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1.
J Biol Chem ; 294(36): 13396-13410, 2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31324722

RESUMO

Cystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR), a chloride channel normally expressed at the surface of epithelial cells. The most frequent mutation, resulting in Phe-508 deletion, causes CFTR misfolding and its premature degradation. Low temperature or pharmacological correctors can partly rescue the Phe508del-CFTR processing defect and enhance trafficking of this channel variant to the plasma membrane (PM). Nevertheless, the rescued channels have an increased endocytosis rate, being quickly removed from the PM by the peripheral protein quality-control pathway. We previously reported that rescued Phe508del-CFTR (rPhe508del) can be retained at the cell surface by stimulating signaling pathways that coax the adaptor molecule ezrin (EZR) to tether rPhe508del-Na+/H+-exchange regulatory factor-1 complexes to the actin cytoskeleton, thereby averting the rapid internalization of this channel variant. However, the molecular basis for why rPhe508del fails to recruit active EZR to the PM remains elusive. Here, using a proteomics approach, we characterized and compared the core components of wt-CFTR- or rPhe508del-containing macromolecular complexes at the surface of human bronchial epithelial cells. We identified calpain 1 (CAPN1) as an exclusive rPhe508del interactor that prevents active EZR recruitment, impairs rPhe508del anchoring to actin, and reduces its stability in the PM. We show that either CAPN1 down-regulation or its chemical inhibition dramatically improves the functional rescue of Phe508del-CFTR in airway cells. These observations suggest that CAPN1 constitutes an appealing target for pharmacological intervention, as part of CF combination therapies restoring Phe508del-CFTR function.

3.
J Cell Sci ; 132(2)2019 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-30630895

RESUMO

Skeletal muscle (SKM) differentiation is a highly regulated process leading to the formation of specialised cells with reorganised compartments and organelles, such as those of the early secretory pathway. During SKM differentiation the Golgi complex (GC) redistributes close to the nuclear envelope and in small distinct peripheral structures distributed throughout the myotube. Concurrently, GC elements closely associate with endoplasmic reticulum-exit sites (ERES). The mechanisms underlying this reorganisation and its relevance for SKM differentiation are poorly understood. Here, we show, by time-lapse imaging studies, that the changes in GC organisation involve GC fragmentation and redistribution of ERES with the formation of tightly associated GC-ERES units. We show that knockdown of GM130 (also known as GOLGA2) or p115 (also known as USO1), two regulators of the early secretory pathway, impairs GC and ERES reorganisation. This in turn results in inhibition of myotube fusion and M-cadherin (also known as CDH15) transport to the sarcolemma. Taken together, our data suggest that the correct reorganisation of the early secretory pathway components plays an important role in SKM differentiation and, thus, associated pathologies.


Assuntos
Autoantígenos/metabolismo , Diferenciação Celular , Proteínas da Matriz do Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Músculo Esquelético/metabolismo , Sarcolema/metabolismo , Via Secretória , Proteínas de Transporte Vesicular/metabolismo , Animais , Autoantígenos/genética , Linhagem Celular , Proteínas da Matriz do Complexo de Golgi/genética , Proteínas de Membrana/genética , Camundongos , Músculo Esquelético/citologia , Sarcolema/genética , Proteínas de Transporte Vesicular/genética
4.
Biochim Biophys Acta Mol Cell Res ; 1865(2): 421-431, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29154949

RESUMO

An attractive possibility to treat Cystic Fibrosis (CF), a severe condition caused by dysfunctional CFTR, an epithelial anion channel, is through the activation of alternative (non-CFTR) anion channels. Anoctamin 1 (ANO1) was demonstrated to be a Ca2+-activated chloride channel (CaCC) and thus of high potential to replace CFTR. Despite that ANO1 is expressed in human lung CF tissue, it is present at the cell surface at very low levels. In addition, little is known about regulation of ANO1 traffic, namely which factors promote its plasma membrane (PM) localization. Here, we generated a novel cellular model, expressing an inducible 3HA-ANO1-eGFP construct, and validated its usage as a microscopy tool to monitor for ANO1 traffic. We demonstrate the robustness and specificity of this cell-based assay, by the identification of siRNAs acting both as ANO1 traffic enhancer and inhibitor, targeting respectively COPB1 and ESYT1 (extended synaptotagmin-1), the latter involved in coupling of the endoplasmic reticulum to the PM at specific microdomains. We further show that knockdown of ESYT1 (and family members ESYT2 and ESYT3) significantly decreased ANO1 current density. This ANO1 cell-based assay constitutes an important tool to be further used in high-throughput screens and drug discovery of high relevance for CF and cancer.


Assuntos
Anoctamina-1/metabolismo , Fibrose Cística/metabolismo , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Sinaptotagminas/metabolismo , Anoctamina-1/genética , Linhagem Celular , Fibrose Cística/genética , Fibrose Cística/patologia , Humanos , Proteínas de Neoplasias/genética , Transporte Proteico , Sinaptotagminas/genética
5.
Elife ; 62017 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-29087936

RESUMO

We present a 3D-fluorescence imaging and classification tool for high throughput analysis of microbial eukaryotes in environmental samples. It entails high-content feature extraction that permits accurate automated taxonomic classification and quantitative data about organism ultrastructures and interactions. Using plankton samples from the Tara Oceans expeditions, we validate its applicability to taxonomic profiling and ecosystem analyses, and discuss its potential for future integration of eukaryotic cell biology into evolutionary and ecological studies.


Assuntos
Ecossistema , Microbiologia Ambiental , Eucariotos/citologia , Eucariotos/fisiologia , Imageamento Tridimensional/métodos , Imagem Óptica/métodos , Eucariotos/classificação
6.
Methods Mol Biol ; 1563: 107-125, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28324605

RESUMO

Laser-mediated dissection methods have been used for many years to micro-irradiate biological samples, but recent technological progress has rendered this technique more precise, powerful, and easy to use. Today pulsed lasers can be operated with diffraction limited, sub-micrometer precision to ablate intracellular structures. Here, we discuss laser nanosurgery setups and the instrumentation in our laboratory. We describe how to use this technique to ablate cytoskeletal elements in living cells. We also show how this technique can be used in multicellular organisms, to micropuncture and/or ablate cells of interest and finally how to monitor a successful laser nanosurgery.


Assuntos
Técnicas Citológicas/métodos , Terapia a Laser/métodos , Microcirurgia/métodos , Nanotecnologia/métodos , Actinas/metabolismo , Animais , Biomarcadores , Calibragem , Técnicas Citológicas/instrumentação , Células HeLa , Humanos , Terapia a Laser/instrumentação , Microscopia/instrumentação , Microscopia/métodos , Microcirurgia/instrumentação , Nanotecnologia/instrumentação , Peixe-Zebra
7.
Nat Commun ; 7: 13887, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-27976684

RESUMO

Genome integrity relies on precise coordination between DNA replication and chromosome segregation. Whereas replication stress attracted much attention, the consequences of mitotic perturbations for genome integrity are less understood. Here, we knockdown 47 validated mitotic regulators to show that a broad spectrum of mitotic errors correlates with increased DNA breakage in daughter cells. Unexpectedly, we find that only a subset of these correlations are functionally linked. We identify the genuine mitosis-born DNA damage events and sub-classify them according to penetrance of the observed phenotypes. To demonstrate the potential of this resource, we show that DNA breakage after cytokinesis failure is preceded by replication stress, which mounts during consecutive cell cycles and coincides with decreased proliferation. Together, our results provide a resource to gauge the magnitude and dynamics of DNA breakage associated with mitotic aberrations and suggest that replication stress might limit propagation of cells with abnormal karyotypes.


Assuntos
Ciclo Celular , Proliferação de Células , Dano ao DNA/genética , Mitose/genética , Linhagem Celular Tumoral , Citocinese/genética , Quebras de DNA , Técnicas de Silenciamento de Genes , Humanos , Processamento de Imagem Assistida por Computador , Microscopia Confocal , Fenótipo , Imagem com Lapso de Tempo
8.
Nat Biotechnol ; 34(12): 1246-1247, 2016 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-27926712

Assuntos
Microscopia
9.
J Cell Biol ; 215(4): 543-558, 2016 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-27872256

RESUMO

Stimulation of cells with epidermal growth factor (EGF) induces internalization and partial degradation of the EGF receptor (EGFR) by the endo-lysosomal pathway. For continuous cell functioning, EGFR plasma membrane levels are maintained by transporting newly synthesized EGFRs to the cell surface. The regulation of this process is largely unknown. In this study, we find that EGF stimulation specifically increases the transport efficiency of newly synthesized EGFRs from the endoplasmic reticulum to the plasma membrane. This coincides with an up-regulation of the inner coat protein complex II (COPII) components SEC23B, SEC24B, and SEC24D, which we show to be specifically required for EGFR transport. Up-regulation of these COPII components requires the transcriptional regulator RNF11, which localizes to early endosomes and appears additionally in the cell nucleus upon continuous EGF stimulation. Collectively, our work identifies a new regulatory mechanism that integrates the degradation and transport of EGFR in order to maintain its physiological levels at the plasma membrane.


Assuntos
Proteínas de Transporte/metabolismo , Endossomos/metabolismo , Receptores ErbB/metabolismo , Proteólise , Transcrição Genética , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/efeitos dos fármacos , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Endossomos/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Transporte Proteico/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transcrição Genética/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
11.
J Cell Sci ; 128(22): 4160-70, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26459637

RESUMO

Newly synthesized proteins are sorted into COPII-coated transport carriers at the endoplasmic reticulum (ER). Assembly of the COPII coat complex, which occurs at ER exit sites (ERES), is initiated by membrane association and GTP loading of SAR1, followed by the recruitment of the SEC23-SEC24 and SEC13-SEC31 subcomplexes. Both of these two subcomplexes stimulate GTP hydrolysis and coat disassembly. This inherent disassembly capacity of COPII complexes needs to be regulated to allow sufficient time for cargo sorting and transport carrier formation. By performing fluorescence recovery after photobleaching (FRAP) and mathematical modeling, we show that p150(glued) (also known as DCTN1), a component of the dynactin complex, stabilizes the COPII pre-budding complex on ER membranes in a microtubule-independent manner. Concentration of the secretory marker ts-O45-G at ERES is reduced in the presence of a C-terminal p150(glued) fragment that prevents binding of endogenous p150(glued) to SEC23. A similar cargo reduction is observed upon p150(glued) knockdown. Taken together, our data suggest that cargo concentration at ERES is regulated by p150(glued) to coordinate protein sorting and transport carrier formation with the subsequent long-range transport towards the Golgi complex along microtubules.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Complexo Dinactina , Recuperação de Fluorescência Após Fotodegradação , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/genética , Modelos Biológicos , Ligação Proteica
13.
Sci Rep ; 5: 12879, 2015 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-26271723

RESUMO

Contact-dependent intercellular transfer (codeIT) of cellular constituents can have functional consequences for recipient cells, such as enhanced survival and drug resistance. Pathogenic viruses, prions and bacteria can also utilize this mechanism to spread to adjacent cells and potentially evade immune detection. However, little is known about the molecular mechanism underlying this intercellular transfer process. Here, we present a novel microscopy-based screening method to identify regulators and cargo of codeIT. Single donor cells, carrying fluorescently labelled endocytic organelles or proteins, are co-cultured with excess acceptor cells. CodeIT is quantified by confocal microscopy and image analysis in 3D, preserving spatial information. An siRNA-based screening using this method revealed the involvement of several myosins and small GTPases as codeIT regulators. Our data indicates that cellular protrusions and tubular recycling endosomes are important for codeIT. We automated image acquisition and analysis to facilitate large-scale chemical and genetic screening efforts to identify key regulators of codeIT.


Assuntos
Comunicação Celular/fisiologia , Membrana Celular/fisiologia , GTP Fosfo-Hidrolases/metabolismo , Junções Intercelulares/fisiologia , Microscopia Confocal/métodos , Miosinas/metabolismo , Membrana Celular/ultraestrutura , Rastreamento de Células/métodos , Células HeLa , Humanos , Dispositivos Lab-On-A-Chip , Imagem Molecular/métodos
14.
Mol Cell Proteomics ; 14(5): 1385-99, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25759509

RESUMO

Several cytoplasmic proteins that are involved in G protein-coupled receptor signaling cascades are known to translocate to the plasma membrane upon receptor activation, such as beta-arrestin2. Based on this example and in order to identify new cytoplasmic proteins implicated in the ON-and-OFF cycle of G protein-coupled receptor, a live-imaging screen of fluorescently labeled cytoplasmic proteins was performed using translocation criteria. The screening of 193 fluorescently tagged human proteins identified eight proteins that responded to activation of the tachykinin NK2 receptor by a change in their intracellular localization. Previously we have presented the functional characterization of one of these proteins, REDD1, that translocates to the plasma membrane. Here we report the results of the entire screening. The process of cell activation was recorded on videos at different time points and all the videos can be visualized on a dedicated website. The proteins BAIAP3 and BIN1, partially translocated to the plasma membrane upon activation of NK2 receptors. Proteins ARHGAP12 and PKM2 translocated toward membrane blebs. Three proteins that associate with the cytoskeleton were of particular interest : PLEKHH2 rearranged from individual dots located near the cell-substrate adhesion surface into lines of dots. The speriolin-like protein, SPATC1L, redistributed to cell-cell junctions. The Chloride intracellular Channel protein, CLIC2, translocated from actin-enriched plasma membrane bundles to cell-cell junctions upon activation of NK2 receptors. CLIC2, and one of its close paralogs, CLIC4, were further shown to respond with the same translocation pattern to muscarinic M3 and lysophosphatidic LPA receptors. This screen allowed us to identify potential actors in signaling pathways downstream of G protein-coupled receptors and could be scaled-up for high-content screening.


Assuntos
Bioensaio , Imagem Molecular/métodos , Receptores da Neurocinina-2/genética , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transporte Proteico , Receptores da Neurocinina-2/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Hormônios Tireóideos/genética , Hormônios Tireóideos/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
15.
Sci Rep ; 5: 9038, 2015 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-25762484

RESUMO

Plasma membrane proteins are essential molecules in the cell which mediate interactions with the exterior milieu, thus representing key drug targets for present pharma. Not surprisingly, protein traffic disorders include a large range of diseases sharing the common mechanism of failure in the respective protein to reach the plasma membrane. However, specific therapies for these diseases are remarkably lacking. Herein, we report a robust platform for drug discovery applied to a paradigmatic genetic disorder affecting intracellular trafficking - Cystic Fibrosis. This platform includes (i) two original respiratory epithelial cellular models incorporating an inducible double-tagged traffic reporter; (ii) a plasma membrane protein traffic assay for high-throughput microscopy screening; and (iii) open-source image analysis software to quantify plasma membrane protein traffic. By allowing direct scoring of compounds rescuing the basic traffic defect, this platform enables an effective drug development pipeline, which can be promptly adapted to any traffic disorder-associated protein and leverage therapy development efforts.


Assuntos
Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala , Microscopia de Fluorescência , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Expressão Gênica , Biblioteca Gênica , Genes Reporter , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Técnicas de Patch-Clamp , Transporte Proteico , RNA Interferente Pequeno , Bibliotecas de Moléculas Pequenas
16.
Nat Biotechnol ; 33(4): 384-9, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25774713

RESUMO

To understand the function of cellular protein networks, spatial and temporal context is essential. Fluorescence correlation spectroscopy (FCS) is a single-molecule method to study the abundance, mobility and interactions of fluorescence-labeled biomolecules in living cells. However, manual acquisition and analysis procedures have restricted live-cell FCS to short-term experiments of a few proteins. Here, we present high-throughput (HT)-FCS, which automates screening and time-lapse acquisition of FCS data at specific subcellular locations and subsequent data analysis. We demonstrate its utility by studying the dynamics of 53 nuclear proteins. We made 60,000 measurements in 10,000 living human cells, to obtain biophysical parameters that allowed us to classify proteins according to their chromatin binding and complex formation. We also analyzed the cell-cycle-dependent dynamics of the mitotic kinase complex Aurora B/INCENP and showed how a rise in Aurora concentration triggers two-step complex formation. We expect that throughput and robustness will make HT-FCS a broadly applicable technology for characterizing protein network dynamics in cells.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Mapeamento de Interação de Proteínas/métodos , Proteoma/metabolismo , Espectrometria de Fluorescência/métodos , Frações Subcelulares/metabolismo , Imagem Molecular/métodos
17.
PLoS Genet ; 11(2): e1004855, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25647241

RESUMO

A fundamental challenge to contemporary genetics is to distinguish rare missense alleles that disrupt protein functions from the majority of alleles neutral on protein activities. High-throughput experimental tools to securely discriminate between disruptive and non-disruptive missense alleles are currently missing. Here we establish a scalable cell-based strategy to profile the biological effects and likely disease relevance of rare missense variants in vitro. We apply this strategy to systematically characterize missense alleles in the low-density lipoprotein receptor (LDLR) gene identified through exome sequencing of 3,235 individuals and exome-chip profiling of 39,186 individuals. Our strategy reliably identifies disruptive missense alleles, and disruptive-allele carriers have higher plasma LDL-cholesterol (LDL-C). Importantly, considering experimental data refined the risk of rare LDLR allele carriers from 4.5- to 25.3-fold for high LDL-C, and from 2.1- to 20-fold for early-onset myocardial infarction. Our study generates proof-of-concept that systematic functional variant profiling may empower rare variant-association studies by orders of magnitude.


Assuntos
Exoma/genética , Estudos de Associação Genética , Infarto do Miocárdio/genética , Receptores de LDL/genética , Alelos , LDL-Colesterol/sangue , LDL-Colesterol/genética , Heterozigoto , Humanos , Mutação de Sentido Incorreto/genética , Infarto do Miocárdio/sangue , Infarto do Miocárdio/patologia , Fenótipo , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA
18.
J Cell Sci ; 128(7): 1279-93, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25717003

RESUMO

The Golgi is a highly organized and dynamic organelle that receives and distributes material from and to the endoplasmic reticulum (ER) and the endocytic pathway. One open question about Golgi organization is whether it is solely based on ER-to-Golgi transport. Here, we analyzed the kinetics of Golgi breakdown in the absence of COPII-dependent ER export with high temporal and spatial resolution using quantitative fluorescence microscopy. We found that Golgi breakdown occurred in two phases. While Golgi enzymes continuously redistributed to the ER, we consistently observed extensive Golgi fragmentation at the beginning of the breakdown, followed by microtubule-dependent formation of a Golgi remnant structure (phase 1). Further Golgi disintegration occurred less uniformly (phase 2). Remarkably, cisternal Golgi morphology was lost early in phase 1 and Golgi fragments instead corresponded to variably sized vesicle clusters. These breakdown intermediates were devoid of COPI-dependent recycling material, but contained typical 'core' Golgi components. Furthermore, Golgi breakdown intermediates were able to disassemble and reassemble following cell division, indicating that they retained important regulatory capabilities. Taken together, these findings support the view that Golgi self-organization exists independently of ER-to-Golgi transport.


Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/genética , Complexo de Golgi/genética , Células HeLa , Humanos , Transporte Proteico , Proteínas de Transporte Vesicular/genética
19.
J Biol Chem ; 290(6): 3654-65, 2015 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-25533462

RESUMO

Previous studies proposed a role for the Na/K-ATPase in unconventional secretion of fibroblast growth factor 2 (FGF2). This conclusion was based upon pharmacological inhibition of FGF2 secretion in the presence of ouabain. However, neither independent experimental evidence nor a potential mechanism was provided. Based upon an unbiased RNAi screen, we now report the identification of ATP1A1, the α1-chain of the Na/K-ATPase, as a factor required for efficient secretion of FGF2. As opposed to ATP1A1, down-regulation of the ß1- and ß3-chains (ATP1B1 and ATP1B3) of the Na/K-ATPase did not affect FGF2 secretion, suggesting that they are dispensable for this process. These findings indicate that it is not the membrane potential-generating function of the Na/K-ATPase complex but rather a so far unidentified role of potentially unassembled α1-chains that is critical for unconventional secretion of FGF2. Consistently, in the absence of ß-chains, we found a direct interaction between the cytoplasmic domain of ATP1A1 and FGF2 with submicromolar affinity. Based upon these observations, we propose that ATP1A1 is a recruitment factor for FGF2 at the inner leaflet of plasma membranes that may control phosphatidylinositol 4,5-bisphosphate-dependent membrane translocation as part of the unconventional secretory pathway of FGF2.


Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Via Secretória , ATPase Trocadora de Sódio-Potássio/metabolismo , Células HeLa , Humanos , Ligação Proteica , Estrutura Terciária de Proteína , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/genética
20.
J Cell Sci ; 127(Pt 21): 4620-33, 2014 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-25189616

RESUMO

The Golgi complex is the central organelle of the secretory pathway. It undergoes dynamic changes during the cell cycle, but how it acquires and maintains its complex structure is unclear. To address this question, we have used laser nanosurgery to deplete BSC1 cells of the Golgi complex and have monitored its biogenesis by quantitative time-lapse microscopy and correlative electron microscopy. After Golgi depletion, endoplasmic reticulum (ER) export is inhibited and the number of ER exit sites (ERES) is reduced and does not increase for several hours. Occasional fusion of small post-ER carriers to form the first larger structures triggers a rapid and drastic growth of Golgi precursors, due to the capacity of these structures to attract more carriers by microtubule nucleation and to stimulate ERES biogenesis. Increasing the chances of post-ER carrier fusion close to ERES by depolymerizing microtubules results in the acceleration of Golgi and ERES biogenesis. Taken together, on the basis of our results, we propose a self-organizing principle of the early secretory pathway that integrates Golgi biogenesis, ERES biogenesis and the organization of the microtubule network by positive-feedback loops.


Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Microtúbulos/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/ultraestrutura , Microscopia Eletrônica , Microtúbulos/ultraestrutura , Imagem com Lapso de Tempo
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