RESUMO
Conformational changes in endocytic proteins are regulators of clathrin-mediated endocytosis. Three clathrin heavy chains associated with clathrin light chains (CLC) assemble into triskelia that link into a geometric lattice that curves to drive endocytosis. Structural changes in CLC have been shown to regulate triskelia assembly in solution, yet the nature of these changes, and their effects on lattice growth, curvature, and endocytosis in cells are unknown. Here, we develop a new correlative fluorescence resonance energy transfer (FRET) and platinum replica electron microscopy method, named FRET-CLEM. With FRET-CLEM, we measure conformational changes in clathrin at thousands of individual morphologically distinct clathrin-coated structures. We discover that the N-terminus of CLC repositions away from the plasma membrane and triskelia vertex as coats curve. Preventing this conformational switch with chemical tools increases lattice sizes and inhibits endocytosis. Thus, a specific conformational switch in the light chain regulates lattice curvature and endocytosis in mammalian cells.
Assuntos
Cadeias Leves de Clatrina , Endocitose , Animais , Cadeias Leves de Clatrina/metabolismo , Membrana Celular/metabolismo , Clatrina/metabolismo , Cadeias Pesadas de Clatrina/metabolismo , Mamíferos/metabolismoRESUMO
BACKGROUND: Hepatitis delta virus (HDV), a satellite virus of hepatitis B virus (HBV), is a small, defective RNA virus strongly associated with the most severe form of hepatitis and progressive chronic liver disease and cirrhosis. Chronic hepatitis D, resulting from HBV/HDV coinfection, is considered to be the most severe form of viral hepatitis and affects 12-20 million people worldwide. Involved in the endocytosis and exocytosis of cellular and viral proteins, clathrin contributes to the pathogenesis and morphogenesis of HDV. Previously, we demonstrated that HDV-I and -II large hepatitis delta antigens (HDAg-L) possess a putative clathrin box that interacts with clathrin heavy chain (CHC) and supports HDV assembly. METHODS: Virus assembly and vesicular trafficking of HDV virus-like particles (VLPs) were evaluated in Huh7 cells expressing HDV-I, -II and -III HDAg-L and hepatitis B surface antigen (HBsAg). To elucidate the interaction motif between HDAg-L and CHC, site-directed mutagenesis was performed to introduce mutations into HDAg-L and CHC and analyzed using coimmunoprecipitation or pull-down assays. RESULTS: Comparable to HDV-I virus-like particles (VLPs), HDV-III VLPs were produced at a similar level and secreted into the medium via clathrin-mediated post-Golgi vesicular trafficking. Mutation at F27 or E33 of CHC abolished the binding of CHC to the C-terminus of HDV-III HDAg-L. Mutation at W207 of HDV-III HDAg-L inhibited its association with CHC and interfered with HDV-III VLP formation. We elucidated mechanism of the binding of HDV-III HDAg-L to CHC and confirmed the pivotal role of clathrin binding in the assembly of genotype III HDV. CONCLUSIONS: A novel W box which was identified at the C terminus of HDV-III HDAg-L is known to differ from the conventional clathrin box but also interacts with CHC. The novel W box of HDAg-L constitutes a new molecular target for anti-HDV-III therapeutics.
Assuntos
Antígenos de Superfície da Hepatite B , Vírus Delta da Hepatite , Clatrina/metabolismo , Cadeias Pesadas de Clatrina/genética , Cadeias Pesadas de Clatrina/metabolismo , Genótipo , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/genética , Vírus Delta da Hepatite/genética , Antígenos da Hepatite delta/química , Antígenos da Hepatite delta/genética , Antígenos da Hepatite delta/metabolismo , Humanos , RNA Viral/metabolismo , Proteínas Virais/genética , Replicação ViralRESUMO
Foot-and-mouth disease virus (FMDV) is a highly contagious virus that causes severe vesicular disease of cloven-hoofed animals. Various endocytosis mechanisms are involved in the entry of FMDV after binding to the integrin and heparan sulfate (HS) receptors. However, the mechanism of FMDV using other unknown receptors to enter the cells remains unclear. Here, we reported that the endocytosis and endosomal pathways are employed by FMDV to invade the Chinese hamster ovary cell line (CHO-677) without the integrin and HS receptors. We demonstrated that the internalization of FMDV into CHO-677 cells was abrogated by chlorpromazine, an inhibitor of clathrin-mediated endocytosis. Knockdown of the clathrin heavy chain decreased the viral protein abundance. Incubation of the CHO-677 cells with the inhibitors of caveolae-mediated endocytosis or transfection by caveolin-1 siRNA also limited FMDV replication. In addition, we determined that the acidic environment and the existence of dynamin were essential for FMDV infection in CHO-677 cells. The endosomal proteins Rab5 (early endosome) and Rab7 (late endosome), but not Rab11 (recycling endosome), were utilized by FMDV during infection. These data provide a new entry model of FMDV by unknown receptors which will help to better understand the pathogenesis mediated by FMDV.
Assuntos
Vírus da Febre Aftosa , Doenças da Boca , Doenças dos Roedores , Cricetinae , Animais , Clatrina/metabolismo , Vírus da Febre Aftosa/genética , Vírus da Febre Aftosa/metabolismo , Células CHO , Caveolina 1/metabolismo , Cricetulus , RNA Interferente Pequeno , Cadeias Pesadas de Clatrina/metabolismo , Clorpromazina , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo , Internalização do Vírus , Endocitose , Dinaminas/metabolismo , Integrinas/metabolismo , Heparitina Sulfato , Proteínas Virais/metabolismo , Doenças da Boca/veterináriaRESUMO
BACKGROUND: Giardia lamblia, a parasitic protist of the Metamonada supergroup, has evolved one of the most diverged endocytic compartment systems investigated so far. Peripheral endocytic compartments, currently known as peripheral vesicles or vacuoles (PVs), perform bulk uptake of fluid phase material which is then digested and sorted either to the cell cytosol or back to the extracellular space. RESULTS: Here, we present a quantitative morphological characterization of these organelles using volumetric electron microscopy and super-resolution microscopy (SRM). We defined a morphological classification for the heterogenous population of PVs and performed a comparative analysis of PVs and endosome-like organelles in representatives of phylogenetically related taxa, Spironucleus spp. and Tritrichomonas foetus. To investigate the as-yet insufficiently understood connection between PVs and clathrin assemblies in G. lamblia, we further performed an in-depth search for two key elements of the endocytic machinery, clathrin heavy chain (CHC) and clathrin light chain (CLC), across different lineages in Metamonada. Our data point to the loss of a bona fide CLC in the last Fornicata common ancestor (LFCA) with the emergence of a protein analogous to CLC (GlACLC) in the Giardia genus. Finally, the location of clathrin in the various compartments was quantified. CONCLUSIONS: Taken together, this provides the first comprehensive nanometric view of Giardia's endocytic system architecture and sheds light on the evolution of GlACLC analogues in the Fornicata supergroup and, specific to Giardia, as a possible adaptation to the formation and maintenance of stable clathrin assemblies at PVs.
Assuntos
Giardia lamblia , Clatrina/metabolismo , Cadeias Pesadas de Clatrina/genética , Cadeias Pesadas de Clatrina/metabolismo , Cadeias Leves de Clatrina/metabolismo , Endocitose , Giardia lamblia/genética , Giardia lamblia/metabolismo , FilogeniaRESUMO
Clathrin heavy chain (Chc) is a constituent of clathrin-coated vesicles and serves important functions in endocytosis and intracellular membrane trafficking but appears to have physiological roles also at the organismal level. Most of what we know about Chc functions originates from studies performed in fungal or vertebrate cells. However, the physiological functions of Chc in insects remain poorly understood. Here, we identified a Chc ortholog from a Locusta migratoria transcriptome database. RT-qPCR revealed that LmChc was constitutively expressed in fifth-instar nymphs. In this developmental stage, LmChc showed the highest expression in the ovary followed by hemolymph, testis, hindgut, midgut, and foregut. In isolated hemocytes, we detected the Chc protein in patches at the plasma membrane. To examine the role of LmChc in L. migratoria during development, RNA interference was performed by injecting dsRNA into the early fifth-instar nymphs. Silencing of LmChc caused a lethal phenotype with molting defect from nymph to adult. In addition, silencing of LmChc resulted in abnormal development of the ovaries, the size of which was significantly smaller than that in controls. Taken together, our results suggest that LmChc is a vital gene in L. migratoria that plays an important role in growth, development, and reproduction. LmChc may be used as an efficient RNAi target gene for developing dsRNA-based biological insecticides to manage insect pests.
Assuntos
Locusta migratoria , Feminino , Animais , Locusta migratoria/metabolismo , Cadeias Pesadas de Clatrina/genética , Cadeias Pesadas de Clatrina/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Muda/genética , Ninfa , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , ReproduçãoRESUMO
Bovine parainfluenza virus 3 (BPIV3) is an important respiratory pathogen of both young and adult cattle. No specific therapies are available for BPIV3. Understanding the viral internalization pathway of BPIV3 will provide new strategies for the development of antiviral treatments. Here, the entry mechanism of BPIV3 into MDBK cells was analyzed using chemical inhibitors and RNA silencing. Our data demonstrated that treatment with an inhibitor targeting the clathrin-mediated pathway or clathrin heavy chain (CHC) knockdown suppressed the entry of BPIV3 into MDBK cells. In contrast, sequestration of cellular cholesterol by nystatin or silencing of caveolin-1 had no effect on viral entry. Moreover, inhibition of critical modulators of macropinocytosis significantly reduced BPIV3 uptake. In addition, fluid-phase uptake was significantly increased in cells infected with BPIV3, which is indicative of virus-induced facilitation of macropinocytosis. These results suggest that BPIV3 enters MDBK cells via macropinocytosis and clathrin- but not caveolar-dependent endocytosis. Furthermore, inhibition of endosomal acidification and activation of cathepsin blocked BPIV3 entry, demonstrating that BPIV3 entered MDBK cells in a acid-dependent manner and required cathepsin L. Finally, we demonstrated that macropinocytosis but not clathrin-mediated endocytosis is dependent on actin dynamics during BPIV3 infection.
Assuntos
Ácidos/metabolismo , Cadeias Pesadas de Clatrina/genética , Clatrina/metabolismo , Endocitose , Vírus da Parainfluenza 3 Bovina/fisiologia , Pinocitose , Internalização do Vírus , Animais , Catepsina L/metabolismo , Bovinos , Linhagem Celular , Cadeias Pesadas de Clatrina/metabolismoRESUMO
Although the treatment of osteosarcoma has improved, the overall survival rate of this common type of osseous malignancies has not changed for four decades. Thus, new targets for better therapeutic regimens are urgently needed. In this study, we found that high expression of clathrin heavy chain (CLTC) was an independent prognostic factor for tumor-free survival (HzR, 3.049; 95% CI, 1.476-6.301) and overall survival (HzR, 2.469; 95% CI, 1.005-6.067) of patients with osteosarcoma. Down-regulation of CLTC resulted in tumor-suppressive effects in vitro and in vivo. Moreover, we found that CLTC was transcriptionally regulated by a transcription factor-specificity protein 1 (SP1), which binds to the CLTC promoter at the -320 to -314-nt and +167 to +173-nt loci. Mechanistic investigations further revealed that CLTC elicited its pro-tumor effects by directly binding to and stabilizing trafficking from the endoplasmic reticulum to the Golgi regulator (TFG). Importantly, overexpression of TFG rescued both the tumor-suppressive effect and inhibition of the TGF-ß and AKT/mTOR pathways caused by CLTC down-regulation, which indicated that the activity of CLTC was TFG-dependent. Immunohistochemistry analysis confirmed that CLTC expression was positively correlated with TFG expression. These findings collectively highlight CLTC as a new prognostic biomarker for patients with osteosarcoma, and the interruption of the SP1/CLTC/TFG axis may serve as a novel therapeutic strategy for osteosarcoma.
Assuntos
Cadeias Pesadas de Clatrina/metabolismo , Regulação Neoplásica da Expressão Gênica , Osteossarcoma/patologia , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Adulto , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Ciclo Celular , Movimento Celular , Proliferação de Células , Cadeias Pesadas de Clatrina/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Osteossarcoma/genética , Osteossarcoma/metabolismo , Prognóstico , Proteínas/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais , Taxa de Sobrevida , Serina-Treonina Quinases TOR/genética , Fator de Crescimento Transformador beta/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto JovemRESUMO
G-protein-coupled receptor-regulated cAMP production from endosomes can specify signaling to the nucleus by moving the source of cAMP without changing its overall amount. How this is possible remains unknown because cAMP gradients dissipate over the nanoscale, whereas endosomes typically localize micrometers from the nucleus. We show that the key location-dependent step for endosome-encoded transcriptional control is nuclear entry of cAMP-dependent protein kinase (PKA) catalytic subunits. These are sourced from punctate accumulations of PKA holoenzyme that are densely distributed in the cytoplasm and titrated by global cAMP into a discrete metastable state, in which catalytic subunits are bound but dynamically exchange. Mobile endosomes containing activated receptors collide with the metastable PKA puncta and pause in close contact. We propose that these properties enable cytoplasmic PKA to act collectively like a semiconductor, converting nanoscale cAMP gradients generated from endosomes into microscale elevations of free catalytic subunits to direct downstream signaling.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Citoplasma/metabolismo , Endossomos/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais/genética , Animais , Domínio Catalítico , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Cadeias Pesadas de Clatrina/antagonistas & inibidores , Cadeias Pesadas de Clatrina/genética , Cadeias Pesadas de Clatrina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Citoplasma/ultraestrutura , Dinamina I/genética , Dinamina I/metabolismo , Endossomos/ultraestrutura , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Holoenzimas/genética , Holoenzimas/metabolismo , Humanos , Luciferases/genética , Luciferases/metabolismo , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Receptores Adrenérgicos beta 2/genéticaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, so understanding its biology and infection mechanisms is critical to facing this major medical challenge. SARS-CoV-2 is known to use its spike glycoprotein to interact with the cell surface as a first step in the infection process. As for other coronaviruses, it is likely that SARS-CoV-2 next undergoes endocytosis, but whether or not this is required for infectivity and the precise endocytic mechanism used are unknown. Using purified spike glycoprotein and lentivirus pseudotyped with spike glycoprotein, a common model of SARS-CoV-2 infectivity, we now demonstrate that after engagement with the plasma membrane, SARS-CoV-2 undergoes rapid, clathrin-mediated endocytosis. This suggests that transfer of viral RNA to the cell cytosol occurs from the lumen of the endosomal system. Importantly, we further demonstrate that knockdown of clathrin heavy chain, which blocks clathrin-mediated endocytosis, reduces viral infectivity. These discoveries reveal that SARS-CoV-2 uses clathrin-mediated endocytosis to gain access into cells and suggests that this process is a key aspect of virus infectivity.
Assuntos
Enzima de Conversão de Angiotensina 2/genética , Cadeias Pesadas de Clatrina/genética , Endocitose/genética , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Internalização do Vírus/efeitos dos fármacos , Células A549 , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Chlorocebus aethiops , Cadeias Pesadas de Clatrina/antagonistas & inibidores , Cadeias Pesadas de Clatrina/metabolismo , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Endossomos/virologia , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Humanos , Hidrazonas/farmacologia , Lentivirus/genética , Lentivirus/metabolismo , Ligação Proteica/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , Transdução de Sinais , Glicoproteína da Espícula de Coronavírus/metabolismo , Sulfonamidas/farmacologia , Tiazolidinas/farmacologia , Células VeroRESUMO
Pseudomyogenic hemangioendothelioma, an uncommon mesenchymal neoplasm composed of plump spindled and/or epithelioid endothelial cells, may present multicentrically and tends to locally recur but rarely metastasizes. Morphologic resemblance to epithelioid sarcoma and other spindle cell neoplasms may result in diagnostic confusion. Molecular characterization of pseudomyogenic hemangioendothelioma has revealed these neoplasms often harbor a rearrangement of the FOSB gene with SERPINE1 or ACTB as recurrent fusion gene partners. Herein, a case of a fibular pseudomyogenic hemangioendothelioma with minimal extension into the adjacent soft tissue arising in a 17 year-old male is presented. The neoplasm exhibited sheets of epithelioid cells with abundant eosinophilic cytoplasm and variably eccentric nuclei. RNA sequencing revealed a novel CLTC-FOSB fusion transcript that was subsequently confirmed by direct sequencing of reverse transcription-polymerase chain reaction products demonstrating an in-frame fusion between exon 17 of the clathrin heavy chain (CLTC) gene and exon 2 of the FOSB (FosB proto-oncogene, AP-1 transcription factor subunit) gene. CLTC-FOSB fusion has not been described in a neoplasm before.
Assuntos
Neoplasias Ósseas/genética , Cadeias Pesadas de Clatrina/genética , Hemangioendotelioma/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas c-fos/genética , Adolescente , Neoplasias Ósseas/patologia , Cadeias Pesadas de Clatrina/metabolismo , Hemangioendotelioma/patologia , Humanos , Masculino , Proteínas de Fusão Oncogênica/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Circular RNAs are involved in the occurrence and development of different types of cancers. We aimed to illustrate the expression profile and mechanism of circ_0074027 in non-small cell lung cancer (NSCLC). Quantitative real-time PCR was employed to detect the expression of circ_0074027, paired like homeodomain 1 (PITX1) mRNA (mPITX1) and microRNA-362-3p (miR-362-3p). Western blot assay was utilized to measure the levels of clathrin heavy chain (CLTC), cyclin D1, BCL2-associated X, apoptosis regulator Bax (Bax), vimentin and matrix metallopeptidase 9. The clonogenicity, apoptosis and metastasis of NSCLC cells were examined by colony formation assay, flow cytometry and transwell migration and invasion assays. The target relationship between miR-362-3p and circ_0074027 or CLTC was predicted by starBase website and was validated by dual-luciferase reporter assay. Murine xenograft assay was applied to explore the function of circ_0074027 in vivo. We found that The enrichment of circ_0074027 and CLTC protein was elevated, and a significant reduction in the expression of miR-362-3p was observed in NSCLC tissues and cells relative to adjacent normal tissues and human bronchial epithelial cells 16HBE. Circ_0074027 possessed a stable circular structure. Circ_0074027 and CLTC could accelerate the colony formation and metastasis and suppress the apoptosis of NSCLC cells. Circ_0074027/miR-362-3p/CLTC axis was first found to regulate the malignance of NSCLC cells. The biological influence caused by circ_0074027 depletion on NSCLC cells was alleviated by the accumulation of CLTC. Circ_0074027 acted as an oncogene to promote the growth of NSCLC tumors in vivo. In conclusion, Circ_0074027 contributed to the progression of NSCLC through promoting the proliferation and motility while hampering the apoptosis of NSCLC cells via miR-362-3p/CLTC axis.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Cadeias Pesadas de Clatrina/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/patologia , MicroRNAs/genética , RNA Circular/genética , Animais , Apoptose , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Movimento Celular , Proliferação de Células , Cadeias Pesadas de Clatrina/genética , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mouse embryonic stem cells (mESCs) display unique mechanical properties, including low cellular stiffness in contrast to differentiated cells, which are stiffer. We have previously shown that mESCs lacking the clathrin heavy chain (Cltc), an essential component for clathrin-mediated endocytosis (CME), display a loss of pluripotency and an enhanced expression of differentiation markers. However, it is not known whether physical properties such as cellular stiffness also change upon loss of Cltc, similar to what is seen in differentiated cells, and if so, how these altered properties specifically impact pluripotency. Using atomic force microscopy (AFM), we demonstrate that mESCs lacking Cltc display higher Young's modulus, indicative of greater cellular stiffness, compared with WT mESCs. The increase in stiffness was accompanied by the presence of actin stress fibers and accumulation of the inactive, phosphorylated, actin-binding protein cofilin. Treatment of Cltc knockdown mESCs with actin polymerization inhibitors resulted in a decrease in the Young's modulus to values similar to those obtained with WT mESCs. However, a rescue in the expression profile of pluripotency factors was not obtained. Additionally, whereas WT mouse embryonic fibroblasts could be reprogrammed to a state of pluripotency, this was inhibited in the absence of Cltc. This indicates that the presence of active CME is essential for the pluripotency of embryonic stem cells. Additionally, whereas physical properties may serve as a simple readout of the cellular state, they may not always faithfully recapitulate the underlying molecular fate.
Assuntos
Cadeias Pesadas de Clatrina/metabolismo , Endocitose , Células-Tronco Embrionárias Murinas/química , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/fisiologia , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Diferenciação Celular , Reprogramação Celular , Cadeias Pesadas de Clatrina/antagonistas & inibidores , Cadeias Pesadas de Clatrina/genética , Módulo de Elasticidade , Camundongos , Microscopia de Força Atômica , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Fosforilação , Profilinas/antagonistas & inibidores , Profilinas/genética , Profilinas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Tiazolidinas/farmacologiaRESUMO
Insulin stimulates glucose transport by triggering regulated delivery of intracellular vesicles containing the GLUT4 glucose transporter to the plasma membrane. This process is defective in diseases such as type 2 diabetes (T2DM). While studies in rodent cells have been invaluable in understanding GLUT4 traffic, evolutionary plasticity must be considered when extrapolating these findings to humans. Recent work has identified species-specific distinctions in GLUT4 traffic, notably the participation of a novel clathrin isoform, CHC22, in humans but not rodents. Here, we discuss GLUT4 sorting in different species and how studies of CHC22 have identified new routes for GLUT4 trafficking. We further consider how different sorting-protein complexes relate to these routes and discuss other implications of these pathways in cell biology and disease.
Assuntos
Cadeias Pesadas de Clatrina/metabolismo , Vesículas Citoplasmáticas/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Animais , Humanos , Modelos Biológicos , UbiquitinaçãoRESUMO
Clathrin function directly derives from its coat structure, and while endocytosis is mediated by clathrin-coated pits, large plaques contribute to cell adhesion. Here, we show that the alternative splicing of a single exon of the clathrin heavy chain gene (CLTC exon 31) helps determine the clathrin coat organization. Direct genetic control was demonstrated by forced CLTC exon 31 skipping in muscle cells that reverses the plasma membrane content from clathrin plaques to pits and by promoting exon inclusion that stimulated flat plaque assembly. Interestingly, mis-splicing of CLTC exon 31 found in the severe congenital form of myotonic dystrophy was associated with reduced plaques in patient myotubes. Moreover, forced exclusion of this exon in WT mice muscle induced structural disorganization and reduced force, highlighting the contribution of this splicing event for the maintenance of tissue homeostasis. This genetic control on clathrin assembly should influence the way we consider how plasticity in clathrin-coated structures is involved in muscle development and maintenance.
Assuntos
Processamento Alternativo/fisiologia , Cadeias Pesadas de Clatrina/metabolismo , Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Adulto , Animais , Membrana Celular/metabolismo , Criança , Endocitose/fisiologia , Éxons/fisiologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Adulto JovemRESUMO
Tumor-associated macrophages affect tumor progression and resistance to immune checkpoint therapy. Here, we identify the chemokine signal regulator FROUNT as a target to control tumor-associated macrophages. The low level FROUNT expression in patients with cancer correlates with better clinical outcomes. Frount-deficiency markedly reduces tumor progression and decreases macrophage tumor-promoting activity. FROUNT is highly expressed in macrophages, and its myeloid-specific deletion impairs tumor growth. Further, the anti-alcoholism drug disulfiram (DSF) acts as a potent inhibitor of FROUNT. DSF interferes with FROUNT-chemokine receptor interactions via direct binding to a specific site of the chemokine receptor-binding domain of FROUNT, leading to inhibition of macrophage responses. DSF monotherapy reduces tumor progression and decreases macrophage tumor-promoting activity, as seen in the case of Frount-deficiency. Moreover, co-treatment with DSF and an immune checkpoint antibody synergistically inhibits tumor growth. Thus, inhibition of FROUNT by DSF represents a promising strategy for macrophage-targeted cancer therapy.
Assuntos
Cadeias Pesadas de Clatrina/metabolismo , Dissulfiram/farmacologia , Neoplasias Pulmonares/patologia , Macrófagos/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Quimiocinas/metabolismo , Progressão da Doença , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Imunoterapia , Cinética , Neoplasias Pulmonares/genética , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Metástase Neoplásica , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Prognóstico , Fatores de RiscoRESUMO
Glucose transporter 4 (GLUT4) is sequestered inside muscle and fat and then released by vesicle traffic to the cell surface in response to postprandial insulin for blood glucose clearance. Here, we map the biogenesis of this GLUT4 traffic pathway in humans, which involves clathrin isoform CHC22. We observe that GLUT4 transits through the early secretory pathway more slowly than the constitutively secreted GLUT1 transporter and localize CHC22 to the ER-to-Golgi intermediate compartment (ERGIC). CHC22 functions in transport from the ERGIC, as demonstrated by an essential role in forming the replication vacuole of Legionella pneumophila bacteria, which requires ERGIC-derived membrane. CHC22 complexes with ERGIC tether p115, GLUT4, and sortilin, and downregulation of either p115 or CHC22, but not GM130 or sortilin, abrogates insulin-responsive GLUT4 release. This indicates that CHC22 traffic initiates human GLUT4 sequestration from the ERGIC and defines a role for CHC22 in addition to retrograde sorting of GLUT4 after endocytic recapture, enhancing pathways for GLUT4 sequestration in humans relative to mice, which lack CHC22.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Vias Biossintéticas , Cadeias Pesadas de Clatrina/metabolismo , Clatrina/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Transporte Proteico , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismoRESUMO
BACKGROUND & AIMS: Upon ligand binding, tyrosine kinase receptors, such as epidermal growth factor receptor (EGFR), are recruited into clathrin-coated pits for internalization by endocytosis, which is relevant for signalling and/or receptor degradation. In liver cells, transforming growth factor-ß (TGF-ß) induces both pro- and anti-apoptotic signals; the latter are mediated by the EGFR pathway. Since EGFR mainly traffics via clathrin-coated vesicles, we aimed to analyse the potential role of clathrin in TGF-ß-induced signalling in liver cells and its relevance in liver cancer. METHODS: Real-Time PCR and immunohistochemistry were used to analyse clathrin heavy-chain expression in human (CLTC) and mice (Cltc) liver tumours. Transient knockdown (siRNA) or overexpression of CLTC were used to analyse its role on TGF-ß and EGFR signalling in vitro. Bioinformatic analysis was used to determine the effect of CLTC and TGFB1 expression on prognosis and overall survival in patients with hepatocellular carcinoma (HCC). RESULTS: Clathrin expression increased during liver tumorigenesis in humans and mice. CLTC knockdown cells responded to TGF-ß phosphorylating SMADs (canonical signalling) but showed impairment in the anti-apoptotic signals (EGFR transactivation). Experiments of loss or gain of function in HCC cells reveal an essential role for clathrin in inhibiting TGF-ß-induced apoptosis and upregulation of its pro-apoptotic target NOX4. Autocrine TGF-ß signalling in invasive HCC cells upregulates CLTC expression, switching its role to pro-tumorigenic. A positive correlation between TGFB1 and CLTC was found in HCC cells and patients. Patients expressing high levels of TGFB1 and CLTC had a worse prognosis and lower overall survival. CONCLUSIONS: This work describes a novel role for clathrin in liver tumorigenesis, favouring non-canonical pro-tumorigenic TGF-ß pathways. CLTC expression in human HCC samples could help select patients that would benefit from TGF-ß-targeted therapy. LAY SUMMARY: Clathrin heavy-chain expression increases during liver tumorigenesis in humans (CLTC) and mice (Cltc), altering the cellular response to TGF-ß in favour of anti-apoptotic/pro-tumorigenic signals. A positive correlation between TGFB1 and CLTC was found in HCC cells and patients. Patients expressing high levels of TGFB1 and CLTC had a worse prognosis and lower overall survival. CLTC expression in HCC human samples could help select patients that would benefit from therapies targeting TGF-ß.
Assuntos
Carcinogênese/genética , Cadeias Pesadas de Clatrina/genética , Cadeias Pesadas de Clatrina/metabolismo , Neoplasias Hepáticas/metabolismo , Transdução de Sinais/genética , Fator de Crescimento Transformador beta1/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Hepatócitos/metabolismo , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Prognóstico , RNA Interferente Pequeno , TransfecçãoRESUMO
Calcium (Ca2+) signaling within the cell nucleus regulates specific cellular events such as gene transcription and cell proliferation. Nuclear and cytosolic Ca2+ levels can be independently regulated, and nuclear translocation of receptor tyrosine kinases (RTKs) is one way to locally activate signaling cascades within the nucleus. Nuclear RTKs, including the epidermal growth factor receptor (EGFR), are important for processes such as transcriptional regulation, DNA-damage repair, and cancer therapy resistance. RTKs can hydrolyze phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) within the nucleus, leading to Ca2+ release from the nucleoplasmic reticulum by inositol 1,4,5-trisphosphate receptors. PI(4,5)P2 hydrolysis is mediated by phospholipase C (PLC). However, it is unknown which nuclear PLC isoform is triggered by EGFR. Here, using subcellular fractionation, immunoblotting and fluorescence, siRNA-based gene knockdowns, and FRET-based biosensor reporter assays, we investigated the role of PLCδ4 in epidermal growth factor (EGF)-induced nuclear Ca2+ signaling and downstream events. We found that EGF-induced Ca2+ signals are inhibited when translocation of EGFR is impaired. Nuclear Ca2+ signals also were reduced by selectively buffering inositol 1,4,5-trisphosphate (InsP3) within the nucleus. EGF induced hydrolysis of nuclear PI(4,5)P2 by the intranuclear PLCδ4, rather than by PLCγ1. Moreover, protein kinase C, a downstream target of EGF, was active in the nucleus of stimulated cells. Furthermore, PLCδ4 and InsP3 modulated cell cycle progression by regulating the expression of cyclins A and B1. These results provide evidence that EGF-induced nuclear signaling is mediated by nuclear PLCδ4 and suggest new therapeutic targets to modulate the proliferative effects of this growth factor.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Núcleo Celular/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Fosfolipase C delta/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cadeias Pesadas de Clatrina/antagonistas & inibidores , Cadeias Pesadas de Clatrina/genética , Cadeias Pesadas de Clatrina/metabolismo , Ciclina A/metabolismo , Ciclina B1/metabolismo , Receptores ErbB/metabolismo , Humanos , Hidrólise , Inositol 1,4,5-Trifosfato/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipase C delta/antagonistas & inibidores , Fosfolipase C delta/genética , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Proteína Quinase C/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
Clathrin regulates mitotic progression, in addition to membrane trafficking. However, the detailed regulatory mechanisms of clathrin during mitosis remain elusive. Here, we demonstrate novel regulation of clathrin during mitotic phase of the cell cycle. Clathrin heavy chain (CHC) was phosphorylated at T606 by its association partner cyclin G-associated kinase (GAK). This phosphorylation was required for proper cell proliferation and tumor growth of cells implanted into nude mice. Immunofluorescence analysis showed that the localization of CHC-pT606 signals changed during mitosis. CHC-pT606 signals localized in the nucleus and at the centrosome during interphase, whereas CHC signals were mostly cytoplasmic. Co-immunoprecipitation suggested that CHC formed a complex with GAK and polo-like kinase 1 (PLK1). Depletion of GAK using siRNA induced metaphase arrest and aberrant localization of CHC-pT606, which abolished Kiz-pT379 (as a phosphorylation target of PLK1) signals on chromatin at metaphase. Taken together, we propose that the GAK_CHC-pT606_PLK1_Kiz-pT379 axis plays a role in proliferation of cancer cells.
Assuntos
Cadeias Pesadas de Clatrina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metáfase/genética , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Centrossomo/metabolismo , Cadeias Pesadas de Clatrina/genética , Feminino , Técnicas de Silenciamento de Genes , Células HeLa , Xenoenxertos , Humanos , Interfase/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/metabolismo , Fuso Acromático/metabolismo , Transfecção , Carga Tumoral/genética , Quinase 1 Polo-LikeRESUMO
Mammalian oocytes segregate chromosomes with a microtubule spindle that lacks centrosomes, but the mechanisms by which acentrosomal spindles are organized and function are largely unclear. In this study, we identify a conserved subcellular structure in mammalian oocytes that forms by phase separation. This structure, which we term the liquid-like meiotic spindle domain (LISD), permeates the spindle poles and forms dynamic protrusions that extend well beyond the spindle. The LISD selectively concentrates multiple microtubule regulatory factors and allows them to diffuse rapidly within the spindle volume. Disruption of the LISD via different means disperses these factors and leads to severe spindle assembly defects. Our data suggest a model whereby the LISD promotes meiotic spindle assembly by serving as a reservoir that sequesters and mobilizes microtubule regulatory factors in proximity to spindle microtubules.