RESUMO
OBJECTIVE: For treatment of medication-related osteonecrosis of the jaw, one proposed approach is the use of a topical agent to block entry of these medications in oral soft tissues. We tested the ability of phosphonoformic acid (PFA), an inhibitor of bisphosphonate entry through certain sodium-dependent phosphate contransporters (SLC20A1, 20A2, 34A1-3) as well as Dynasore, a macropinocytosis inhibitor, for their abilities to prevent zoledronate-induced (ZOL) death in human gingival fibroblasts (HGFs). METHODOLOGY: MTT assay dose-response curves were performed to determine non-cytotoxic levels of both PFA and Dynasore. In the presence of 50 µM ZOL, optimized PFA and Dynasore doses were tested for their ability to restore HGF viability. To determine SLC expression in HGFs, total HGF RNA was subjected to quantitative real-time RT-PCR. Confocal fluorescence microscopy was employed to see if Dynasore inhibited macropinocytotic HGF entry of AF647-ZOL. Endosomal acidification in the presence of Dynasore was measured by live cell imaging utilizing LysoSensor Green DND-189. As a further test of Dynasore's ability to interfere with ZOL-containing endosomal maturation, perinuclear localization of mature endosomes containing AF647-ZOL or TRITC-dextran as a control were assessed via confocal fluorescence microscopy with CellProfiler™ software analysis of the resulting photomicrographs. RESULTS: 0.5 mM PFA did not rescue HGFs from ZOL-induced viability loss at 72 hours while 10 and 30 µM geranylgeraniol did partially rescue. HGFs did not express the SLC transporters as compared to the expression in positive control tissues. 10 µM Dynasore completely prevented ZOL-induced viability loss. In the presence of Dynasore, AF647-ZOL and FITC-dextran co-localized in endosomes. Endosomal acidification was inhibited by Dynasore and perinuclear localization of both TRITC-dextran- and AF647-ZOL-containing endosomes was inhibited by 30 µM Dynasore. CONCLUSION: Dynasore prevents ZOL-induced viability loss in HGFs by partially interfering with macropinocytosis and by inhibiting the endosomal maturation pathway thought to be needed for ZOL delivery to the cytoplasm.
Assuntos
Sobrevivência Celular , Difosfonatos , Endossomos , Fibroblastos , Gengiva , Hidrazonas , Imidazóis , Ácido Zoledrônico , Ácido Zoledrônico/farmacologia , Humanos , Fibroblastos/efeitos dos fármacos , Gengiva/efeitos dos fármacos , Gengiva/citologia , Difosfonatos/farmacologia , Imidazóis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Hidrazonas/farmacologia , Células Cultivadas , Fatores de Tempo , Reação em Cadeia da Polimerase em Tempo Real , Conservadores da Densidade Óssea/farmacologia , Reprodutibilidade dos Testes , Microscopia Confocal , Relação Dose-Resposta a Droga , Pinocitose/efeitos dos fármacosRESUMO
Introduction: Cytomegaloviruses (CMVs) extensively reorganize the membrane system of the cell and establish a new structure as large as the cell nucleus called the assembly compartment (AC). Our previous studies on murine CMV (MCMV)-infected fibroblasts indicated that the inner part of the AC contains rearranged early endosomes, recycling endosomes, endosomal recycling compartments and trans-Golgi membrane structures that are extensively tubulated, including the expansion and retention of tubular Rab10 elements. An essential process that initiates Rab10-associated tubulation is cargo sorting and retrieval mediated by SNX27, Retromer, and ESCPE-1 (endosomal SNX-BAR sorting complex for promoting exit 1) complexes. Objective: The aim of this study was to investigate the role of SNX27:Retromer:ESCPE-1 complexes in the biogenesis of pre-AC in MCMV-infected cells and subsequently their role in secondary envelopment and release of infectious virions. Results: Here we show that SNX27:Retromer:ESCPE1-mediated tubulation is essential for the establishment of a Rab10-decorated subset of membranes within the pre-AC, a function that requires an intact F3 subdomain of the SNX27 FERM domain. Suppression of SNX27-mediated functions resulted in an almost tenfold decrease in the release of infectious virions. However, these effects cannot be directly linked to the contribution of SNX27:Retromer:ESCPE-1-dependent tubulation to the secondary envelopment, as suppression of these components, including the F3-FERM domain, led to a decrease in MCMV protein expression and inhibited the progression of the replication cycle. Conclusion: This study demonstrates a novel and important function of membrane tubulation within the pre-AC associated with the control of viral protein expression.
Assuntos
Endossomos , Nexinas de Classificação , Replicação Viral , Endossomos/metabolismo , Endossomos/virologia , Animais , Camundongos , Humanos , Nexinas de Classificação/metabolismo , Nexinas de Classificação/genética , Fibroblastos/virologia , Fibroblastos/metabolismo , Muromegalovirus/fisiologia , Muromegalovirus/genética , Linhagem Celular , Montagem de Vírus , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genética , Citomegalovirus/fisiologia , Citomegalovirus/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genéticaRESUMO
The majority of viruses classified as pandemic threats are enveloped viruses which enter the cell through receptor-mediated endocytosis and take advantage of endosomal acidification to activate their fusion machinery. Here we report that the endosomal fusion of low pH-requiring viruses is highly dependent on TRPM7, a widely expressed TRP channel that is located on the plasma membrane and in intracellular vesicles. Using several viral infection systems expressing the envelope glycoproteins of various viruses, we find that loss of TRPM7 protects cells from infection by Lassa, LCMV, Ebola, Influenza, MERS, SARS-CoV-1, and SARS-CoV-2. TRPM7 ion channel activity is intrinsically necessary to acidify virus-laden endosomes but is expendable for several other endosomal acidification pathways. We propose a model wherein TRPM7 ion channel activity provides a countercurrent of cations from endosomal lumen to cytosol necessary to sustain the pumping of protons into these virus-laden endosomes. This study demonstrates the possibility of developing a broad-spectrum, TRPM7-targeting antiviral drug to subvert the endosomal fusion of low pH-dependent enveloped viruses.
Assuntos
Endossomos , Canais de Cátion TRPM , Internalização do Vírus , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPM/genética , Endossomos/metabolismo , Endossomos/virologia , Concentração de Íons de Hidrogênio , Humanos , Animais , Células HEK293 , SARS-CoV-2/fisiologia , SARS-CoV-2/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Ebolavirus/fisiologia , Ebolavirus/metabolismo , Vírus da Coriomeningite Linfocítica/fisiologia , Chlorocebus aethiops , Envelope Viral/metabolismo , Vírus Lassa/metabolismo , Vírus Lassa/fisiologiaRESUMO
The insulin-like growth factor II/mannose 6-phosphate (IGF-II/M6P) receptor is a multifunctional glycoprotein not only play roles in IGF-II degradation and pro-TGFß activation but binding to and transport M6P-bearing lysosomal enzymes from the trans-Golgi network (TGN) or the cell surface to lysosomes. At present, information regarding a retrograde transport of IGF-II/M6P receptor from endosomes to the TGN is still limited. We show here that a continuous ligand-dependent activation of sphingosine 1-phosphate receptor type 3 (S1P3R) on the endosomal membranes is required for subsequent recycling back of cargo-unloaded IGF-II/M6P receptors to the TGN. We have further clarified that Gq coupled with S1P3R plays a critical role in the activation of casein kinase 2, which phosphorylates and keeps PACS1 connector protein active for the association with IGF-II/M6P receptors, which enables transport carrier formation with the aid of other adaptor proteins toward the TGN. These findings shed light on the molecular mechanism underlying how continuous activation of the S1P receptor and subsequent downstream Gq signaling regulates the retrograde transport of the empty IGF-II/M6P receptors back to the TGN.
Assuntos
Endossomos , Lisofosfolipídeos , Transporte Proteico , Receptor IGF Tipo 2 , Transdução de Sinais , Esfingosina , Rede trans-Golgi , Rede trans-Golgi/metabolismo , Endossomos/metabolismo , Humanos , Receptor IGF Tipo 2/metabolismo , Lisofosfolipídeos/metabolismo , Esfingosina/metabolismo , Esfingosina/análogos & derivados , Fator de Crescimento Insulin-Like II/metabolismo , Peptídeos Semelhantes à InsulinaRESUMO
In this issue of Developmental Cell, Jiang et al. report that the Arabidopsis HOPS tethering complex subunit VPS41 acts to catalyze the formation of a degradation pathway composed of a hybrid of autophagosomes and late endosomes.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Autofagossomos , Autofagia , Endossomos , Vacúolos , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Autofagossomos/metabolismo , Autofagia/fisiologia , Endossomos/metabolismo , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMO
Truncating genetic variants of SORL1, encoding the endosome recycling receptor SORLA, have been accepted as causal of Alzheimer's disease (AD). However, most genetic variants observed in SORL1 are missense variants, for which it is complicated to determine the pathogenicity level because carriers come from pedigrees too small to be informative for penetrance estimations. Here, we describe three unrelated families in which the SORL1 coding missense variant rs772677709, that leads to a p.Y1816C substitution, segregates with Alzheimer's disease. Further, we investigate the effect of SORLA p.Y1816C on receptor maturation, cellular localization, and trafficking in cell-based assays. Under physiological circumstances, SORLA dimerizes within the endosome, allowing retromer-dependent trafficking from the endosome to the cell surface, where the luminal part is shed into the extracellular space (sSORLA). Our results showed that the p.Y1816C mutant impairs SORLA homodimerization in the endosome, leading to decreased trafficking to the cell surface and less sSORLA shedding. These trafficking defects of the mutant receptor can be rescued by the expression of the SORLA 3Fn-minireceptor. Finally, we find that iPSC-derived neurons with the engineered p.Y1816C mutation have enlarged endosomes, a defining cytopathology of AD. Our studies provide genetic as well as functional evidence that the SORL1 p.Y1816C variant is causal for AD. The partial penetrance of the mutation suggests this mutation should be considered in clinical genetic screening of multiplex early-onset AD families.
Assuntos
Doença de Alzheimer , Endossomos , Proteínas Relacionadas a Receptor de LDL , Proteínas de Membrana Transportadoras , Linhagem , Humanos , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Endossomos/metabolismo , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas Relacionadas a Receptor de LDL/metabolismo , Feminino , Masculino , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mutação de Sentido Incorreto , Transporte Proteico , Multimerização Proteica , Idoso , Pessoa de Meia-Idade , Células HEK293RESUMO
In the realm of intracellular drug delivery, overcoming the barrier of endosomal entrapment stands as a critical factor influencing the effectiveness of nanodrug delivery systems. This study focuses on the synthesis of an acid-sensitive fatty acid derivative called imidazole-stearic acid (IM-SA). Leveraging the proton sponge effect attributed to imidazole groups, IM-SA was anticipated to play a pivotal role in facilitating endosomal escape. Integrated into the lipid core of solid lipid nanoparticles (SLNs), IM-SA was paired with hyaluronic acid (HA) coating on the surface of SLNs loading with curcumin (CUR). The presence of IM-SA and HA endowed HA-IM-SLNs@CUR with dual functionalities, enabling the promotion of endosomal escape, and specifical targeting of liver cancer. HA-IM-SLNs@CUR exhibited a particle size of â¼228â¯nm, with impressive encapsulation efficiencies (EE) of 87.5â¯% ± 2.3â¯% for CUR. Drugs exhibit significant pH sensitive release behavior. Cellular experiments showed that HA-IM-SLN@CUR exhibits enhanced drug delivery capability. The incorporation of IM-SA significantly improved the endosomal escape of HA-IM-SLN@CUR, facilitating accelerated intracellular drug release and increasing intracellular drug concentration, exhibiting excellent growth inhibitory effects on HepG2 cells. Animal experiments revealed a 3.4-fold increase in CUR uptake at the tumor site with HA-IM-SLNs@CUR over the free CUR, demonstrating remarkable tumor homing potential with the tumor growth inhibition rate of 97.2â¯%. These findings indicated the significant promise of HA-IM-SLNs@CUR in the realm of cancer drug delivery.
Assuntos
Antineoplásicos , Curcumina , Endossomos , Nanopartículas , Tamanho da Partícula , Curcumina/farmacologia , Curcumina/química , Humanos , Nanopartículas/química , Animais , Endossomos/metabolismo , Endossomos/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/administração & dosagem , Células Hep G2 , Liberação Controlada de Fármacos , Camundongos , Lipídeos/química , Sistemas de Liberação de Medicamentos , Proliferação de Células/efeitos dos fármacos , Fígado/metabolismo , Fígado/efeitos dos fármacos , Propriedades de Superfície , Portadores de Fármacos/química , Ácidos Esteáricos/química , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Imidazóis/química , Imidazóis/farmacologia , Camundongos Nus , Ácido Hialurônico/química , Camundongos Endogâmicos BALB C , LipossomosRESUMO
Secondary envelopment of the human cytomegalovirus (HCMV) is a critical but not well-understood process that takes place at the cytoplasmic viral assembly complex (cVAC) where nucleocapsids acquire their envelope by budding into cellular membranes containing viral glycoproteins. Previous studies presented controversial results regarding the composition of the viral envelope, suggesting trans-Golgi and endosomal origins, as well as intersections with the exosomal and endocytic pathways. Here, we investigated the role of endocytic membranes for the secondary envelopment of HCMV by using wheat germ agglutinin (WGA) pulse labeling to label glycoproteins at the plasma membrane and to follow their trafficking during HCMV infection by light microscopy and transmission electron microscopy (TEM). WGA labeled different membrane compartments within the cVAC, including early endosomes, multivesicular bodies, trans-Golgi, and recycling endosomes. Furthermore, TEM analysis showed that almost 90% of capsids undergoing secondary envelopment and 50% of enveloped capsids were WGA-positive within 90 min. Our data reveal extensive remodeling of the endocytic compartment in the late stage of HCMV infection, where the endocytic compartment provides an optimized environment for virion morphogenesis and serves as the primary membrane source for secondary envelopment. Furthermore, we show that secondary envelopment is a rapid process in which endocytosed membranes are transported from the plasma membrane to the cVAC within minutes to be utilized by capsids for envelopment.
Assuntos
Membrana Celular , Citomegalovirus , Endocitose , Montagem de Vírus , Humanos , Citomegalovirus/fisiologia , Citomegalovirus/metabolismo , Membrana Celular/metabolismo , Membrana Celular/virologia , Envelope Viral/metabolismo , Endossomos/virologia , Endossomos/metabolismo , Aglutininas do Germe de Trigo/metabolismo , Infecções por Citomegalovirus/virologia , Infecções por Citomegalovirus/metabolismoRESUMO
Essentially all plasma membrane proteins are glycosylated, and their activity is regulated by tuning their cell surface dynamics. This is achieved by glycan-binding proteins of the galectin family that either retain glycoproteins within lattices or drive their endocytic uptake via the clathrin-independent glycolipid-lectin (GL-Lect) mechanism. Here, we have used immunofluorescence-based assays to analyze how lattice and GL-Lect mechanisms affect the internalization of the cell adhesion and migration glycoprotein α5ß1 integrin. In retinal pigment epithelial (RPE-1) cells, internalized α5ß1 integrin is found in small peripheral endosomes under unperturbed conditions. Pharmacological compounds were used to competitively inhibit one of the galectin family members, galectin-3 (Gal3), or to inhibit the expression of glycosphingolipids, both of which are the fabric of the GL-Lect mechanism. We found that under acute inhibition conditions, endocytic uptake of α5ß1 integrin was strongly reduced, in agreement with previous studies on the GL-Lect driven internalization of the protein. In contrast, upon prolonged inhibitor treatment, the uptake of α5ß1 integrin was increased, and the protein was now internalized by alternative pathways into large perinuclear endosomes. Our findings suggest that under these prolonged inhibitor treatment conditions, α5ß1 integrin containing galectin lattices are dissociated, leading to an altered endocytic compartmentalization.
Assuntos
Endocitose , Galectina 3 , Integrina alfa5beta1 , Humanos , Galectina 3/metabolismo , Integrina alfa5beta1/metabolismo , Linhagem Celular , Endossomos/metabolismo , Adesão Celular , Galectinas/metabolismo , Proteínas SanguíneasRESUMO
Glaesserella parasuis cytolethal distending toxin (GpCDT) can induce cell cycle arrest and apoptosis. Our laboratory's previous work demonstrated that GTPase 4b (Rab4b) is a key host protein implicated in GpCDT-induced cytotoxicity. This study investigated the probable involvement of Rab4b in the process. Our study used CRISPR/Cas9 technology to create a Rab4b-knockout cell line. The results showed greater resistance to GpCDT-induced cell cytotoxicity. In contrast, forced Rab4b overexpression increased GpCDT-induced cytotoxicity. Further immunoprecipitation study reveals that GpCDT may bind with Rab4b. In PK-15 cells, GpCDT is transported to the early endosomes and late endosomes, while after knocking out Rab4b, GpCDT cannot be transported to the early endosome via vesicles. Rab4b appears essential for GpCDT-induced cytotoxicity in PK-15 cells.
Assuntos
Toxinas Bacterianas , Proteínas rab4 de Ligação ao GTP , Animais , Linhagem Celular , Toxinas Bacterianas/toxicidade , Proteínas rab4 de Ligação ao GTP/metabolismo , Proteínas rab4 de Ligação ao GTP/genética , Suínos , Endossomos/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Apoptose/efeitos dos fármacosRESUMO
BACKGROUND: The involvement of tetraspanins in cancer development has been widely implicated. In this study, the function and molecular mechanisms of tetraspanin 3 (TSPAN3) in non-small cell lung cancer (NSCLC) cells were explored. METHODS: Tissue samples from patients diagnosed with NSCLC were analyzed by immunohistochemistry, western blotting, and real-time polymerase chain reaction (PCR) to indicate the involvement of TSPAN3 in cancer progression. In the meantime, we also performed exhaustive mechanistic studies using A549 and H460 cells in vitro through a variety of methods including western blotting, real-time PCR, immunofluorescent staining, coimmunoprecipitation, cell proliferation assay, and nocodazole (NZ) washout assay. Proper statistical analysis was implemented wherever necessary in this study. RESULTS: TSPAN3 was found to be highly expressed in lung cancer cells and tissues. Moreover, high levels of TSPAN3 positively correlated with poor differentiation, lymph node involvement, advanced pathological tumor-node-metastasis stage, and poor prognosis in patients with NSCLC. TSPAN3 showed potential to promote the proliferation of NSCLC cells in vitro and in vivo. Specifically, TSPAN3 was found to interact with ß1 integrin via the LEL domain, thereby facilitating the sorting of ß1 integrin into Rab11a endosomes and promoting ß1 integrin recycling and upregulation. CONCLUSIONS: Our findings reveal TSPAN3 may represent a potentially valuable therapeutic target for NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Proliferação de Células , Integrina beta1 , Neoplasias Pulmonares , Tetraspaninas , Humanos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/genética , Proliferação de Células/genética , Tetraspaninas/metabolismo , Tetraspaninas/genética , Integrina beta1/metabolismo , Integrina beta1/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Feminino , Masculino , Linhagem Celular Tumoral , Pessoa de Meia-Idade , Animais , Células A549 , Camundongos Nus , Endossomos/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genética , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Signaling pathways activated by secreted Wnt ligands play an essential role in tissue development and the progression of diseases, like cancer. Secretion of the lipid-modified Wnt proteins is tightly regulated by a repertoire of intracellular factors. For instance, a membrane protein, Evi, interacts with the Wnt ligand in the ER, and it is essential for its further trafficking and release in the extracellular space. After dissociating from the Wnt, the Wnt-unbound Evi is recycled back to the ER via Golgi. However, where in this trafficking path Wnt proteins dissociate from Evi remains unclear. Here, we have used the Drosophila wing epithelium to trace the route of the Evi-Wg (Wnt homolog) complex leading up to their separation. In these polarized cells, Wg is first trafficked to the apical surface; however, the secretion of Wg is believed to occurs post-internalization via recycling. Our results show that the Evi-Wg complex is internalized from the apical surface and transported to the retromer-positive endosomes. Furthermore, using antibodies that specifically label the Wnt-unbound Evi, we show that Evi and Wg separation occurs post-internalization in the acidic endosomes. These results refine our understanding of the polarized trafficking of Wg and highlight the importance of Wg endocytosis in its secondary secretion.
Assuntos
Proteínas de Drosophila , Endossomos , Transporte Proteico , Proteína Wnt1 , Animais , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Endocitose/fisiologia , Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Asas de Animais/metabolismo , Proteína Wnt1/metabolismo , Proteína Wnt1/genéticaRESUMO
Deleterious mutations in the lipopolysaccharide responsive beige-like anchor protein (LRBA) gene cause severe childhood immune dysregulation. The complexity of the symptoms involving multiple organs and the broad range of unpredictable clinical manifestations of LRBA deficiency complicate the choice of therapeutic interventions. Although LRBA has been linked to Rab11-dependent trafficking of the immune checkpoint protein CTLA-4, its precise cellular role remains elusive. We show that LRBA, however, only slightly colocalizes with Rab11. Instead, LRBA is recruited by members of the small GTPase Arf protein family to the TGN and to Rab4+ endosomes, where it controls intracellular traffic. In patient-derived fibroblasts, loss of LRBA led to defects in the endosomal pathway promoting the accumulation of enlarged endolysosomes and lysosome secretion. Thus, LRBA appears to regulate flow through the endosomal system on Rab4+ endosomes. Our data strongly suggest functions of LRBA beyond CTLA-4 trafficking and provide a conceptual framework to develop new therapies for LRBA deficiency.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Endossomos , Homeostase , Lisossomos , Proteínas rab de Ligação ao GTP , Proteínas rab4 de Ligação ao GTP , Humanos , Endossomos/metabolismo , Lisossomos/metabolismo , Proteínas rab4 de Ligação ao GTP/metabolismo , Proteínas rab4 de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Transporte Proteico , Fibroblastos/metabolismo , Fatores de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/genética , Rede trans-Golgi/metabolismo , Células HeLa , Células HEK293 , Fator 1 de Ribosilação do ADPRESUMO
The cAMP cascade is increasingly recognized to transduce physiological effects locally through spatially limited cAMP gradients. However, little is known about how adenylyl cyclase enzymes that initiate cAMP gradients are localized. Here we address this question in physiologically relevant striatal neurons and investigate how AC localization impacts downstream signaling function. We show that the major striatal AC isoforms are differentially sorted between ciliary and extraciliary domains of the plasma membrane, and that one isoform, AC9, is uniquely concentrated in endosomes. We identify key sorting determinants in the N-terminal cytoplasmic domain responsible for isoform-specific localization. We further show that AC9-containing endosomes accumulate activated dopamine receptors and form an elaborately intertwined network with juxtanuclear PKA stores bound to Golgi membranes. Finally, we provide evidence that endosomal localization enables AC9 to selectively elevate PKA activity in the nucleus relative to the cytoplasm. Together, these results reveal a precise spatial landscape of the cAMP cascade in neurons and a key role of AC localization in directing downstream PKA signaling to the nucleus.
Assuntos
Adenilil Ciclases , Proteínas Quinases Dependentes de AMP Cíclico , AMP Cíclico , Dopamina , Endossomos , Neurônios , Transdução de Sinais , Adenilil Ciclases/metabolismo , Animais , Dopamina/metabolismo , Neurônios/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Endossomos/metabolismo , Membrana Celular/metabolismo , Camundongos , Corpo Estriado/metabolismo , Corpo Estriado/citologia , Receptores Dopaminérgicos/metabolismo , Complexo de Golgi/metabolismo , Núcleo Celular/metabolismo , Humanos , Células HEK293RESUMO
BACKGROUND: Intracellular tracking is commonly used in trafficking research. Until today, the respective techniques have remained complex, and complicated, mostly transgenic target protein changes are necessary, often requiring expensive equipment and expert knowledge. METHODS: We present a novel method, which we term "cell-sonar", that enables the user to track expression changes of specific protein markers that serve as points of interaction. Our study provides comparable analyses of expression changes of these marker proteins by in-cell Western analyses in two otherwise isogenic cell lines that only differ in the overexpression of the tracked target protein. Using the overexpressed human adult muscle-type nicotinic acetylcholine receptor as an example, we demonstrate that cell-sonar can cover multiple intracellular compartments such as the endoplasmic reticulum, the pathway between it and the Golgi apparatus, and the endocytic pathway. RESULTS: We provide evidence for receptor maturation in the Golgi and storage in recycling endosomes, rather than the fate of increased insertion into the plasma membrane. Additionally, we demonstrate with the implementation of nicotine that the receptor's destiny is exasperated up to secondary degradation. CONCLUSIONS: Cell-sonar is an affordable, easy-to-implement, and cheap method that can be adapted to a broad variety of proteins and cellular pathways of interest to researchers.
Assuntos
Complexo de Golgi , Via Secretória , Humanos , Complexo de Golgi/metabolismo , Retículo Endoplasmático/metabolismo , Receptores Nicotínicos/metabolismo , Transporte Proteico , Endossomos/metabolismo , Linhagem CelularRESUMO
The neurovascular unit (NVU) is a complex multicellular structure that helps maintain cerebral homeostasis and blood-brain barrier (BBB) integrity. While extensive evidence links NVU alterations to cerebrovascular diseases and neurodegeneration, the underlying molecular mechanisms remain unclear. Here, we use zebrafish embryos carrying a mutation in Scavenger Receptor B2, a highly conserved endolysosomal protein expressed predominantly in Radial Glia Cells (RGCs), to investigate the interplay among different NVU components. Through live imaging and genetic manipulations, we demonstrate that compromised acidification of the endolysosomal compartment in mutant RGCs leads to impaired Notch3 signaling, thereby inducing excessive neurogenesis and reduced glial differentiation. We further demonstrate that alterations to the neuron/glia balance result in impaired VEGF and Wnt signaling, leading to severe vascular defects, hemorrhages, and a leaky BBB. Altogether, our findings provide insights into NVU formation and function and offer avenues for investigating diseases involving white matter defects and vascular abnormalities.
Assuntos
Barreira Hematoencefálica , Lisossomos , Neurogênese , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Lisossomos/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Células Ependimogliais/metabolismo , Células Ependimogliais/patologia , Endossomos/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Receptores Notch/metabolismo , Receptores Notch/genética , Neuroglia/metabolismo , Neuroglia/patologia , Diferenciação Celular , Células-Tronco/metabolismo , Via de Sinalização Wnt , Mutação , Neovascularização Fisiológica , Animais Geneticamente Modificados , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/irrigação sanguínea , Transdução de Sinais , AngiogêneseRESUMO
Baculovirus is an obligate parasitic virus of the phylum Arthropoda. Baculovirus including Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has been widely used in the laboratory and industrial preparation of proteins or protein complexes. Due to its large packaging capacity and non-replicative and non-integrative natures in mammals, baculovirus has been proposed as a gene therapy vector for transgene delivery. However, the mechanism of baculovirus transduction in mammalian cells has not been fully illustrated. Here, we employed a cell surface protein-focused CRISPR screen to identify host dependency factors for baculovirus transduction in mammalian cells. The screening experiment uncovered a series of baculovirus host factors in human cells, including exostosin-like glycosyltransferase 3 (EXTL3) and NPC intracellular cholesterol transporter 1 (NPC1). Further investigation illustrated that EXTL3 affected baculovirus attachment and entry by participating in heparan sulfate biosynthesis. In addition, NPC1 promoted baculovirus transduction by mediating membrane fusion and endosomal escape. Moreover, in vivo, baculovirus transduction in Npc1-/+ mice showed that disruption of Npc1 gene significantly reduced baculovirus transduction in mouse liver. In summary, our study revealed the functions of EXTL3 and NPC1 in baculovirus attachment, entry, and endosomal escape in mammalian cells, which is useful for understanding baculovirus transduction in human cells.
Assuntos
N-Acetilglucosaminiltransferases , Proteína C1 de Niemann-Pick , Nucleopoliedrovírus , Animais , Nucleopoliedrovírus/genética , Nucleopoliedrovírus/fisiologia , Humanos , Camundongos , N-Acetilglucosaminiltransferases/metabolismo , N-Acetilglucosaminiltransferases/genética , Células HEK293 , Endossomos/metabolismo , Heparitina Sulfato/metabolismo , Internalização do Vírus , Transdução Genética , Células Sf9 , Fígado/metabolismo , Fígado/virologia , Sistemas CRISPR-CasRESUMO
The engineering of pH-sensitive therapeutic antibodies, particularly for improving effectiveness and specificity in acidic solid-tumor microenvironments, has recently gained traction. While there is a justified need for pH-dependent immunotherapies, current engineering techniques are tedious and laborious, requiring repeated rounds of experiments under different pH conditions. Inexpensive computational techniques to predict the effectiveness of His pH-switches require antibody-antigen complex structures, but these are lacking in most cases. To circumvent these requirements, we introduce a sequence-based in silico method for predicting His mutations in the variable region of antibodies, which could lead to pH-biased antigen binding. This method, called Sequence-based Identification of pH-sensitive Antibody Binding (SIpHAB), was trained on 3D-structure-based calculations of 3,490 antibody-antigen complexes with solved experimental structures. SIpHAB was parametrized to enhance preferential binding either toward or against the acidic pH, for selective targeting of solid tumors or for antigen release in the endosome, respectively. Applications to nine antibody-antigen systems with previously reported binding preferences at different pHs demonstrated the utility and enrichment capabilities of this high-throughput computational tool. SIpHAB, which only requires knowledge of the antibody primary amino-acid sequence, could enable a more efficient triage of pH-sensitive antibody candidates than could be achieved conventionally. An online webserver for running SipHAB is available freely at https://mm.nrc-cnrc.gc.ca/software/siphab/runner/.
Assuntos
Endossomos , Neoplasias , Engenharia de Proteínas , Concentração de Íons de Hidrogênio , Humanos , Engenharia de Proteínas/métodos , Neoplasias/imunologia , Neoplasias/terapia , Endossomos/metabolismo , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Microambiente Tumoral/imunologia , AnimaisRESUMO
ATG9A is the only integral membrane protein among core autophagy-related (ATG) proteins. We previously found that ATG9A does not co-assemble into synaptophysin-positive vesicles, but rather, localizes to a distinct pool of vesicles within synapsin condensates in both fibroblasts and nerve terminals. The endocytic origin of these vesicles further suggests the existence of different intracellular sorting or segregation mechanisms for ATG9A and synaptophysin in cells. However, the precise underlying mechanism remains largely unknown. In this follow-up study, we investigated the endosomal localization of these two proteins by exploiting the advantages of a Rab5 mutant that induces the formation of enlarged endosomes. Notably, ATG9A and synaptophysin intermix perfectly and do not segregate on giant endosomes, indicating that the separation of these two proteins is not solely caused by the inherent properties of the proteins, but possibly by other unknown factors.
Assuntos
Proteínas Relacionadas à Autofagia , Endossomos , Mutação , Sinaptofisina , Proteínas rab5 de Ligação ao GTP , Endossomos/metabolismo , Mutação/genética , Sinaptofisina/metabolismo , Sinaptofisina/genética , Proteínas rab5 de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Animais , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Humanos , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , CamundongosRESUMO
Cells release intraluminal vesicles in multivesicular bodies as exosomes to communicate with other cells. Although recent studies suggest an intimate link between exosome biogenesis and autophagy, the detailed mechanism is not fully understood. Here we employed comprehensive RNA interference screening for autophagy-related factors and discovered that Rubicon, a negative regulator of autophagy, is essential for exosome release. Rubicon recruits WIPI2d to endosomes to promote exosome biogenesis. Interactome analysis of WIPI2d identified the ESCRT components that are required for intraluminal vesicle formation. Notably, we found that Rubicon is required for an age-dependent increase of exosome release in mice. In addition, small RNA sequencing of serum exosomes revealed that Rubicon determines the fate of exosomal microRNAs associated with cellular senescence and longevity pathways. Taken together, our current results suggest that the Rubicon-WIPI axis functions as a key regulator of exosome biogenesis and is responsible for age-dependent changes in exosome quantity and quality.