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1.
EMBO J ; 42(2): e112287, 2023 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-36644906

RESUMO

Proteins exit from endosomes through tubular carriers coated by retromer, a complex that impacts cellular signaling, lysosomal biogenesis and numerous diseases. The coat must overcome membrane tension to form tubules. We explored the dynamics and driving force of this process by reconstituting coat formation with yeast retromer and the BAR-domain sorting nexins Vps5 and Vps17 on oriented synthetic lipid tubules. This coat oligomerizes bidirectionally, forming a static tubular structure that does not exchange subunits. High concentrations of sorting nexins alone constrict membrane tubes to an invariant radius of 19 nm. At lower concentrations, oligomers of retromer must bind and interconnect the sorting nexins to drive constriction. Constricting less curved membranes into tubes, which requires more energy, coincides with an increased surface density of retromer on the sorting nexin layer. Retromer-mediated crosslinking of sorting nexins at variable densities may thus tune the energy that the coat can generate to deform the membrane. In line with this, genetic ablation of retromer oligomerization impairs endosomal protein exit in yeast and human cells.


Assuntos
Saccharomyces cerevisiae , Nexinas de Classificação , Humanos , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Nexinas de Classificação/genética , Nexinas de Classificação/metabolismo , Constrição , Endossomos/metabolismo
2.
PLoS Genet ; 19(1): e1010571, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36689473

RESUMO

Drosophila trachea is a premier model to study tube morphogenesis. After the formation of continuous tubes, tube maturation follows. Tracheal tube maturation starts with an apical secretion pulse that deposits extracellular matrix components to form a chitin-based apical luminal matrix (aECM). This aECM is then cleared and followed by the maturation of taenidial folds. Finally, air fills the tubes. Meanwhile, the cellular junctions are maintained to ensure tube integrity. Previous research has identified several key components (ER, Golgi, several endosomes) of protein trafficking pathways that regulate the secretion and clearance of aECM, and the maintenance of cellular junctions. The Osiris (Osi) gene family is located at the Triplo-lethal (Tpl) locus on chromosome 3R 83D4-E3 and exhibits dosage sensitivity. Here, we show that three Osi genes (Osi9, Osi15, Osi19), function redundantly to regulate adherens junction (AJ) maintenance, luminal clearance, taenidial fold formation, tube morphology, and air filling during tube maturation. The localization of Osi proteins in endosomes (Rab7-containing late endosomes, Rab11-containing recycling endosomes, Lamp-containing lysosomes) and the reduction of these endosomes in Osi mutants suggest the possible role of Osi genes in tube maturation through endosome-mediated trafficking. We analyzed tube maturation in zygotic rab11 and rab7 mutants, respectively, to determine whether endosome-mediated trafficking is required. Interestingly, similar tube maturation defects were observed in rab11 but not in rab7 mutants, suggesting the involvement of Rab11-mediated trafficking, but not Rab7-mediated trafficking, in this process. To investigate whether Osi genes regulate tube maturation primarily through the maintenance of Rab11-containing endosomes, we overexpressed rab11 in Osi mutant trachea. Surprisingly, no obvious rescue was observed. Thus, increasing endosome numbers is not sufficient to rescue tube maturation defects in Osi mutants. These results suggest that Osi genes regulate other aspects of endosome-mediated trafficking, or regulate an unknown mechanism that converges or acts in parallel with Rab11-mediated trafficking during tube maturation.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Traqueia/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Endossomos/metabolismo , Proteínas de Drosophila/genética
3.
Methods Mol Biol ; 2623: 3-23, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36602676

RESUMO

Filamentous fungi have been used for studying long-distance transport of cargoes driven by cytoplasmic dynein. Aspergillus nidulans is a well-established genetic model organism used for studying dynein function and regulation in vivo. Here, we describe how we grow A. nidulans strains for live-cell imaging and how we observe the dynein-mediated distribution of early endosomes and secretory vesicles. Using an on-stage incubator and culture chambers for inverted microscopes, we can image fungal hyphae that naturally attach to the bottom of the chambers, using wide-field epifluorescence microscopes or the new Zeiss LSM 980 (with Airyscan 2) microscope. In addition to methods for preparing cells for imaging, a procedure for A. nidulans transformation is also described.


Assuntos
Aspergillus nidulans , Dineínas , Dineínas/metabolismo , Aspergillus nidulans/genética , Transporte Biológico , Endossomos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo
5.
Nat Commun ; 14(1): 295, 2023 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-36653346

RESUMO

The formation of the protein corona is a well-known effect when nanoparticles (NP) are exposed to biological environments. The protein corona is the most important factor, which determines the rate and route of endocytosis, and decisively impacts cellular processes and even the release of the active pharmaceutical ingredient from the nanoparticles. While many studies concentrate on the effect of the protein corona formation extracellularly or the uptake consequences, little is known about the fate of the protein corona inside of cells. Here, we reconstruct for the first time the separation of the protein corona from the NPs by the cell and their further fate. Ultimately, the NPs and protein corona are separated from each other and end up in morphologically different cellular compartments. The cell directs the NPs towards recycling endosomes, whereas the protein corona gathers in multivesicular bodies. From this, we conclude that the NPs are prepared for subsequent exocytosis, while the protein corona remains in the cell and is finally metabolized there.


Assuntos
Nanopartículas , Coroa de Proteína , Coroa de Proteína/metabolismo , Nanopartículas/metabolismo , Endocitose , Transporte Biológico , Endossomos/metabolismo
6.
Cancer Lett ; 555: 216049, 2023 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-36608865

RESUMO

Triple-negative breast cancer (TNBC) is one of the most malignant tumors with poor prognosis. Methuosis is a new type of nonapoptotic cell death characterized by the accumulation of cytoplasmic vacuoles. In this study, we synthesized and screened a series of N-phenyl-4-pyrimidinediamine derivatives in TNBC cells, finding that DZ-514 was the best compound with high toxicity independent of the inhibition of BCL6. DZ-514 decreased cell viability, inhibited cell cycle progression, and induced caspase-independent cell death in TNBC cells. Interestingly, DZ-514 induced cytoplasm vacuolation, which could be blocked by Baf A1, the V-ATPase inhibitor. Furthermore, we found that DZ-514-induced vacuoles were derived from macropinosomes rather than autophagosomes. Most importantly, methuosis induced by DZ-514 was partially mediated by activating the ROS-MKK4-p38 axis. Finally, we demonstrated that DZ-514 significantly inhibited tumor growth in an HCC1806 xenograft mouse model. These findings revealed that the novel methuosis inducer DZ-514 could be developed for TNBC treatment.


Assuntos
Neoplasias de Mama Triplo Negativas , Humanos , Animais , Camundongos , Neoplasias de Mama Triplo Negativas/patologia , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular Tumoral , Morte Celular , Apoptose , Endossomos/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proliferação de Células
7.
Prog Mol Biol Transl Sci ; 194: 19-48, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36631193

RESUMO

Ligands, agonists, or antagonists use receptor-mediated endocytosis (RME) to reach their intracellular targets. After the internalization of ligand-receptor complexes, it traffics through different subcellular organelles such as early endosome, recycling endosome, lysosome, etc. Further, after the ligand binding to the receptor, different second messengers are generated, such as cGMP, cAMP, IP3, etc. Several methods have been used, such as radioligand binding assay, western blotting, co-immunoprecipitation (co-IP), qRT-PCR, immunofluorescence and confocal microscopy, microRNA/siRNA, and bioassays to understand the various events, such as internalization, subcellular trafficking, signaling, metabolic degradation, etc. This chapter briefly discusses the key principles and methods used to study internalization, subcellular trafficking, signaling, and metabolic degradation of numerous receptors.


Assuntos
Endocitose , Transdução de Sinais , Humanos , Ligantes , Transporte Proteico , Endossomos/metabolismo
8.
Prog Mol Biol Transl Sci ; 194: 67-78, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36631201

RESUMO

Transferrin receptor (TFRC) is a transmembrane protein that plays a crucial role in mediating homeostasis of iron in the cell. The binding of transferrin (that is bound to iron) to TFRC at the cell membrane generally starts endocytosis of TFRC-transferrin complex, which leads to formation of vesicles that are positive for TFRC. These vesicles travel to the early endosomes and later to the endocytic recycling compartment. Release of iron occurs in the early endosomes because of acidic pH. Major fraction of the transferrin and TFRC is transported back to the cell membrane; however, a minor fraction of it is transported to lysosomes through the process of autophagy. Optineurin (OPTN) is a multi-functional adaptor protein that plays a pivotal role in the control of TFRC trafficking, recycling and autophagy dependent degradation. Optineurin also plays a role in cargo-selective and non-selective autophagy. Here, we review our understanding of the function of OPTN in regulating TFRC trafficking, recycling and autophagy dependent degradation. We also discuss the mechanisms by which certain disease-associated mutations of OPTN alter these processes.


Assuntos
Endocitose , Receptores da Transferrina , Humanos , Transporte Proteico , Receptores da Transferrina/metabolismo , Transporte Biológico , Ferro/metabolismo , Transferrinas/metabolismo , Endossomos/metabolismo
9.
J Cell Biol ; 222(3)2023 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-36524956

RESUMO

The functional significance of Kinesin-1 autoinhibition has been unclear. Kinesin-1 transports multiple cargoes including cytoplasmic dynein to microtubule plus ends. From a genetic screen for Aspergillus mutants defective in dynein-mediated early endosome transport, we identified a kinesin-1 mutation kinAK895* at the C-terminal IAK motif involved in autoinhibition. The kinA∆IAK and kinAK895E mutants exhibited a similar defect in dynein-mediated early endosome transport, verifying the importance of kinesin-1 autoinhibition in dynein-mediated transport. Kinesin-1 autoinhibition is not critical for dynein accumulation at microtubule plus ends or for the secretory vesicle cargoes of kinesin-1 to reach the hyphal tip. However, it facilitates dynein to initiate early endosome transport. This is unrelated to a direct competition between dynein and kinesin-1 on early endosomes because kinesin-3 rather than kinesin-1 drives the plus-end-directed early endosome movement. This effect of kinesin-1 autoinhibition on dynein-mediated early endosome transport is related to cargo adapter-mediated dynein activation but at a step beyond the switching of dynein from its autoinhibited conformation.


Assuntos
Aspergillus nidulans , Dineínas , Proteínas Fúngicas , Cinesinas , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Transporte Biológico , Dineínas/genética , Dineínas/metabolismo , Endossomos/genética , Endossomos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Cinesinas/genética , Cinesinas/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo
10.
Life Sci Alliance ; 6(3)2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36585258

RESUMO

Nutrient deprivation ("starvation") is a major catabolic stress faced by mammalian cells in both pathological and physiological situations. Starvation induces autophagosome biogenesis in the immediate vicinity of ER and leads to lysosome spatial repositioning, but little is known about the consequences of nutritional stress on endosomes. Here, we report that starvation induces tethering of endosomal tubules to ER subregions, fostering autophagosome assembly. We show that this endosomal membrane generation is regulated by sorting nexin 1 (SNX1) protein and is important for the autophagic response. These newly formed SNX1 endosomal tubules establish connections with ER subdomains engaged in early autophagic machinery mobilization. Such endosome-ER transient tethers are regulated by a local dialog between SNX2, an endosomal partner of SNX1, and VAPB, an ER protein associated with autophagy initiation stage regulation. We propose that in a very early response to starvation, SNX1 and SNX2 cooperation induces and regulates endosomal membrane tubulation towards VAPB-positive ER subdomains involved in autophagosome biogenesis, highlighting the contribution of early endosomes in the cellular response to nutritional stress.


Assuntos
Proteínas de Transporte , Proteínas de Transporte Vesicular , Animais , Proteínas de Transporte/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Endossomos/metabolismo , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Mamíferos/metabolismo
11.
J Cell Sci ; 136(2)2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36546731

RESUMO

Contractile vacuoles (CVs), enigmatic osmoregulatory organelles, share common characteristics, such as a requirement for RAB11 and high levels of V-ATPase. These commonalities suggest a conserved evolutionary origin for the CVs with implications for understanding of the last common ancestor of eukaryotes and eukaryotic diversification more broadly. A taxonomically broader sampling of CV-associated machinery is required to address this question further. We used a transcriptomics-based approach to identify CV-associated gene products in Dictyostelium discoideum. This approach was first validated by assessing a set of known CV-associated gene products, which were significantly upregulated following hypo-osmotic exposure. Moreover, endosomal and vacuolar gene products were enriched in the upregulated gene set. An upregulated SNARE protein (NPSNB) was predominantly plasma membrane localised and enriched in the vicinity of CVs, supporting the association with this organelle found in the transcriptomic analysis. We therefore confirm that transcriptomic approaches can identify known and novel players in CV function, in our case emphasizing the role of endosomal vesicle fusion machinery in the D. discoideum CV and facilitating future work to address questions regarding the deep evolution of eukaryotic organelles.


Assuntos
Dictyostelium , Vacúolos , Vacúolos/genética , Vacúolos/metabolismo , Dictyostelium/genética , Dictyostelium/metabolismo , Endossomos/genética , Endossomos/metabolismo , Transporte Biológico , Membrana Celular/metabolismo
12.
Methods Mol Biol ; 2557: 349-364, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36512226

RESUMO

The Golgi-associated retrograde protein (GARP) complex is proposed to tether endosome-derived transport vesicles, but the exact function and mechanism of GARP action are not completely understood. To uncover the GARP function in human cells, we employ CRISPR/Cas9 strategy and knock out (KO) the unique VPS54 subunit of the GARP complex. In this chapter, we describe the detailed method of generating CRISPR/Cas9-mediated VPS54-KO in hTERT-RPE1 cells, rescue of resulting KO cells with stable near-endogenous expression of myc-tagged VPS54, and validation of KO and rescued (KO-R) cells using Western blot and immunofluorescence approaches. This approach is helpful in uncovering new functions of the GARP and other vesicle tethering complexes.


Assuntos
Complexo de Golgi , Proteínas de Transporte Vesicular , Humanos , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Complexo de Golgi/metabolismo , Endossomos/metabolismo , Linhagem Celular , Vesículas Transportadoras/metabolismo
13.
Methods Mol Biol ; 2557: 595-618, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36512240

RESUMO

High-level microscopy enables the comprehensive study of dynamic intracellular processes. Here we describe a toolkit of combinatorial approaches for fixed cell imaging and live cell imaging to investigate the interactions along the trans-Golgi network (TGN)-endosome-lysosome transport axis, which underlie the maturation of endosomal compartments and degradative flux. For fixed cell approaches, we specifically highlight how choices of permeabilization conditions, antibody selection, and antibody multiplexing affect interpretation of results. For live cell approaches, we emphasize the use of sensors that read out pH and degradative capacity in combination with endosomal identity for elucidating dynamic compartment changes.


Assuntos
Endossomos , Rede trans-Golgi , Rede trans-Golgi/metabolismo , Transporte Proteico/fisiologia , Endossomos/metabolismo , Lisossomos/metabolismo , Neurônios
14.
Proc Natl Acad Sci U S A ; 120(1): e2211258120, 2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36577063

RESUMO

The retromer is a heteromeric protein complex that localizes to endosomal membranes and drives the formation of endosomal tubules that recycle membrane protein cargoes. In plants, the retromer plays essential and canonical functions in regulating the transport of vacuolar storage proteins and the recycle of endocytosed plasma membrane proteins (PM); however, the mechanisms underlying the regulation of assembly, protein stability, and membrane recruitment of the plant retromer complex remain to be elucidated. In this study, we identify a plant-unique endosomal regulator termed BLISTER (BLI), which colocalizes and associates with the retromer complex by interacting with the retromer core subunits VPS35 and VPS29. Depletion of BLI perturbs the assembly and membrane recruitment of the retromer core VPS26-VPS35-VPS29 trimer. Consequently, depletion of BLI disrupts retromer-regulated endosomal trafficking function, including transport of soluble vacuolar proteins and recycling of endocytosed PIN-FORMED (PIN) proteins from the endosomes back to the PM. Moreover, genetic analysis in Arabidopsis thaliana mutants reveals BLI and core retromer interact genetically in the regulation of endosomal trafficking. Taken together, we identified BLI as a plant-specific endosomal regulator, which functions in retromer pathway to modulate the recycling of endocytosed PM proteins and the trafficking of soluble vacuolar cargoes.


Assuntos
Arabidopsis , Proteínas de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transporte Proteico , Endossomos/metabolismo , Vacúolos/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Arabidopsis/metabolismo , Nexinas de Classificação/metabolismo
15.
J Cell Biol ; 222(1)2023 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-36282215

RESUMO

Arl8b, an Arf-like GTP-binding protein, regulates cargo trafficking and positioning of lysosomes. However, it is unknown whether Arl8b regulates lysosomal cargo sorting. Here, we report that Arl8b binds to the Rab4 and Rab14 interaction partner, RUN and FYVE domain-containing protein (RUFY) 1, a known regulator of cargo sorting from recycling endosomes. Arl8b determines RUFY1 endosomal localization through regulating its interaction with Rab14. RUFY1 depletion led to a delay in CI-M6PR retrieval from endosomes to the TGN, resulting in impaired delivery of newly synthesized hydrolases to lysosomes. We identified the dynein-dynactin complex as an RUFY1 interaction partner, and similar to a subset of activating dynein adaptors, the coiled-coil region of RUFY1 was required for interaction with dynein and the ability to mediate dynein-dependent organelle clustering. Our findings suggest that Arl8b and RUFY1 play a novel role on recycling endosomes, from where this machinery regulates endosomes to TGN retrieval of CI-M6PR and, consequently, lysosomal cargo sorting.


Assuntos
Fatores de Ribosilação do ADP , Proteínas Adaptadoras de Transdução de Sinal , Dineínas , Endossomos , Lisossomos , Proteínas rab de Ligação ao GTP , Humanos , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Complexo Dinactina/metabolismo , Dineínas/metabolismo , Endossomos/metabolismo , Células HeLa , Lisossomos/metabolismo , Transporte Proteico , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo
16.
J Biol Chem ; 299(1): 102775, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36493904

RESUMO

Phosphatidylinositol (3,5)-bisphosphate [PtdIns(3,5)P2] is a critical signaling phospholipid involved in endolysosome homeostasis. It is synthesized by a protein complex composed of PIKfyve, Vac14, and Fig4. Defects in PtdIns(3,5)P2 synthesis underlie a number of human neurological disorders, including Charcot-Marie-Tooth disease, child onset progressive dystonia, and others. However, neuron-specific functions of PtdIns(3,5)P2 remain less understood. Here, we show that PtdIns(3,5)P2 pathway is required to maintain neurite thickness. Suppression of PIKfyve activities using either pharmacological inhibitors or RNA silencing resulted in decreased neurite thickness. We further find that the regulation of neurite thickness by PtdIns(3,5)P2 is mediated by NSG1/NEEP21, a neuron-specific endosomal protein. Knockdown of NSG1 expression also led to thinner neurites. mCherry-tagged NSG1 colocalized and interacted with proteins in the PtdIns(3,5)P2 machinery. Perturbation of PtdIns(3,5)P2 dynamics by overexpressing Fig4 or a PtdIns(3,5)P2-binding domain resulted in mislocalization of NSG1 to nonendosomal locations, and suppressing PtdIns(3,5)P2 synthesis resulted in an accumulation of NSG1 in EEA1-positive early endosomes. Importantly, overexpression of NSG1 rescued neurite thinning in PtdIns(3,5)P2-deficient CAD neurons and primary cortical neurons. Our study uncovered the role of PtdIns(3,5)P2 in the morphogenesis of neurons, which revealed a novel aspect of the pathogenesis of PtdIns(3,5)P2-related neuropathies. We also identified NSG1 as an important downstream protein of PtdIns(3,5)P2, which may provide a novel therapeutic target in neurological diseases.


Assuntos
Neuritos , Fosfatidilinositóis , Criança , Humanos , Fosfatidilinositóis/metabolismo , Neuritos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Endossomos/metabolismo , Neurônios/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas de Membrana/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Flavoproteínas/genética , Flavoproteínas/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo
17.
Mol Ther ; 30(2): 855-867, 2022 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-34547467

RESUMO

Cell-penetrating peptides (CPPs) hold great promise for intracellular delivery of therapeutic proteins. However, endosomal entrapment of transduced cargo is a major bottleneck hampering their successful application. While developing a transducible zinc finger protein-based artificial transcription factor targeting the expression of endothelin receptor A, we identified interaction between the CPP and the endosomal membrane or endosomal entanglement as a main culprit for endosomal entrapment. To achieve endosomal disentanglement, we utilized endosome-resident proteases to sever the artificial transcription factor from its CPP upon arrival inside the endosome. Using this approach, we greatly enhanced the correct subcellular localization of the disentangled artificial transcription factor, significantly increasing its biological activity and distribution in vivo. With rational engineering of proteolytic sensitivity, we propose a new design principle for transducible therapeutic proteins, helping CPPs attain their full potential as delivery vectors for therapeutic proteins.


Assuntos
Peptídeos Penetradores de Células , Receptores de Endotelina , Peptídeos Penetradores de Células/metabolismo , Endossomos/metabolismo , Receptores de Endotelina/metabolismo , Fatores de Transcrição/metabolismo
18.
Int J Mol Sci ; 23(24)2022 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-36555247

RESUMO

Gap junction protein connexin 43 (Cx43) plays a critical role in gap junction communication in rat hepatocytes. However, those located between hepatocytes are easily internalized following exposure to poisons. Herein, we investigated the potential of buffalo rat liver 3A (BRL 3A) cells to generate annular gap junctions (AGJs) proficient at alleviating cadmium (Cd) cytotoxic injury through degradation via an endosome-lysosome pathway. Our results showed that Cd-induced damage of liver microtubules promoted Cx43 internalization and increased Cx43 phosphorylation at Ser373 site. Furthermore, we established that Cd induced AGJs generation in BRL 3A cells, and AGJs were subsequently degraded through the endosome-lysosome pathway. Overall, our results suggested that Cx43 internalization and the generation of AGJs were cellular protective mechanisms to alleviate Cd toxicity in rat hepatocytes.


Assuntos
Cádmio , Conexina 43 , Ratos , Animais , Cádmio/toxicidade , Cádmio/metabolismo , Conexina 43/metabolismo , Hepatócitos/metabolismo , Lisossomos/metabolismo , Endossomos/metabolismo , Junções Comunicantes/metabolismo
19.
Science ; 378(6625): eabq5209, 2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36520888

RESUMO

Cells respond to fluctuating nutrient supply by adaptive changes in organelle dynamics and in metabolism. How such changes are orchestrated on a cell-wide scale is unknown. We show that endosomal signaling lipid turnover by MTM1, a phosphatidylinositol 3-phosphate [PI(3)P] 3-phosphatase mutated in X-linked centronuclear myopathy in humans, controls mitochondrial morphology and function by reshaping the endoplasmic reticulum (ER). Starvation-induced endosomal recruitment of MTM1 impairs PI(3)P-dependent contact formation between tubular ER membranes and early endosomes, resulting in the conversion of ER tubules into sheets, the inhibition of mitochondrial fission, and sustained oxidative metabolism. Our results unravel an important role for early endosomal lipid signaling in controlling ER shape and, thereby, mitochondrial form and function to enable cells to adapt to fluctuating nutrient environments.


Assuntos
Retículo Endoplasmático , Endossomos , Mitocôndrias , Dinâmica Mitocondrial , Fosfatos de Fosfatidilinositol , Fosfatidilinositóis , Proteínas Tirosina Fosfatases não Receptoras , Humanos , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Mitocôndrias/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositóis/metabolismo , Transdução de Sinais , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas Tirosina Fosfatases não Receptoras/metabolismo
20.
PLoS One ; 17(12): e0278848, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36520788

RESUMO

TRPML3 (mucolipin 3, MCOLN3) is an endolysosomal cation channel belonging to the TRPML subfamily of transient receptor potential channels. Gain-of-function mutations in the Trpml3 gene cause deafness, circling behavior and coat color dilution in mice due to cell death of TRPML3-expressing hair cells of the inner ear or skin melanocytes, respectively. Furthermore, TRPML3 was found to play a role in the long term survival of cochlear hair cells (its absence contributing to presbycusis), in specialized giant lysosomes that neonatal (birth to weaning) enterocytes used for the uptake and digestion of maternal milk nutrients, and in the expulsion of exosome-encased bacteria such as uropathogenic E. coli, infecting bladder epithelial cells. Recently, TRPML3 was found to be expressed at high levels in alveolar macrophages and loss of TRPML3 results in a lung emphysema phenotype, confirmed in two independently engineered Trpml3 knockout lines. TRPML3 is not ubiquitously expressed like its relative TRPML1 and thus cellular expression of TRPML3 on a whole-tissue level remains, with the exceptions mentioned above, largely elusive. To overcome this problem, we generated a τGFP reporter mouse model for TRPML3 and compared expression data obtained from this model by immunofluorescence on tissue sections with immunohistochemistry using TRPML3 antibodies and in situ hybridization. We thus uncovered expression in several organs and distinct cell types. We confirmed TRPML3 expression in both neonatal and adult alveolar macrophages, in melanocytes of hair follicles and glabrous skin, in principle cells of the collecting duct of the neonatal and adult kidney, and in olfactory sensory neurons of the olfactory epithelium, including its fibres protruding to the glomeruli of the olfactory bulb. Additionally, we localized TRPML3 in several glands including parathyroid, thyroid, salivary, adrenal, and pituitary gland, testes and ovaries, suggestive of potential roles for the channel in secretion or uptake of different hormones.


Assuntos
Glândulas Endócrinas , Canais de Potencial de Receptor Transitório , Camundongos , Animais , Escherichia coli/metabolismo , Canais de Potencial de Receptor Transitório/genética , Endossomos/metabolismo , Células Ciliadas Auditivas/fisiologia , Modelos Animais de Doenças
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