells, affects Mena-dependent regulation of actin dynamics and cell behavior. While other Mena isoforms promote actin polymerization and drive membrane protrusion, we find that Mena11a decreases actin polymerization and growth factor-stimulated membrane protrusion at lamellipodia. Ectopic Mena11a expression slows mesenchymal-like cell motility, while isoform-specific depletion of endogenous Mena11a in epithelial-like tumor cells perturbs cell:cell junctions and increases membrane protrusion and overall cell motility. Mena11a can dampen membrane protrusion and reduce actin polymerization in the absence of other Mena isoforms, indicating that it is not simply an inactive Mena isoform. We identify a phosphorylation site within 11a that is required for some Mena11a-specific functions. RNA-seq data analysis from patient cohorts demonstrates that the difference between mRNAs encoding constitutive Mena sequences and those containing the 11a exon correlates with metastasis in colorectal cancer, suggesting that 11a exon exclusion contributes to invasive phenotypes and leads to poor clinical outcomes.
Assuntos
Citoesqueleto de Actina , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Processamento Alternativo , Animais , Biomarcadores/metabolismo , Adesão Celular , Comunicação Celular , Membrana Celular/metabolismo , Movimento Celular , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Receptores ErbB/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Pulmão/embriologia , Pulmão/metabolismo , Células MCF-7 , Camundongos , Fenótipo , Fosforilação , Pseudópodes/patologia , Alvéolos Pulmonares/metabolismo , Pele/embriologia , Pele/metabolismo , Resultado do Tratamento , Regulação para Cima , CicatrizaçãoRESUMO
During breast cancer progression, alternative mRNA splicing produces functionally distinct isoforms of Mena, an actin regulator with roles in cell migration and metastasis. Aggressive tumor cell subpopulations express Mena(INV), which promotes tumor cell invasion by potentiating EGF responses. However, the mechanism by which this occurs is unknown. Here we report that Mena associates constitutively with the tyrosine phosphatase PTP1B and mediates a novel negative feedback mechanism that attenuates receptor tyrosine kinase signaling. On EGF stimulation, complexes containing Mena and PTP1B are recruited to the EGFR, causing receptor dephosphorylation and leading to decreased motility responses. Mena also interacts with the 5' inositol phosphatase SHIP2, which is important for the recruitment of the Mena-PTP1B complex to the EGFR. When Mena(INV) is expressed, PTP1B recruitment to the EGFR is impaired, providing a mechanism for growth factor sensitization to EGF, as well as HGF and IGF, and increased resistance to EGFR and Met inhibitors in signaling and motility assays. In sum, we demonstrate that Mena plays an important role in regulating growth factor-induced signaling. Disruption of this attenuation by Mena(INV) sensitizes tumor cells to low-growth factor concentrations, thereby increasing the migration and invasion responses that contribute to aggressive, malignant cell phenotypes.
Assuntos
Proteínas dos Microfilamentos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Actinas/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Adesão Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Proteínas do Citoesqueleto , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Feminino , Humanos , Metástase Neoplásica , Fosforilação , Isoformas de Proteínas , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
We present use of a synthetic, injectable matrix metalloproteinase (MMP)-responsive, bioactive hydrogel as an in situ forming scaffold to deliver thymosin ß4 (Tß4), a pro-angiogenic and pro-survival factor, along with vascular cells derived from human embryonic stem cells (hESC) in ischemic injuries to the heart in a rat model. The gel was found to substitute the degrading extracellular matrix in the infarcted myocardium of rats and to promote structural organization of native endothelial cells, while some of the delivered hESC-derived vascular cells formed de novo capillaries in the infarct zone. Magnetic resonance imaging (MRI) revealed that the microvascular grafts effectively preserved contractile performance 3 d and 6 wk after myocardial infarction, attenuated left ventricular dilation, and decreased infarct size as compared to infarcted rats treated with PBS injection as a control (3 d ejection fraction, + â¼7%, P < 0.001; 6 wk ejection faction, + â¼12%, P < 0.001). Elevation in vessel density was observed in response to treatment, which may be due in part to elevations in human (donor)-derived cytokines EGF, VEGF and HGF (1 d). Thus, a clinically relevant matrix for dual delivery of vascular cells and drugs may be useful in engineering sustained tissue preservation and potentially regenerating ischemic cardiac tissue.
Assuntos
Células-Tronco Embrionárias/fisiologia , Infarto do Miocárdio/patologia , Miocárdio/patologia , Engenharia Tecidual/métodos , Transplantes , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Sobrevivência Celular , Células-Tronco Embrionárias/citologia , Humanos , Hidrogéis/química , Hidrogéis/metabolismo , Teste de Materiais , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Miócitos Cardíacos/patologia , Ratos , Regeneração/fisiologia , Timosina/administração & dosagem , Timosina/metabolismoRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human malignancies. To investigate the cellular origin(s) of this cancer, we determined the effect of PDAC-relevant gene mutations in distinct cell types of the adult pancreas. We show that a subpopulation of Pdx1-expressing cells is susceptible to oncogenic K-Ras-induced transformation without tissue injury, whereas insulin-expressing endocrine cells are completely refractory to transformation under these conditions. However, chronic pancreatic injury can alter their endocrine fate and allow them to serve as the cell of origin for exocrine neoplasia. These results suggest that one mechanism by which inflammation and/or tissue damage can promote neoplasia is by altering the fate of differentiated cells that are normally refractory to oncogenic stimulation.
Assuntos
Carcinoma Ductal Pancreático/genética , Transformação Celular Neoplásica/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Adulto , Animais , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Modelos Animais de Doenças , Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/biossíntese , Transdução de SinaisRESUMO
The airway epithelium is comprised of specialized cell types that play key roles in protecting the lungs from environmental insults. The cellular composition of the murine respiratory epithelium is established during development and different cell types populate specific regions along the airway. Here we show that E2f4-deficiency leads to an absence of ciliated cells from the entire airway epithelium and the epithelium of the submucosal glands in the paranasal sinuses. This defect is particularly striking in the nasal epithelium of E2f4-/- mice where ciliated cells are replaced by columnar secretory cells that produce mucin-like substances. In addition, in the proximal lung, E2f4 loss causes a reduction in Clara cell marker expression indicating that Clara cell development is also affected. These defects arise during embryogenesis and, in the nasal epithelium, appear to be independent of any changes in cell proliferation, the principal process regulated by members of the E2f family of transcription factors. We therefore conclude that E2f4 is required to determine the appropriate development of the airway epithelium. Importantly, the combination of no ciliated cells and excess mucous cells can account for the chronic rhinitis and increased susceptibility to opportunistic infections that causes the postnatal lethality of E2f4 mutant mice.
Assuntos
Fator de Transcrição E2F4/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Mucosa Respiratória/embriologia , Animais , Proliferação de Células , Cílios/patologia , Cílios/ultraestrutura , Fator de Transcrição E2F4/deficiência , Fator de Transcrição E2F4/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucosa Respiratória/patologia , Mucosa Respiratória/ultraestruturaRESUMO
Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are key actin regulators that localize at regions of dynamic actin remodeling, including cellular protrusions and cell-cell and cell-matrix junctions. Several studies have suggested that Ena/VASP proteins are involved in the formation and function of cellular junctions. Here, we establish the importance of Ena/VASP in endothelial junctions in vivo by analysis of Ena/VASP-deficient animals. In the absence of Ena/VASP, the vasculature exhibits patterning defects and lacks structural integrity, leading to edema, hemorrhaging, and late stage embryonic lethality. In endothelial cells, we find that Ena/VASP activity is required for normal F-actin content, actomyosin contractility, and proper response to shear stress. These findings demonstrate that Ena/VASP is critical for actin cytoskeleton remodeling events involved in the maintenance of functional endothelia.
Assuntos
Moléculas de Adesão Celular/fisiologia , Células Endoteliais/fisiologia , Proteínas dos Microfilamentos/fisiologia , Fosfoproteínas/fisiologia , Actinas/fisiologia , Actomiosina/fisiologia , Animais , Aorta/citologia , Aorta/embriologia , Vasos Sanguíneos/patologia , Vasos Sanguíneos/ultraestrutura , Moléculas de Adesão Celular/deficiência , Células Cultivadas , Citoesqueleto/fisiologia , Edema/genética , Edema/patologia , Embrião de Mamíferos , Endotélio Vascular/citologia , Feminino , Coração/embriologia , Hemorragia/genética , Hemorragia/patologia , Humanos , Imuno-Histoquímica , Junções Intercelulares/metabolismo , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/deficiência , Fosfoproteínas/deficiência , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Gravidez , Veias Umbilicais/citologia , Veias Umbilicais/embriologiaRESUMO
The Conserved Oligomeric Golgi (COG) complex is an eight-subunit (Cog1-8) peripheral Golgi protein involved in membrane trafficking and glycoconjugate synthesis. COG appears to participate in retrograde vesicular transport and is required to maintain normal Golgi structure and function. COG mutations interfere with normal transport, distribution, and/or stability of Golgi proteins associated with glycoconjugate synthesis and trafficking, and lead to failure of spermatogenesis in Drosophila melanogaster, misdirected migration of gonadal distal tip cells in Caenorhabditis elegans, and type II congenital disorders of glycosylation in humans. The mechanism by which COG influences Golgi structure and function is unclear. Immunogold electron microscopy was used to visualize the intraGolgi distribution of a functional, hemagglutinin epitope-labeled COG subunit, Cog1-HA, that complements the Cog1-deficiency in Cog1-null Chinese hamster ovary cells. COG was found to be localized primarily on or in close proximity to the tips and rims of the Golgi's cisternae and their associated vesicles and on vesicles and vesiculo-tubular structures seen on both the cis and trans-Golgi Network faces of the cisternal stacks, in some cases on COPI containing vesicles. These findings support the proposal that COG is directly involved in controlling vesicular retrograde transport of Golgi resident proteins throughout the Golgi apparatus.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Complexo de Golgi/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/ultraestrutura , Animais , Células CHO , Células Cultivadas , Complexo I de Proteína do Envoltório/ultraestrutura , Cricetinae , Cricetulus , Complexo de Golgi/ultraestrutura , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Mutação/genética , Fragmentos de Peptídeos/metabolismo , Fenótipo , Transporte Proteico , Rede trans-Golgi/ultraestruturaRESUMO
Malignant tumors generate new blood vessels by secreting growth factors, particularly members of the vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) family. Overall, the new blood vessels that form are hyperpermeable to plasma proteins, a property that is thought to be important for generating new stroma. However, tumor blood vessels are structurally heterogeneous and include microvessels of at least the following distinct types: mother vessels (MV), glomeruloid microvascular proliferations (GMP), arterio-venous-like vascular malformations and capillaries. Our goal was to determine whether macromolecular tracers leaked from all or from only a subset of these vessel types and to elucidate the extravasation pathways. As blood vessels are only a minor component of tumors, and therefore, difficult to study in situ, we used an adenoviral vector to express VEGF-A164, the most important member of the VPF/VEGF family, in mouse tissues. So expressed, VEGF-A164 induces large numbers of surrogate vessels of each type found in tumors in a highly reproducible manner. Overall permeability to plasma proteins was assessed qualitatively with Evan's blue dye and quantitatively with a dual tracer method employing radioactive albumin. Leaky vessels were identified by confocal microscopy (FITC-dextran) and by electron microscopy (ferritin). MV, and to a lesser extent GMP, were found to be hyperpermeable but capillaries and vascular malformations were not. Ferritin extravasated primarily by two trans-cellular routes, vesiculo-vacuolar organelles (VVOs) and fenestrae. This occurred despite a considerable reduction in VVO frequency as VVO membranes translocated to the plasma membrane during MV formation. However, reduction in the number and complexity of VVOs was offset by extensive endothelial cell thinning and a greatly shortened extravasation pathway. Extrapolating these findings to tumors predicts that only a subset of tumor vessels, MV and GMP, is hyperpermeable, and that measures of overall vessel permeability greatly underestimate the permeability of individual MV and GMP.
Assuntos
Vasos Sanguíneos/fisiologia , Neoplasias Experimentais/irrigação sanguínea , Permeabilidade , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Sequência de Bases , Primers do DNA , Camundongos , Microscopia Confocal , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
The conserved oligomeric Golgi (COG) complex is a heterooctameric complex that regulates intraGolgi trafficking and the integrity of the Golgi compartment in eukaryotic cells. Here, we describe a patient with a mild form of congenital disorder of glycosylation type II (CDG-II) that is caused by a deficiency in the Cog1 subunit of the complex. This patient has a defect in both N- and O-glycosylation. Mass spectrometric analysis of the structures of the N-linked glycans released from glycoproteins from the patient's serum revealed a reduction in sialic acid and galactose residues. Peanut agglutinin (PNA) lectin staining revealed a decrease in sialic acids on core 1 mucin type O-glycans, indicating a combined defect in N- and O-glycosylation. Sequence analysis of the COG1 cDNA and gene identified a homozygous insertion of a single nucleotide (2659-2660insC), which is predicted to lead to a premature translation stop and truncation of the C terminus of the Cog1 protein by 80 amino acids. This mutation destabilizes several other COG subunits and alters their subcellular localization and hence the overall integrity of the COG complex. This results in reduced levels and/or altered Golgi localization of alpha-mannosidase II and beta-1,4 galactosyltransferase I, which links it to the glycosylation deficiency. Transfection of primary fibroblasts of this patient with the full length hemagglutinin-tagged Cog1 indeed restored beta-1,4 galactosyltransferase Golgi localization. We propose naming this disorder CDG-II/Cog1, or CDG-II caused by Cog1 deficiency.
Assuntos
Erros Inatos do Metabolismo dos Carboidratos/genética , Erros Inatos do Metabolismo dos Carboidratos/metabolismo , Proteínas de Transporte/genética , Complexo de Golgi/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas Adaptadoras de Transporte Vesicular , Sequência de Bases , Proteínas Sanguíneas/química , Proteínas de Transporte/química , DNA/genética , Análise Mutacional de DNA , Feminino , Glicoproteínas/sangue , Glicoproteínas/química , Glicosilação , Humanos , Técnicas In Vitro , Lactente , Recém-Nascido , Proteínas de Membrana/química , Polissacarídeos/química , Subunidades Proteicas , TransfecçãoRESUMO
The conserved oligomeric Golgi (COG) complex is thought to function in intra-Golgi retrograde trafficking mediated by coat protein I vesicles, a pathway essential for the proper structure and function of the Golgi apparatus. Previous work suggested that COG might act as a tethering factor to mediate the initial attachment between coat protein I vesicles and Golgi membranes. Here, we present extensive in vitro co-translation and immunoprecipitation experiments leading to a new model for the overall architecture of the mammalian COG complex. The eight COG subunits (Cog1-8) are found to form two heterotrimeric subassemblies (Cog2/3/4 and Cog5/6/7) linked by a heterodimer composed of the remaining subunits (Cog1/8). This model is in excellent agreement with in vivo data presented in an accompanying paper (Oka, T., Vasile, E., Penman, M., Novina, C. D., Dykxhoorn, D. M., Ungar, D., Hughson, F. M., and Krieger, M. (2005) J. Biol. Chem. 280, 32736-32745).
Assuntos
Complexo de Golgi/química , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Transporte Biológico , Proteínas de Transporte/metabolismo , Cromatografia em Gel , Cromatografia por Troca Iônica , Dimerização , Eletroforese em Gel de Poliacrilamida , Complexo de Golgi/metabolismo , Humanos , Immunoblotting , Imunoprecipitação , Proteínas de Membrana/metabolismo , Modelos Biológicos , Plasmídeos/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Proteínas/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
The conserved oligomeric Golgi (COG) complex is an eight-subunit (Cog1-8) peripheral Golgi protein involved in Golgi-associated membrane trafficking and glycoconjugate synthesis. We have analyzed the structure and function of COG using Cog1 or Cog2 null Chinese hamster ovary cell mutants, fibroblasts from a patient with Cog7-deficient congenital disorders of glycosylation, and stable Cog5-deficient HeLa cells generated by RNA interference. Although the dilation of some Golgi cisternae in Cog5-deficient cells resembled that observed in Cog1- or Cog2-deficient cells, their global glycosylation defects (less severe) and intracellular processing and function of low density lipoprotein receptors (essentially normal) differed from Cog1- and Cog2-deficient cells. Immunoblotting, gel filtration, and immunofluorescence microscopy analyses of the COG-deficient cells and cell extracts indicated that 1) Cog2-4 and Cog5-7 form stable subcomplexes, 2) Cog1 mediates Golgi association of a Cog2-4 plus Cog8 subcomplex, 3) Cog8 associates stably with both Cog5-7 and Cog1-4 subcomplexes, and thus 4) Cog8 helps assemble the Cog1-4 and Cog5-7 subcomplexes into the complete COG complex. This model of the subunit organization of COG is in excellent agreement with in vitro data presented in an accompanying paper (Ungar, D., Oka, T., Vasile, E., Krieger, M., and Hughson, F. M. (2005) J. Biol. Chem. 280, 32729-32735). Only one or two of the seven Cog1- or Cog2-dependent Golgi membrane proteins called GEARs are also sensitive to Cog5 or Cog7 deficiency, indicating that the COG subunits play distinctive roles in controlling Golgi structure and function.
Assuntos
Proteínas de Transporte/genética , Proteínas de Membrana/genética , Proteínas Adaptadoras de Transporte Vesicular , Animais , Arachis , Células CHO , Proteínas de Transporte/química , Cromatografia em Gel , Cricetinae , Fibroblastos/metabolismo , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Lectinas/química , Proteínas de Membrana/química , Microscopia Eletrônica , Microscopia de Fluorescência , Modelos Biológicos , Mutação , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Interferência de RNA , TransfecçãoRESUMO
At 39.5 degrees C in the temperature-sensitive, conditional-lethal mutant ldlG, glycoprotein processing is disrupted and secretion is blocked. The ultrastructure of the Golgi apparatus in ldlG cells was examined using immunofluorescence and immunoelectron microscopy. At 34 degrees C the structure of the Golgi apparatus was normal, whereas after incubation at 39.5 degrees C for 12 h it disassembled into dispersed vesicles. These reassembled into stacks when cells were returned to 34 degrees C for 6 h. At both 34 and 39.5 degrees C, all Golgi markers examined were present at wild-type levels except GM130, which was undetectable (<5% of control). Transfection with GM130 corrected the mutant phenotypes. Although the endogenous gene encoding NSF is apparently normal in ldlG cells, all mutant phenotypes were corrected by transfection with NSF, suggesting that NSF functioned as an extragenic suppressor. These findings provide additional support for a role of GM130 in determining the properties of the Golgi apparatus and for NSF in influencing GM130 stability and function. They also suggest that, at 34 degrees C, detectable levels of GM130 are not required for normal Golgi morphology and function, but that GM130 - or a GM130-dependent protein(s) - does play a role in protecting the Golgi, and thus the cells, from stress at higher temperatures.
Assuntos
Genes Letais , Complexo de Golgi/ultraestrutura , Proteínas de Membrana/genética , Mutação , Animais , Autoantígenos , Northern Blotting , Western Blotting , Células CHO , Clonagem Molecular , Cricetinae , DNA Complementar , Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleases/metabolismo , TemperaturaRESUMO
The murine class B, type I scavenger receptor mSR-BI, a high density lipoprotein (HDL) receptor that mediates selective uptake of HDL lipids, contains 11 potential N-linked glycosylation sites and unknown numbers of both endoglycosidase H-sensitive and -resistant oligosaccharides. We have examined the consequences of mutating each of these sites (Asn --> Gln or Thr --> Ala) on post-translational processing of mSR-BI, cell surface expression, and HDL binding and lipid transport activities. All 11 sites were glycosylated; however, disruption of only two (Asn-108 and Asn-173) substantially altered expression and function. There was very little detectable post-translational processing of these two mutants to endoglycosidase H resistance and very low cell surface expression, suggesting that oligosaccharide modification at these sites apparently plays an important role in endoplasmic reticulum folding and/or intracellular transport. Strikingly, although the low levels of the 108 and 173 mutants that were expressed on the cell surface exhibited a marked reduction in their ability to transfer lipids from HDL to cells, they nevertheless bound nearly normal amounts of HDL. Indeed, the affinity of (125)I-HDL binding to the 173 mutant was similar to that of the wild-type receptor. Thus, N-linked glycosylation can influence both the intracellular transport and lipid-transporter activity of SR-BI. The ability to uncouple the HDL binding and lipid transport activities of mSR-BI by in vitro mutagenesis should provide a powerful tool for further analysis of the mechanism of SR-BI-mediated selective lipid uptake.