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1.
Nat Commun ; 15(1): 5715, 2024 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-38977659

RESUMO

Mitochondria are maternally inherited, but the mechanisms underlying paternal mitochondrial elimination after fertilization are far less clear. Using Drosophila, we show that special egg-derived multivesicular body vesicles promote paternal mitochondrial elimination by activating an LC3-associated phagocytosis-like pathway, a cellular defense pathway commonly employed against invading microbes. Upon fertilization, these egg-derived vesicles form extended vesicular sheaths around the sperm flagellum, promoting degradation of the sperm mitochondrial derivative and plasma membrane. LC3-associated phagocytosis cascade of events, including recruitment of a Rubicon-based class III PI(3)K complex to the flagellum vesicular sheaths, its activation, and consequent recruitment of Atg8/LC3, are all required for paternal mitochondrial elimination. Finally, lysosomes fuse with strings of large vesicles derived from the flagellum vesicular sheaths and contain degrading fragments of the paternal mitochondrial derivative. Given reports showing that in some mammals, the paternal mitochondria are also decorated with Atg8/LC3 and surrounded by multivesicular bodies upon fertilization, our findings suggest that a similar pathway also mediates paternal mitochondrial elimination in other flagellated sperm-producing organisms.


Assuntos
Proteínas de Drosophila , Fertilização , Mitocôndrias , Corpos Multivesiculares , Fagocitose , Espermatozoides , Animais , Mitocôndrias/metabolismo , Masculino , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Feminino , Espermatozoides/metabolismo , Corpos Multivesiculares/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Óvulo/metabolismo , Lisossomos/metabolismo , Cauda do Espermatozoide/metabolismo , Mitofagia
2.
ACS Nano ; 18(24): 15651-15660, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38830824

RESUMO

Lipid bilayers possess the capacity for self-assembly due to the amphipathic nature of lipid molecules, which have both hydrophobic and hydrophilic regions. When confined, lipid bilayers exhibit astonishing versatility in their forms, adopting diverse shapes that are challenging to observe through experimental means. Exploiting this adaptability, lipid structures motivate the development of bio-inspired mechanomaterials and integrated nanobio-interfaces that could seamlessly merge with biological entities, ultimately bridging the gap between synthetic and biological systems. In this work, we demonstrate how, in numerical simulations of multivesicular bodies, a fascinating evolution unfolds from an initial semblance of order toward states of higher entropy over time. We observe dynamic rearrangements in confined vesicles that reveal unexpected limit shapes of distinct geometric patterns. We identify five structures as the basic building blocks that systematically repeat under various conditions of size and composition. Moreover, we observe more complex and less frequent shapes that emerge in confined spaces. Our results provide insights into the dynamics of multivesicular systems, offering a richer understanding of how confined lipid bodies spontaneously self-organize.


Assuntos
Corpos Multivesiculares , Corpos Multivesiculares/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Entropia , Interações Hidrofóbicas e Hidrofílicas
3.
Int J Biol Macromol ; 273(Pt 2): 133112, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38880454

RESUMO

Tumor metastasis is the leading cause of cancer-related death in patients with colorectal cancer (CRC). Heterogeneous nuclear ribonucleoproteins (hnRNPs) are RNA-binding proteins, involved in the tumorigenesis and metastasis of various cancers. However, the molecular mechanisms of hnRNPs in CRC metastasis remain unclear. This study aims to uncover the pivotal roles and molecular mechanisms of hnRNPs in CRC metastasis. Clinical database analysis suggested that the expression of hnRNP-Associated with Lethal Yellow (RALY, an important member of hnRNPs) was strongly correlated with the aggressiveness and survival of CRC patients. Gain- and loss-of-function studies demonstrated that RALY promotes the production of exosomes by increasing the formation of multivesicular bodies (MVBs) and enhancing the fusion of MVBs with the plasma membrane. Notably, RALY directly interacts with phospholipase D2 (PLD2) to enable exosome biogenesis, and cooperates with RBM15b to control PLD2 mRNA stability in an m6A-dependent manner. RALY-mediated exosome secretion activates pro-tumor macrophages and further facilitates CRC metastasis, while rescue experiments in vivo further confirmed that RALY-mediated exosome biogenesis facilitates CRC metastasis. Collectively, our findings demonstrate that RALY promotes exosome biogenesis and facilitates colorectal cancer metastasis by upregulating PLD2 and enhancing exosome production in an m6A-dependent manner, suggesting potential therapeutic strategies for combating CRC metastasis.


Assuntos
Neoplasias Colorretais , Exossomos , Metástase Neoplásica , Proteínas de Ligação a RNA , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/genética , Exossomos/metabolismo , Humanos , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Animais , Camundongos , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Corpos Multivesiculares/metabolismo , Fosfolipase D/metabolismo , Fosfolipase D/genética
4.
Biol Open ; 13(5)2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38682696

RESUMO

Arf GTPase-activating proteins (ArfGAPs) mediate the hydrolysis of GTP bound to ADP-ribosylation factors. ArfGAPs are critical for cargo sorting in the Golgi-to-ER traffic. However, the role of ArfGAPs in sorting into intralumenal vesicles (ILVs) in multivesicular bodies (MVBs) in post-Golgi traffic remains unclear. Exosomes are extracellular vesicles (EVs) of endosomal origin. CD63 is an EV marker. CD63 is enriched ILVs in MVBs of cells. However, the secretion of CD63 positive EVs has not been consistent with the data on CD63 localization in MVBs, and how CD63-containing EVs are formed is yet to be understood. To elucidate the mechanism of CD63 transport to ILVs, we focused on CD63 localization in MVBs and searched for the ArfGAPs involved in CD63 localization. We observed that ADAP1 and ARAP1 depletion inhibited CD63 localization to enlarged endosomes after Rab5Q79L overexpression. We tested epidermal growth factor (EGF) and CD9 localization in MVBs. We observed that ADAP1 and ARAP1 depletion inhibited CD9 localization in enlarged endosomes but not EGF. Our results indicate ADAP1 and ARAP1, regulate incorporation of CD63 and CD9, but not EGF, in overlapped and different MVBs. Our work will contribute to distinguish heterogenous ILVs and exosomes by ArfGAPs.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Ativadoras de GTPase , Corpos Multivesiculares , Tetraspanina 30 , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Fatores de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/genética , Endossomos/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Células HeLa , Corpos Multivesiculares/metabolismo , Transporte Proteico , Tetraspanina 30/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo
5.
J Cell Biol ; 223(8)2024 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-38668767

RESUMO

The microtubule cytoskeleton consists of microtubule subsets with distinct compositions of microtubule-associated proteins, which instruct the position and traffic of subcellular organelles. In the endocytic pathway, these microtubule-associated cues are poorly understood. Here, we report that in MDCK cells, endosomes with multivesicular body (MVB) and late endosome (LE) markers localize preferentially to microtubules coated with septin GTPases. Compared with early endosomes, CD63-containing MVBs/LEs are largely immotile on septin-coated microtubules. In vitro reconstitution assays revealed that the motility of isolated GFP-CD63 endosomes is directly inhibited by microtubule-associated septins. Quantification of CD63-positive endosomes containing the early endosome antigen (EEA1), the Rab7 effector and dynein adaptor RILP or Rab27a, showed that intermediary EEA1- and RILP-positive GFP-CD63 preferentially associate with septin-coated microtubules. Septin knockdown enhanced GFP-CD63 motility and decreased the percentage of CD63-positive MVBs/LEs with lysobiphosphatidic acid without impacting the fraction of EEA1-positive CD63. These results suggest that MVB maturation involves immobilization on septin-coated microtubules, which may facilitate multivesiculation and/or organelle-organelle contacts.


Assuntos
Microtúbulos , Corpos Multivesiculares , Septinas , Animais , Cães , Células Madin Darby de Rim Canino , Microtúbulos/química , Microtúbulos/metabolismo , Corpos Multivesiculares/química , Corpos Multivesiculares/metabolismo , Septinas/química , Septinas/metabolismo , Tetraspanina 30/metabolismo , Citoesqueleto/química , Citoesqueleto/metabolismo , Endocitose
6.
J Mol Cell Cardiol ; 190: 35-47, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38593639

RESUMO

BACKGROUND: Exosomes released by cardiomyocytes are essential mediators of intercellular communications within the heart, and various exosomal proteins and miRNAs are associated with cardiovascular diseases. However, whether the endosomal sorting complex required for transport (ESCRT) and its key component Alix is required for exosome biogenesis within cardiomyocyte remains poorly understood. METHODS: Super-resolution imaging was performed to investigate the subcellular location of Alix and multivesicular body (MVB) in primary cardiomyocytes. Cardiomyocyte-specific Alix-knockout mice were generated using AAV9/CRISPR/Cas9-mediated in vivo gene editing. A stable Alix-knockdown H9c2 cardiomyocyte line was constructed through lentiviral-mediated delivery of short hairpin RNA. In order to determine the role of Alix in controlling exosome biogenesis, exosomes from cardiomyocyte-specific Alix-knockout mice plasma and Alix-knockdown H9c2 culture medium were isolated and examined by western blot, NTA analysis and transmission electron microscopy. Biochemical and immunofluorescence analysis were performed to determine the role of ESCRT machinery in regulating MVB formation. Lastly, transverse aortic constriction (TAC)-induced cardiac pressure overload model was established to further explore the role of Alix-mediated exosome biogenesis under stress conditions. RESULTS: A significant proportion of Alix localized to the MVB membrane within cardiomyocytes. Genetic deletion of Alix in murine heart resulted in a reduction of plasma exosome content without affecting cardiac structure or contractile function. Consistently, the downregulation of Alix in H9c2 cardiomyocyte line also suppressed the biogenesis of exosomes. We found the defective ESCRT machinery and suppressed MVB formation upon Alix depletion caused compromised exosome biogenesis. Remarkably, TAC-induced cardiac pressure overload led to increased Alix, MVB levels, and elevated plasma exosome content, which could be totally abolished by Alix deletion. CONCLUSION: These results establish Alix as an essential and stress-sensitive regulator of cardiac exosome biogenesis and the findings may yield valuable therapeutic implications.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte , Exossomos , Camundongos Knockout , Miócitos Cardíacos , Estresse Fisiológico , Miócitos Cardíacos/metabolismo , Animais , Exossomos/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Camundongos , Corpos Multivesiculares/metabolismo , Linhagem Celular , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Ratos
7.
Proc Natl Acad Sci U S A ; 121(17): e2317680121, 2024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38635626

RESUMO

The endosomal sorting complex required for transport (ESCRT) machinery constitutes multisubunit protein complexes that play an essential role in membrane remodeling and trafficking. ESCRTs regulate a wide array of cellular processes, including cytokinetic abscission, cargo sorting into multivesicular bodies (MVBs), membrane repair, and autophagy. Given the versatile functionality of ESCRTs, and the intricate organizational structure of the ESCRT machinery, the targeted modulation of distinct ESCRT complexes is considerably challenging. This study presents a pseudonatural product targeting IST1-CHMP1B within the ESCRT-III complexes. The compound specifically disrupts the interaction between IST1 and CHMP1B, thereby inhibiting the formation of IST1-CHMP1B copolymers essential for normal-topology membrane scission events. While the compound has no impact on cytokinesis, MVB sorting, or biogenesis of extracellular vesicles, it rapidly inhibits transferrin receptor recycling in cells, resulting in the accumulation of transferrin in stalled sorting endosomes. Stalled endosomes become decorated by lipidated LC3, suggesting a link between noncanonical LC3 lipidation and inhibition of the IST1-CHMP1B complex.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos , Endossomos/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Transporte Proteico , Corpos Multivesiculares/metabolismo
8.
Int J Mol Sci ; 25(5)2024 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-38474091

RESUMO

Ubiquitin-like modifier-activating enzyme 6 (UBA6) is a member of the E1 enzyme family, which initiates the ubiquitin-proteasome system (UPS). The UPS plays critical roles not only in protein degradation but also in various cellular functions, including neuronal signaling, myocardial remodeling, immune cell differentiation, and cancer development. However, the specific role of UBA6 in cellular functions is not fully elucidated in comparison with the roles of the UPS. It has been known that the E1 enzyme is associated with the motility of cancer cells. In this study, we verified the physiological roles of UBA6 in lung cancer cells through gene-silencing siRNA targeting UBA6 (siUBA6). The siUBA6 treatment attenuated the migration of H1975 cells, along with a decrease in lysosomal Ca2+ release. While autophagosomal proteins remained unchanged, lysosomal proteins, including TRPML1 and TPC2, were decreased in siUBA6-transfected cells. Moreover, siUBA6 induced the production of multivesicular bodies (MVBs), accompanied by an increase in MVB markers in siUBA6-transfected H1975 cells. Additionally, the expression of the exosomal marker CD63 and extracellular vesicles was increased by siUBA6 treatment. Our findings suggest that knock-down of UBA6 induces lysosomal TRPML1 depletion and inhibits endosomal trafficking to lysosome, and subsequently, leads to the accumulation of MVBs and enhanced exosomal secretion in lung cancer cells.


Assuntos
Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/metabolismo , Lisossomos/metabolismo , Corpos Multivesiculares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo
9.
J Cell Biol ; 223(4)2024 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-38319250

RESUMO

Endosomes are specialized organelles that function in the secretory and endocytic protein sorting pathways. Endocytosed cell surface receptors and transporters destined for lysosomal degradation are sorted into intraluminal vesicles (ILVs) at endosomes by endosomal sorting complexes required for transport (ESCRT) proteins. The endosomes (multivesicular bodies, MVBs) then fuse with the lysosome. During endosomal maturation, the number of ILVs increases, but the size of endosomes does not decrease despite the consumption of the limiting membrane during ILV formation. Vesicle-mediated trafficking is thought to provide lipids to support MVB biogenesis. However, we have uncovered an unexpected contribution of a large bridge-like lipid transfer protein, Vps13, in this process. Here, we reveal that Vps13-mediated lipid transfer at ER-endosome contact sites is required for the ESCRT pathway. We propose that Vps13 may play a critical role in supplying lipids to the endosome, ensuring continuous ESCRT-mediated sorting during MVB biogenesis.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Endocitose , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/genética , Lipídeos , Corpos Multivesiculares , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transporte Proteico
10.
FEBS J ; 291(13): 2849-2875, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38401056

RESUMO

The apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) is an essential enzyme of the base excision repair pathway of non-distorting DNA lesions. In response to genotoxic treatments, APE1 is highly secreted (sAPE1) in association with small-extracellular vesicles (EVs). Interestingly, its presence in the serum of patients with hepatocellular or non-small-cell-lung cancers may represent a prognostic biomarker. The mechanism driving APE1 to associate with EVs is unknown, but is of paramount importance in better understanding the biological roles of sAPE1. Because APE1 lacks an endoplasmic reticulum-targeting signal peptide, it can be secreted through an unconventional protein secretion endoplasmic reticulum-Golgi-independent pathway, which includes an endosome-based secretion of intraluminal vesicles, mediated by multivesicular bodies (MVBs). Using HeLa and A549 cell lines, we investigated the role of endosomal sorting complex required for transport protein pathways (either-dependent or -independent) in the constitutive or trichostatin A-induced secretion of sAPE1, by means of manumycin A and GW 4869 treatments. Through an in-depth biochemical analysis of late-endosomes (LEs) and early-endosomes (EEs), we observed that the distribution of APE1 on density gradient corresponded to that of LE-CD63, LE-Rab7, EE-EEA1 and EE-Rab 5. Interestingly, the secretion of sAPE1, induced by cisplatin genotoxic stress, involved an autophagy-based unconventional secretion requiring MVBs. The present study enlightens the central role played by MVBs in the secretion of sAPE1 under various stimuli, and offers new perspectives in understanding the biological relevance of sAPE1 in cancer cells.


Assuntos
Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Transporte Proteico , Humanos , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Células HeLa , Endossomos/metabolismo , Células A549 , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Corpos Multivesiculares/metabolismo , Reparo por Excisão , Ácidos Hidroxâmicos
11.
Cell Mol Gastroenterol Hepatol ; 17(4): 589-605, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38190941

RESUMO

BACKGROUND: A peculiar feature of the hepatitis E virus (HEV) is its reliance on the exosomal route for viral release. Genomic replication is mediated via the viral polyprotein pORF1, yet little is known about its subcellular localization. METHODS: Subcellular localization of pORF1 and its subdomains, generated and cloned based on a structural prediciton of the viral replicase, was analyzed via confocal laser scanning microscopy. Exosomes released from cells were isolated via ultracentrifugation and analyzed by isopycnic density gradient centrifugation. This was followed by fluorimetry or Western blot analyses or reverse transcriptase-polymerase chain reaction to analyze separated particles in more detail. RESULTS: We found pORF1 to be accumulating within the endosomal system, most dominantly to multivesicular bodies (MVBs). Expression of the polyprotein's 7 subdomains revealed that the papain-like cysteine-protease (PCP) is the only domain localizing like the full-length protein. A PCP-deficient pORF1 mutant lost its association to MVBs. Strikingly, both pORF1 and PCP can be released via exosomes. Similarly, genomic RNA still is released via exosomes in the absence of pORF2/3. CONCLUSIONS: Taken together, we found that pORF1 localizes to MVBs in a PCP-dependent manner, which is followed by exosomal release. This reveals new aspects of HEV life cycle, because replication and release could be coupled at the endosomal interface. In addition, this may mediate capsid-independent spread or may facilitate the spread of viral infection, because genomes entering the cell during de novo infection readily encounter exosomally transferred pORF1.


Assuntos
Vírus da Hepatite E , Corpos Multivesiculares/metabolismo , Proteínas/metabolismo , Poliproteínas/metabolismo , Peptídeo Hidrolases/metabolismo
12.
Trends Cell Biol ; 34(2): 90-108, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-37507251

RESUMO

Exosomes are specialized cargo delivery vesicles secreted from cells by fusion of multivesicular bodies (MVBs) with the plasma membrane (PM). While the function of exosomes during physiological and pathological events has been extensively reported, there remains a lack of understanding of the mechanisms that regulate exosome biogenesis, secretion, and internalization. Recent technological and methodological advances now provide details about MVB/exosome structure as well as the pathways of exosome biogenesis, secretion, and uptake. In this review, we outline our current understanding of these processes and highlight outstanding questions following on recent discoveries in the field.


Assuntos
Exossomos , Humanos , Exossomos/metabolismo , Membrana Celular/metabolismo , Corpos Multivesiculares/metabolismo , Transporte Biológico
13.
Hepatol Int ; 18(1): 273-288, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37330971

RESUMO

BACKGROUND AND AIMS: The important role of extracellular vesicles (EVs) in liver fibrosis has been confirmed. However, EVs derived from liver sinusoidal endothelial cells (LSECs) in the activation of hepatic stellate cells (HSCs) and liver fibrosis is still unclear. Our previous work demonstrated that Aldosterone (Aldo) may have the potential to regulate EVs from LSECs via autophagy pathway. Thus, we aim to investigate the role of Aldo in the regulation of EVs derived from LSECs. APPROACH AND RESULTS: Using an Aldo-continuous pumping rat model, we observed that Aldo-induced liver fibrosis and capillarization of LSECs. In vitro, transmission electron microscopy (TEM) revealed that stimulation of Aldo led to the upregulation of autophagy and degradation of multivesicular bodies (MVBs) in LSECs. Mechanistically, Aldo upregulated ATP6V0A2, which promoted lysosomal acidification and subsequent autophagy in LSECs. Inhibiting autophagy with si-ATG5 adeno-associated virus (AAV) in LSECs effectively mitigated Aldo-induced liver fibrosis in rats. RNA sequencing and nanoparticle tracking (NTA) analyses of EVs derived from LSECs indicated that Aldo result in a decrease in both the quantity and quality of EVs. We also observed a reduction in the protective miRNA-342-5P in EVs derived from Aldo-treated LSECs, which may play a critical role in HSCs activation. Target knockdown of EV secretion with si-RAB27a AAV in LSECs led to the development of liver fibrosis and HSC activation in rats. CONCLUSION: Aldo-induced Autophagic degradation of MVBs in LSECs promotes a decrease in the quantity and quality of EVs derived from LSECs, resulting in the activation of HSCs and liver fibrosis under hyperaldosteronism. Modulating the autophagy level of LSECs and their EV secretion may represent a promising therapeutic approach for treating liver fibrosis.


Assuntos
Aldosterona , Células Endoteliais , Ratos , Animais , Aldosterona/metabolismo , Aldosterona/farmacologia , Células Endoteliais/patologia , Corpos Multivesiculares/metabolismo , Fígado/patologia , Cirrose Hepática/metabolismo , Células Estreladas do Fígado/patologia , Autofagia
14.
Traffic ; 25(1): e12921, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-37926552

RESUMO

ESCRTs (Endosomal Sorting Complex Required for Transports) are a modular set of protein complexes with membrane remodeling activities that include the formation and release of intraluminal vesicles (ILVs) to generate multivesicular endosomes. While most of the 12 ESCRT-III proteins are known to play roles in ILV formation, IST1 has been associated with a wider range of endosomal remodeling events. Here, we extend previous studies of IST1 function in endosomal trafficking and confirm that IST1, along with its binding partner CHMP1B, contributes to scission of early endosomal carriers. Functionally, depleting IST1 impaired delivery of transferrin receptor from early/sorting endosomes to the endocytic recycling compartment and instead increased its rapid recycling to the plasma membrane via peripheral endosomes enriched in the clathrin adaptor AP-1. IST1 is also important for export of mannose 6-phosphate receptor from early/sorting endosomes. Examination of IST1 binding partners on endosomes revealed that IST1 interacts with the MIT domain-containing sorting nexin SNX15, a protein previously reported to regulate endosomal recycling. Our kinetic and spatial analyses establish that SNX15 and IST1 occupy a clathrin-containing subdomain on the endosomal perimeter distinct from those previously implicated in cargo retrieval or degradation. Using live-cell microscopy, we see that SNX15 and CHMP1B alternately recruit IST1 to this subdomain or the base of endosomal tubules. These findings indicate that IST1 contributes to a subset of recycling pathways from the early/sorting endosome.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Transporte Proteico , Endossomos/metabolismo , Corpos Multivesiculares/metabolismo , Transporte Biológico
15.
Nat Commun ; 14(1): 6883, 2023 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-37898620

RESUMO

Exosomes are secreted to the extracellular milieu when multivesicular endosomes (MVEs) dock and fuse with the plasma membrane. However, MVEs are also known to fuse with lysosomes for degradation. How MVEs are directed to the plasma membrane for exosome secretion rather than to lysosomes is unclear. Here we report that a conversion of phosphatidylinositol-3-phosphate (PI(3)P) to phosphatidylinositol-4-phosphate (PI(4)P) catalyzed sequentially by Myotubularin 1 (MTM1) and phosphatidylinositol 4-kinase type IIα (PI4KIIα) on the surface of MVEs mediates the recruitment of the exocyst complex. The exocyst then targets the MVEs to the plasma membrane for exosome secretion. We further demonstrate that disrupting PI(4)P generation or exocyst function blocked exosomal secretion of Programmed death-ligand 1 (PD-L1), a key immune checkpoint protein in tumor cells, and led to its accumulation in lysosomes. Together, our study suggests that the PI(3)P to PI(4)P conversion on MVEs and the recruitment of the exocyst direct the exocytic trafficking of MVEs for exosome secretion.


Assuntos
Exossomos , Exossomos/metabolismo , Endossomos/metabolismo , Fosfatidilinositóis/metabolismo , Corpos Multivesiculares/metabolismo
16.
J Extracell Vesicles ; 12(9): e12356, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37700095

RESUMO

Exosomes play crucial roles in local and distant cellular communication and are involved in various physiological and pathological processes. Tumour-derived exosomes are pivotal to tumorigenesis, but the precise mechanisms underlying their secretion remain elusive. In particular, the SNARE proteins that mediate the fusion of multivesicular bodies (MVBs) with the plasma membrane (PM) in tumour cells are subject to debate. In this study, we identified syntaxin-4, SNAP-23, and VAMP-7 as the SNAREs responsible for exosome secretion in MCF-7 breast cancer cells and found that a SNARE complex consisting of these SNAREs can drive membrane fusion in vitro. Deletion of any of these SNAREs in MCF-7 cells did not affect MVB biogenesis and transportation, indicating their specific involvement in MVB-PM fusion. In addition, syntaxin-4, SNAP-23, and VAMP-7 play equivalent roles in exosome secretion in both HeLa cervical cancer cells and A375 melanoma cells, suggesting their conserved function in exosome secretion. Furthermore, deletion of VAMP-7 in 4T1 mammary carcinoma cells efficiently inhibited exosome secretion and led to significant attenuation of tumour growth and lung metastasis in mouse models, implying that VAMP-7 may hold promise as a novel therapeutic target for breast cancer.


Assuntos
Exossomos , Vesículas Extracelulares , Animais , Camundongos , Humanos , Corpos Multivesiculares , Membrana Celular , Proteínas Qa-SNARE
17.
F1000Res ; 12: 884, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37635943

RESUMO

Charged multivesicular body protein 2B is a subunit of the endosomal sorting complex required for transport III (ESRCT-III), a complex implicated in the lysosomal degradation pathway and formation of multivesicular bodies. Mutations to the CHMP2B gene can result in abnormal protein aggregates in neurons and is therefore predicted to be associated in neurodegenerative diseases, including across the ALS-FTD spectrum. Through our standardized experimental protocol which compares read-outs in knockout cell lines and isogenic parental controls, this study aims to enhance the reproducibility of research on this target by characterizing eight commercial antibodies against charged multivesicular body protein 2b using Western Blot, immunoprecipitation, and immunofluorescence. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.


Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Humanos , Corpos Multivesiculares , Reprodutibilidade dos Testes , Western Blotting , Imunofluorescência , Imunoprecipitação , Anticorpos
18.
Nat Commun ; 14(1): 5031, 2023 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-37596294

RESUMO

Mitochondrial quality control is critical for cardiac homeostasis as these organelles are responsible for generating most of the energy needed to sustain contraction. Dysfunctional mitochondria are normally degraded via intracellular degradation pathways that converge on the lysosome. Here, we identified an alternative mechanism to eliminate mitochondria when lysosomal function is compromised. We show that lysosomal inhibition leads to increased secretion of mitochondria in large extracellular vesicles (EVs). The EVs are produced in multivesicular bodies, and their release is independent of autophagy. Deletion of the small GTPase Rab7 in cells or adult mouse heart leads to increased secretion of EVs containing ubiquitinated cargos, including intact mitochondria. The secreted EVs are captured by macrophages without activating inflammation. Hearts from aged mice or Danon disease patients have increased levels of secreted EVs containing mitochondria indicating activation of vesicular release during cardiac pathophysiology. Overall, these findings establish that mitochondria are eliminated in large EVs through the endosomal pathway when lysosomal degradation is inhibited.


Assuntos
Vesículas Extracelulares , Lisossomos , Animais , Camundongos , Mitocôndrias , Transporte Biológico , Corpos Multivesiculares
19.
Proc Natl Acad Sci U S A ; 120(31): e2221522120, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37487085

RESUMO

Cataract is a leading ocular disease causing global blindness. The mechanism of cataractogenesis has not been well defined. Here, we demonstrate that the heat shock protein 90ß (HSP90ß) plays a fundamental role in suppressing cataractogenesis. HSP90ß is the most dominant HSP in normal lens, and its constitutive high level of expression is largely derived from regulation by Sp1 family transcription factors. More importantly, HSP90ß is significantly down-regulated in human cataract patients and in aging mouse lenses, whereas HSP90ß silencing in zebrafish causes cataractogenesis, which can only be rescued by itself but not other HSP90 genes. Mechanistically, HSP90ß can directly interact with CHMP4B, a newly-found client protein involved in control of cytokinesis. HSP90ß silencing causes upregulation of CHMP4B and another client protein, the tumor suppressor p53. CHMP4B upregulation or overexpression induces excessive division of lens epithelial cells without proper differentiation. As a result, these cells were triggered to undergo apoptosis due to activation of the p53/Bak-Bim pathway, leading to cataractogenesis and microphthalmia. Silence of both HSP90ß and CHMP4B restored normal phenotype of zebrafish eye. Together, our results reveal that HSP90ß is a critical inhibitor of cataractogenesis through negative regulation of CHMP4B and the p53-Bak/Bim pathway.


Assuntos
Catarata , Proteínas de Choque Térmico HSP90 , Proteína Supressora de Tumor p53 , Animais , Humanos , Camundongos , Envelhecimento/genética , Catarata/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Corpos Multivesiculares/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
20.
Elife ; 122023 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-37489754

RESUMO

Fusion of multivesicular bodies (MVBs) with the plasma membrane results in the secretion of intraluminal vesicles (ILVs), or exosomes. The sorting of one exosomal cargo RNA, miR223, is facilitated by the RNA-binding protein, YBX1 (Shurtleff et al., 2016). We found that miR223 specifically binds a 'cold shock' domain (CSD) of YBX1 through a 5' proximal sequence motif UCAGU that may represent a binding site or structural feature required for sorting. Prior to sorting into exosomes, most of the cytoplasmic miR223 resides in mitochondria. An RNA-binding protein localized to the mitochondrial matrix, YBAP1, appears to serve as a negative regulator of miR223 enrichment into exosomes. miR223 levels decreased in the mitochondria and increased in exosomes after loss of YBAP1. We observed YBX1 shuttle between mitochondria and endosomes in live cells. YBX1 also partitions into P body granules in the cytoplasm (Liu et al., 2021). We propose a model in which miR223 and likely other miRNAs are stored in mitochondria and are then mobilized by YBX1 to cytoplasmic phase condensate granules for capture into invaginations in the endosome that give rise to exosomes.


Assuntos
Exossomos , MicroRNAs , Exossomos/metabolismo , Endossomos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Corpos Multivesiculares/metabolismo , Mitocôndrias/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
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