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1.
Eur J Pharmacol ; 882: 173288, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32561291

RESUMO

In December 2019, many pneumonia cases with unidentified sources appeared in Wuhan, Hubei, China, with clinical symptoms like viral pneumonia. Deep sequencing analysis of samples from lower respiratory tract revealed a novel coronavirus, called 2019 novel coronavirus (2019-nCoV). Currently there is a rapid global spread. World Health Organization declare the disease a pandemic condition. The pathologic source of this disease was a new RNA virus from Coronaviridae family, which was named COVID-19. SARS-CoV-2 entry starts with the binding of the spike glycoprotein expressed on the viral envelope to ACE2 on the alveolar surface followed by clathrin-dependent endocytosis of the SARS-CoV-2 and ACE2 complex. SARS-CoV-2 enters the cells through endocytosis process, which is possibly facilitated, via a pH dependent endosomal cysteine protease cathepsins. Once inside the cells, SARS-CoV-2 exploits the endogenous transcriptional machinery of alveolar cells to replicate and spread through the entire lung. Endosomal acidic pH for SARS-CoV-2 processing and internalization is critical. After entering the cells, it possibly activates or hijack many intracellular pathways in favor of its replication. In the current opinion article, we will explain the possible involvement of unfolded protein response as a cellular stress response to the SARS-CoV-2 infection.


Assuntos
Células Epiteliais Alveolares/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Retículo Endoplasmático/efeitos dos fármacos , Ionóforos/farmacologia , Pneumonia Viral/tratamento farmacológico , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/virologia , Betacoronavirus/metabolismo , Vesículas Revestidas por Clatrina/efeitos dos fármacos , Vesículas Revestidas por Clatrina/metabolismo , Infecções por Coronavirus/virologia , Endocitose/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Humanos , Ionóforos/uso terapêutico , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos
2.
Nat Commun ; 10(1): 4974, 2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31672988

RESUMO

Clathrin light chains (CLCa and CLCb) are major constituents of clathrin-coated vesicles. Unique functions for these evolutionary conserved paralogs remain elusive, and their role in clathrin-mediated endocytosis in mammalian cells is debated. Here, we find and structurally characterize a direct and selective interaction between CLCa and the long isoform of the actin motor protein myosin VI, which is expressed exclusively in highly polarized tissues. Using genetically-reconstituted Caco-2 cysts as proxy for polarized epithelia, we provide evidence for coordinated action of myosin VI and CLCa at the apical surface where these proteins are essential for fission of clathrin-coated pits. We further find that myosin VI and Huntingtin-interacting protein 1-related protein (Hip1R) are mutually exclusive interactors with CLCa, and suggest a model for the sequential function of myosin VI and Hip1R in actin-mediated clathrin-coated vesicle budding.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cadeias Leves de Clatrina/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Actinas/metabolismo , Células CACO-2 , Técnicas de Cultura de Células , Cadeias Leves de Clatrina/ultraestrutura , Cistos , Endocitose , Humanos , Espectroscopia de Ressonância Magnética , Cadeias Pesadas de Miosina/ultraestrutura , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas
3.
Dev Cell ; 50(4): 494-508.e11, 2019 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-31430451

RESUMO

Clathrin-mediated endocytosis (CME) is key to maintaining the transmembrane protein composition of cells' limiting membranes. During mammalian CME, a reversible phosphorylation event occurs on Thr156 of the µ2 subunit of the main endocytic clathrin adaptor, AP2. We show that this phosphorylation event starts during clathrin-coated pit (CCP) initiation and increases throughout CCP lifetime. µ2Thr156 phosphorylation favors a new, cargo-bound conformation of AP2 and simultaneously creates a binding platform for the endocytic NECAP proteins but without significantly altering AP2's cargo affinity in vitro. We describe the structural bases of both. NECAP arrival at CCPs parallels that of clathrin and increases with µ2Thr156 phosphorylation. In turn, NECAP recruits drivers of late stages of CCP formation, including SNX9, via a site distinct from where NECAP binds AP2. Disruption of the different modules of this phosphorylation-based temporal regulatory system results in CCP maturation being delayed and/or stalled, hence impairing global rates of CME.


Assuntos
Complexo 2 de Proteínas Adaptadoras/genética , Subunidades alfa do Complexo de Proteínas Adaptadoras/genética , Endocitose/genética , Nexinas de Classificação/genética , Complexo 2 de Proteínas Adaptadoras/metabolismo , Clatrina/genética , Clatrina/metabolismo , Vesículas Revestidas por Clatrina/genética , Vesículas Revestidas por Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/genética , Invaginações Revestidas da Membrana Celular/metabolismo , Humanos , Fosforilação/genética , Ligação Proteica/genética
4.
Int J Mol Sci ; 20(16)2019 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-31408934

RESUMO

Recent findings have revealed the role of membrane traffic in the signaling of transforming growth factor-ß (TGF-ß). These findings originate from the pivotal function of TGF-ß in development, cell proliferation, tumor metastasis, and many other processes essential in malignancy. Actin and unconventional myosin have crucial roles in subcellular trafficking of receptors; research has also revealed a growing number of unconventional myosins that have crucial roles in TGF-ß signaling. Unconventional myosins modulate the spatial organization of endocytic trafficking and tether membranes or transport them along the actin cytoskeletons. Current models do not fully explain how membrane traffic forms a bridge between TGF-ß and the downstream effectors that produce its functional responsiveness, such as cell migration. In this review, we present a brief overview of the current knowledge of the TGF-ß signaling pathway and the molecular components that comprise the core pathway as follows: ligands, receptors, and Smad mediators. Second, we highlight key role(s) of myosin motor-mediated protein trafficking and membrane domain segregation in the modulation of the TGF-ß signaling pathway. Finally, we review future challenges and provide future prospects in this field.


Assuntos
Miosinas/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Vesículas Revestidas por Clatrina/metabolismo , Endocitose , Humanos , Microdomínios da Membrana/metabolismo , Transporte Proteico
5.
Physiol Genomics ; 51(8): 323-332, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31172864

RESUMO

Atrial fibrillation is a significant worldwide contributor to cardiovascular morbidity and mortality. Few studies have investigated the differences in gene expression between the left and right atrial appendages, leaving their characterization largely unexplored. In this study, differential gene expression was investigated in atrial fibrillation and sinus rhythm using left and right atrial appendages from the same patients. RNA sequencing was performed on the left and right atrial appendages from five sinus rhythm (SR) control patients and five permanent AF case patients. Differential gene expression in both the left and right atrial appendages was analyzed using the Bioconductor package edgeR. A selection of differentially expressed genes, with relevance to atrial fibrillation, were further validated using quantitative RT-PCR. The distribution of the samples assessed through principal component analysis showed distinct grouping between left and right atrial appendages and between SR controls and AF cases. Overall 157 differentially expressed genes were identified to be downregulated and 90 genes upregulated in AF. Pathway enrichment analysis indicated a greater involvement of left atrial genes in the Wnt signaling pathway whereas right atrial genes were involved in clathrin-coated vesicle and collagen formation. The differing expression of genes in both left and right atrial appendages indicate that there are different mechanisms for development, support and remodeling of AF within the left and right atria.


Assuntos
Apêndice Atrial/fisiopatologia , Fibrilação Atrial/genética , Análise de Sequência de RNA/métodos , Transcriptoma/genética , Idoso , Idoso de 80 Anos ou mais , Fibrilação Atrial/patologia , Vesículas Revestidas por Clatrina/metabolismo , Estudos de Coortes , Colágeno/metabolismo , Ponte de Artéria Coronária , Regulação para Baixo/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética , Regulação para Cima/genética , Via de Sinalização Wnt/genética
6.
Cell Rep ; 26(12): 3380-3390.e5, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30893609

RESUMO

Chlamydial infection requires the formation of a membrane-bound vacuole, termed the inclusion, that undergoes extensive interactions with select host organelles. The importance of the Inc protein CT229 in the formation and maintenance of the chlamydial inclusion was recently highlighted by studies demonstrating that its absence during infection results in reduced bacterial replication, premature inclusion lysis, and host cell death. Previous reports have indicated that CT229 binds Rab GTPases; however, the physiological implications of this interaction are unknown. Here, we show that CT229 regulates host multivesicular trafficking by recruiting multiple Rab GTPases and their cognate effectors to the inclusion. We demonstrate that CT229 specifically modulates clathrin-coated vesicle trafficking and regulates the trafficking of transferrin and the mannose-6-phosphate receptor, both of which are crucial for proper chlamydial development. This study highlights CT229 as a master regulator of multiple host vesicular trafficking pathways essential for chlamydial infection.


Assuntos
Proteínas de Bactérias/metabolismo , Infecções por Chlamydia/metabolismo , Chlamydia trachomatis/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Vacúolos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas de Bactérias/genética , Transporte Biológico Ativo , Infecções por Chlamydia/genética , Infecções por Chlamydia/patologia , Chlamydia trachomatis/genética , Vesículas Revestidas por Clatrina/genética , Vesículas Revestidas por Clatrina/microbiologia , Células HeLa , Humanos , Corpos de Inclusão/genética , Corpos de Inclusão/metabolismo , Corpos de Inclusão/microbiologia , Vacúolos/genética , Vacúolos/microbiologia , Proteínas rab de Ligação ao GTP/genética
7.
Auris Nasus Larynx ; 46(5): 790-796, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30739815

RESUMO

Objective The endocytosis of cationized feritin (CF) via a clathrin-mediated pathway is regulated by a signaling network. Marginal cells showed the active endocytosis of CF via a clathrin-mediated pathway. The internalization of receptors through this clathrin-mediated pathway is an important regulatory event in signal transduction. Numerous kinases are involved in endocytosis, and each endocytic route is subjected to high-order regulation by cellular signaling mechanisms. In this study, we investigated whether ROCK and MLCK signaling cascades and G-proteins regulate the endocytosis of CF in marginal cells of the stria vascularis. Methods CF was infused into the cochlear duct with pertussis toxin (PTX),Clostridium botulinum C3 toxin (BTX), guanosine(g-thio)-triphosphate (GTP-γS), ML-7, Y-27632. Endocytic activity was measured at 30 min after the start of infusion under an electron microscope. Results In marginal cells, CF was internalized via a clathrin-mediated pathway that depends on F-actin and microtubules (MT). Its processes were controlled by myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK), but not affected by G-protein-coupled receptor (GPCR) or the RhoA signaling cascade. Conclusion Our previous study showed that the main endocytotic pathway of microperoxidase (MPO) did not depend on the Rho/ROCK molecular switch or actin/myosin motor system, but was mainly regulated by the RhoA signaling cascade. The present study results indicate that these signaling cascades regulating CF internalization completely differ from the cascades for MPO internalization.


Assuntos
Vesículas Revestidas por Clatrina/metabolismo , Endocitose/fisiologia , Ferritinas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Estria Vascular/metabolismo , Quinases Associadas a rho/metabolismo , ADP Ribose Transferases/farmacologia , Amidas/farmacologia , Animais , Azepinas/farmacologia , Toxinas Botulínicas/farmacologia , Ducto Coclear , Endocitose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteínas de Ligação ao GTP/antagonistas & inibidores , Cobaias , Microscopia Eletrônica , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Fosfatase de Miosina-de-Cadeia-Leve/antagonistas & inibidores , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Naftalenos/farmacologia , Toxina Pertussis/farmacologia , Piridinas/farmacologia , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , Estria Vascular/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/metabolismo
8.
PLoS Biol ; 17(2): e3000180, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30811478
9.
Elife ; 82019 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-30688648

RESUMO

The fibroblast growth factor FGF21 was labeled with molecularly defined gold nanoparticles (AuNPs), applied to human adipocytes, and imaged by cryo-electron tomography (cryo-ET). Most AuNPs were in pairs about 80 Å apart, on the outer cell surface. Pairs of AuNPs were also abundant inside the cells in clathrin-coated vesicles and endosomes. AuNPs were present but no longer paired in multivesicular bodies. FGF21 could thus be tracked along the endocytotic pathway. The methods developed here to visualize signaling coupled to endocytosis can be applied to a wide variety of cargo and may be extended to studies of other intracellular transactions.


Assuntos
Membrana Celular/química , Endocitose/genética , Endossomos/química , Fatores de Crescimento de Fibroblastos/química , Movimento Celular/genética , Vesículas Revestidas por Clatrina/química , Vesículas Revestidas por Clatrina/metabolismo , Tomografia com Microscopia Eletrônica , Fatores de Crescimento de Fibroblastos/isolamento & purificação , Ouro/química , Humanos , Nanopartículas Metálicas/química , Transporte Proteico/genética , Transdução de Sinais , Propriedades de Superfície
10.
Blood Adv ; 3(2): 168-183, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30670533

RESUMO

In the earliest phase of inflammation, histamine and other agonists rapidly mobilize P-selectin to the apical membranes of endothelial cells, where it initiates rolling adhesion of flowing neutrophils. Clustering of P-selectin in clathrin-coated pits facilitates rolling. Inflammatory cytokines typically signal by regulating gene transcription over a period of hours. We found that neutrophils rolling on P-selectin secreted the cytokine oncostatin M (OSM). The released OSM triggered signals through glycoprotein 130 (gp130)-containing receptors on endothelial cells that, within minutes, further clustered P-selectin and markedly enhanced its adhesive function. Antibodies to OSM or gp130, deletion of the gene encoding OSM in hematopoietic cells, or conditional deletion of the gene encoding gp130 in endothelial cells inhibited neutrophil rolling on P-selectin in trauma-stimulated venules of the mouse cremaster muscle. In a mouse model of P-selectin-dependent deep vein thrombosis, deletion of OSM in hematopoietic cells or of gp130 in endothelial cells markedly inhibited adhesion of neutrophils and monocytes and the rate and extent of thrombus formation. Our results reveal a paracrine-signaling mechanism by which neutrophil-released OSM rapidly influences endothelial cell function during physiological and pathological inflammation.


Assuntos
Endotélio Vascular/metabolismo , Neutrófilos/metabolismo , Oncostatina M/metabolismo , Selectina-P/metabolismo , Trombose/etiologia , Trombose/metabolismo , Vasculite/etiologia , Vasculite/metabolismo , Animais , Biomarcadores , Adesão Celular , Comunicação Celular , Vesículas Revestidas por Clatrina/metabolismo , Receptor gp130 de Citocina/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Células Endoteliais/metabolismo , Humanos , Imunofenotipagem , Migração e Rolagem de Leucócitos , Camundongos , Camundongos Knockout , Modelos Biológicos , Neutrófilos/imunologia , Selectina-P/genética , Ligação Proteica , Transdução de Sinais , Trombose/patologia , Vasculite/patologia
11.
Biochem Cell Biol ; 97(3): 315-324, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30383978

RESUMO

Endocytic organelles maintain their acidic pH using the V-type ATPase proton pump. However, proton accumulation across the membrane generates a voltage and requires the movement of an additional ion, known as a counterion, to dissipate charge buildup. The role of counterion movement in endosomes is not clear, but a subpopulation of early endosomes, clathrin-coated vesicles (CCVs), has previously been shown to use external chloride (Cl-) to allow V-ATPase-dependent acidification. We aimed to determine the identity and function of this presumed Cl- transporting protein. Our sample of highly enriched bovine brain CCVs exhibited V-type ATPase-facilitated acidification in the presence of external Cl-, independent of the monovalent cations present. While unsuccessful at identifying the mechanism of anion transport, we used glutamate-facilitated acidification, density gradients, and mass spectrometry to show that most brain CCVs are synaptic vesicles, complementing results from earlier studies that argued similarity only on the basis on protein content. The source of Cl--dependent acidification in brain CCVs may be vGLUT1, a synaptic vesicle glutamate transporter with known Cl- permeability, although CCVs in other tissues are likely to utilize different proteins to facilitate acidification.


Assuntos
Encéfalo/metabolismo , Cloretos/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Animais , Bovinos , Concentração de Íons de Hidrogênio
12.
Cell Microbiol ; 21(3): e12961, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30291809

RESUMO

Heme is a major source of iron for pathogens of humans, and its use is critical in determining the outcome of infection and disease. Cryptococcus neoformans is an encapsulated fungal pathogen that causes life-threatening infections in immunocompromised individuals. C. neoformans effectively uses heme as an iron source, but the underlying mechanisms are poorly defined. Non-iron metalloporphyrins (MPPs) are toxic analogues of heme and are thought to enter microbial cells via endogenous heme acquisition systems. We therefore carried out a mutant screen for susceptibility against manganese MPP (MnMPP) to identify new components for heme uptake in C. neoformans. We identified several genes involved in signalling, DNA repair, sugar metabolism, and trafficking that play important roles in susceptibility to MnMPP and in the use of heme as an iron source. We focused on investigating the role of clathrin-mediated endocytosis (CME) and found that several components of CME including Chc1, Las17, Rvs161, and Rvs167 are required for growth on heme and hemoglobin and for endocytosis and intracellular trafficking of these molecules. We show that the hemoglobin uptake process in C. neoformans involves clathrin heavy chain, Chc1, which appears to colocalise with hemoglobin-containing vesicles and to potentially assist in proper delivery of hemoglobin to the vacuole. Additionally, C. neoformans strains lacking Chc1, Las17, Rvs161, or Rvs167 were defective in the elaboration of several key virulence factors, and a las17 mutant was avirulent in a mouse model of cryptococcosis. Overall, this study unveils crucial functions of CME in the use of heme iron by C. neoformans and reveals a role for CME in fungal pathogenesis.


Assuntos
Vesículas Revestidas por Clatrina/metabolismo , Clatrina/metabolismo , Cryptococcus neoformans/metabolismo , Endocitose , Heme/metabolismo , Hemoglobinas/metabolismo , Animais , Clatrina/genética , Vesículas Revestidas por Clatrina/genética , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/genética , Cryptococcus neoformans/crescimento & desenvolvimento , Testes Genéticos , Ferro/metabolismo , Manganês/toxicidade , Camundongos , Fatores de Virulência/metabolismo
13.
EBioMedicine ; 36: 229-240, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30279141

RESUMO

BACKGROUND: Epidermal growth factor receptor (EGFR) signalling is critical in epithelial cancer development. Human rhomboid family-1 (RHBDF1) facilitates the secretion of TGFα, an EGFR ligand, in breast cancer; however, the underlying mechanism remains unclear. We evaluated the role for RHBDF1 in clathrin-coated vesicle (CCV)-dependent pro-TGFα membrane trafficking in breast cancer cells upon stimulation by G-protein coupled receptor (GPCR) agonists. METHODS: RHBDF1 was silenced in various breast cancer cells using shRNA. TGFα levels, subcellular localization, and secretion were evaluated using ELISA, immunofluorescent staining, and coimmunoprecipitation. Phosphorylation and expression of relevant proteins were measured by western blotting. RHBDF1-dependent cell viability and invasion were measured. FINDINGS: RHBDF1 mediates GPCR agonist-induced EGFR phosphorylation by promoting TGFα secretion in various types of breast cancer cells. RHBDF1 not only mediates ADAM17-dependent shedding of TGFα, but is essential in membrane trafficking of pro-TGFα. RHBDF1 silencing results in blocking of clathrin uncoating from CCV, a crucial step for the plasma membrane release of pro-TGFα. Interaction of RHBDF1 with auxilin-2, a CCV protein, determines the recruitment of HSC70 to CCV to facilitate clathrin uncoating. RHBDF1 function is required for the proliferation and mobility of breast cancer cells upon stimulation by Sphingosine 1 Phosphate (S1P), a GPCR agonist. We demonstrate a significant correlation between RHBDF1 overexpression and EGFR activation in breast cancer tissues. INTERPRETATION: RHBDF1 is an indispensable component of the protein trafficking machinery involved in GPCR-mediated EGFR transactivation, and is an attractive therapeutic target for cancer. FUND: National Natural Science Foundation of China (81,672,740 to ZSZ, 81,272,356 and 81,330,029 to LYL).


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Vesículas Revestidas por Clatrina/metabolismo , Proteínas de Membrana/metabolismo , Fator de Crescimento Transformador alfa/metabolismo , Proteína ADAM17/metabolismo , Auxilinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Receptores ErbB/metabolismo , Feminino , Proteínas de Choque Térmico HSC70/metabolismo , Humanos , Ligantes , Modelos Biológicos , Ligação Proteica , Transporte Proteico , RNA Interferente Pequeno/genética
14.
Mol Biol Cell ; 29(24): 2959-2968, 2018 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-30188768

RESUMO

New methods in stem cell 3D organoid tissue culture, advanced imaging, and big data image analytics now allow tissue-scale 4D cell biology, but currently available analytical pipelines are inadequate for handing and analyzing the resulting gigabytes and terabytes of high-content imaging data. We expressed fluorescent protein fusions of clathrin and dynamin2 at endogenous levels in genome-edited human embryonic stem cells, which were differentiated into hESC-derived intestinal epithelial organoids. Lattice light-sheet imaging with adaptive optics (AO-LLSM) allowed us to image large volumes of these organoids (70 × 60 × 40 µm xyz) at 5.7 s/frame. We developed an open-source data analysis package termed pyLattice to process the resulting large (∼60 Gb) movie data sets and to track clathrin-mediated endocytosis (CME) events. CME tracks could be recorded from ∼35 cells at a time, resulting in ∼4000 processed tracks per movie. On the basis of their localization in the organoid, we classified CME tracks into apical, lateral, and basal events and found that CME dynamics is similar for all three classes, despite reported differences in membrane tension. pyLattice coupled with AO-LLSM makes possible quantitative high temporal and spatial resolution analysis of subcellular events within tissues.


Assuntos
Vesículas Revestidas por Clatrina/metabolismo , Clatrina/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Processamento de Imagem Assistida por Computador/métodos , Intestinos/citologia , Animais , Big Data , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Dinamina II/metabolismo , Endocitose/fisiologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Camundongos , Organoides/citologia , Organoides/diagnóstico por imagem , Organoides/metabolismo
15.
Nat Commun ; 9(1): 3825, 2018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-30237420

RESUMO

It is generally assumed that cells interrogate the mechanical properties of their environment by pushing and pulling on the extracellular matrix (ECM). For instance, acto-myosin-dependent contraction forces exerted at focal adhesions (FAs) allow the cell to actively probe substrate elasticity. Here, we report that a subset of long-lived and flat clathrin-coated structures (CCSs), also termed plaques, are contractility-independent mechanosensitive signaling platforms. We observed that plaques assemble in response to increasing substrate rigidity and that this is independent of FAs, actin and myosin-II activity. We show that plaque assembly depends on αvß5 integrin, and is a consequence of frustrated endocytosis whereby αvß5 tightly engaged with the stiff substrate locally stalls CCS dynamics. We also report that plaques serve as platforms for receptor-dependent signaling and are required for increased Erk activation and cell proliferation on stiff environments. We conclude that CCSs are mechanotransduction structures that sense substrate rigidity independently of cell contractility.


Assuntos
Vesículas Revestidas por Clatrina/metabolismo , Endocitose , Mecanotransdução Celular , Linhagem Celular , Proliferação de Células , Vesículas Revestidas por Clatrina/ultraestrutura , Humanos , Sistema de Sinalização das MAP Quinases , Receptores de Vitronectina/metabolismo
16.
Sci Rep ; 8(1): 12241, 2018 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-30115966

RESUMO

Intracellular dynamics of an abnormal isoform of prion protein (PrPSc) are tightly associated with prion propagation. However, the machineries involved in the intracellular trafficking of PrPSc are not fully understood. Our previous study suggested that PrPSc in persistently prion-infected cells dynamically circulates between endocytic-recycling compartments (ERCs) and peripheral regions of the cells. To investigate these machineries, we focused on retrograde transport from endosomes to the trans-Golgi network, which is one of the pathways involved in recycling of molecules. PrPSc was co-localized with components of clathrin-coated vesicles (CCVs) as well as those of the retromer complex, which are known as machineries for retrograde transport. Fractionation of intracellular compartments by density gradient centrifugation showed the presence of PrPSc and the components of CCVs in the same fractions. Furthermore, PrPSc was detected in CCVs isolated from intracellular compartments of prion-infected cells. Knockdown of clathrin interactor 1, which is one of the clathrin adaptor proteins involved in retrograde transport, did not change the amount of PrPSc, but it altered the distribution of PrPSc from ERCs to peripheral regions, including late endosomes/lysosomes. These data demonstrated that some PrPSc is transported from endosomes to ERCs by CCVs, which might be involved in the recycling of PrPSc.


Assuntos
Vesículas Revestidas por Clatrina/metabolismo , Proteínas PrPSc/metabolismo , Animais , Linhagem Celular Tumoral , Endossomos/metabolismo , Camundongos , Transporte Proteico
17.
Methods Mol Biol ; 1847: 1-11, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30129005

RESUMO

Here, we describe a purification protocol for isolating clathrin-coated vesicles (CCVs) from adult rat brain by using differential centrifugation coupled with Ficoll-sucrose and D2O-sucrose density gradient centrifugation and an additional linear sucrose step gradient at the end to separate CCVs from contaminating membranes present in the crude microsomal fraction.


Assuntos
Encéfalo/metabolismo , Fracionamento Celular , Vesículas Revestidas por Clatrina/metabolismo , Animais , Fracionamento Celular/métodos , Centrifugação com Gradiente de Concentração/métodos , Ratos , Frações Subcelulares
18.
Methods Mol Biol ; 1847: 23-35, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30129007

RESUMO

Endocytosis mediates the cellular uptake of nutrients, modulates signaling by regulating levels of cell surface receptors, and is usurped by pathogens during infection. Endocytosis activity is known to vary during the cell cycle, in particular during mitosis. Importantly, different experimental conditions can lead to opposite results and conclusions, thereby emphasizing the need for a careful design of protocols. For example, experiments using serum-starvation, ice-cold steps or using mitotic arrest produced by chemicals widely used to synchronize cells (nocodazole, RO-3306, or S-trityl-L-cysteine) induce a blockage of clathrin-mediated endocytosis during mitosis not observed in unperturbed, dividing cells. In addition, perturbations produced by mRNA interference or dominant-negative mutant overexpression affect endocytosis long before cells are being assayed. Here, we describe simple experimental procedures to assay endocytosis along the cell cycle with minimal perturbations.


Assuntos
Bioensaio , Ciclo Celular , Endocitose/fisiologia , Bioensaio/métodos , Biomarcadores , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular , Vesículas Revestidas por Clatrina/metabolismo , Imunofluorescência , Microscopia Confocal , Mutação
19.
Methods Mol Biol ; 1847: 37-50, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30129008

RESUMO

Nowadays, live fluorescent microscopes allow us to study the dynamics of cellular processes in living cells with high spatial and temporal resolution. Since the implementation of this methodology to the field of clathrin-mediated endocytosis (CME), this approach has revolutionized our molecular understanding of clathrin-driven cellular uptake. Conventional live cell microscopy approaches are used to determine the precise functions of specific proteins or lipids in orchestrating CME. Here, we will describe, in depth, the procedure to investigate the contribution of membrane tension in regulating clathrin-dependent endocytosis. We will explain two alternative methods to manipulate membrane tension while performing live fluorescence microscopy: cellular swelling through osmotic shock and cellular stretching of cells grown on stretchable silicon inserts.


Assuntos
Membrana Celular/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Clatrina/metabolismo , Endocitose/fisiologia , Animais , Linhagem Celular Tumoral , Membrana Celular/química , Fragilidade Osmótica , Pressão Osmótica
20.
Methods Mol Biol ; 1847: 51-64, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30129009

RESUMO

Clathrin-mediated endocytosis (CME) is a universal and evolutionarily conserved process that enables the internalization of numerous cargo proteins, including receptors for nutrients and signaling molecules, as well as synaptic vesicle reformation. Multiple genetic and chemical approaches have been developed to interfere with this process. However, many of these tools do not selectively block CME, for example by targeting components shared with clathrin-independent endocytosis pathways or by interfering with other cellular processes that indirectly affect CME.Clathrin, via interactions of endocytic proteins with its terminal domain (TD), serves as a central interaction hub for coat assembly in CME. Here, we describe an ELISA-based, high-throughput screening method used to identify small molecules that inhibit these interactions. In addition, we provide protocols for the purification of recombinant protein domains used for screening, e.g., the clathrin TD and the amphiphysin B/C domain. The screen has been applied successfully in the past, and ultimately led to the discovery of the Pitstop® family of inhibitors, but remains in use to evaluate the inhibitory potency of derivatives of these compounds, and to screen for completely novel inhibitor families.


Assuntos
Clatrina/antagonistas & inibidores , Descoberta de Drogas , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Animais , Clatrina/química , Clatrina/genética , Clatrina/isolamento & purificação , Vesículas Revestidas por Clatrina/efeitos dos fármacos , Vesículas Revestidas por Clatrina/metabolismo , Relação Dose-Resposta a Droga , Descoberta de Drogas/métodos , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Humanos , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo
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