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1.
Mol Cell ; 64(4): 790-802, 2016 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-27840028

RESUMO

Recent studies have revealed the importance of Ki-67 and the chromosome periphery in chromosome structure and segregation, but little is known about this elusive chromosome compartment. Here we used correlative light and serial block-face scanning electron microscopy, which we term 3D-CLEM, to model the entire mitotic chromosome complement at ultra-structural resolution. Prophase chromosomes exhibit a highly irregular surface appearance with a volume smaller than metaphase chromosomes. This may be because of the absence of the periphery, which associates with chromosomes only after nucleolar disassembly later in prophase. Indeed, the nucleolar volume almost entirely accounts for the extra volume found in metaphase chromosomes. Analysis of wild-type and Ki-67-depleted chromosomes reveals that the periphery comprises 30%-47% of the entire chromosome volume and more than 33% of the protein mass of isolated mitotic chromosomes determined by quantitative proteomics. Thus, chromatin makes up a surprisingly small percentage of the total mass of metaphase chromosomes.


Assuntos
Cromatina/ultraestrutura , Cromossomos/ultraestrutura , Metáfase , Microscopia Eletrônica de Varredura/métodos , Prófase , Linhagem Celular Transformada , Nucléolo Celular/química , Nucléolo Celular/ultraestrutura , Cromatina/química , Cromossomos/química , Expressão Gênica , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Epitélio Pigmentado da Retina/química , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/ultraestrutura
2.
Biochem J ; 479(18): 1985-1997, 2022 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-36065754

RESUMO

Approximately 15% of all cancer patients harbor mutated KRAS. Direct inhibitors of KRAS have now been generated and are beginning to make progress through clinical trials. These include a suite of inhibitors targeting the KRASG12C mutation commonly found in lung cancer. We investigated emergent resistance to representative examples of different classes of Ras targeted therapies. They all exhibited rapid reactivation of Ras signaling within days of exposure and adaptive responses continued to change over long-term treatment schedules. Whilst the gene signatures were distinct for each inhibitor, they commonly involved up-regulation of upstream nodes promoting mutant and wild-type Ras activation. Experiments to reverse resistance unfortunately revealed frequent desensitization to members of a panel of anti-cancer therapeutics, suggesting that salvage approaches are unlikely to be feasible. Instead, we identified triple inhibitor combinations that resulted in more durable responses to KRAS inhibitors and that may benefit from further pre-clinical evaluation.


Assuntos
Neoplasias Pulmonares , Proteínas Proto-Oncogênicas p21(ras) , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais
3.
PLoS Pathog ; 16(11): e1009016, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33216805

RESUMO

The opportunistic pathogen Streptococcus pneumoniae has dual lifestyles: one of an asymptomatic colonizer in the human nasopharynx and the other of a deadly pathogen invading sterile host compartments. The latter triggers an overwhelming inflammatory response, partly driven via pore forming activity of the cholesterol dependent cytolysin (CDC), pneumolysin. Although pneumolysin-induced inflammation drives person-to-person transmission from nasopharynx, the primary reservoir for pneumococcus, it also contributes to high mortality rates, creating a bottleneck that hampers widespread bacterial dissemination, thus acting as a double-edged sword. Serotype 1 ST306, a widespread pneumococcal clone, harbours a non-hemolytic variant of pneumolysin (Ply-NH). Performing crystal structure analysis of Ply-NH, we identified Y150H and T172I as key substitutions responsible for loss of its pore forming activity. We uncovered a novel inter-molecular cation-π interaction, governing formation of the transmembrane ß-hairpins (TMH) in the pore state of Ply, which can be extended to other CDCs. H150 in Ply-NH disrupts this interaction, while I172 provides structural rigidity to domain-3, through hydrophobic interactions, inhibiting TMH formation. Loss of pore forming activity enabled improved cellular invasion and autophagy evasion, promoting an atypical intracellular lifestyle for pneumococcus, a finding that was corroborated in in vivo infection models. Attenuation of inflammatory responses and tissue damage promoted tolerance of Ply-NH-expressing pneumococcus in the lower respiratory tract. Adoption of this altered lifestyle may be necessary for ST306 due to its limited nasopharyngeal carriage, with Ply-NH, aided partly by loss of its pore forming ability, facilitating a benign association of SPN in an alternative, intracellular host niche.


Assuntos
Adaptação Fisiológica , Inflamação/microbiologia , Mutação com Perda de Função , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/fisiologia , Estreptolisinas/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/microbiologia , Colesterol/metabolismo , Citoplasma/microbiologia , Feminino , Humanos , Camundongos , Modelos Estruturais , Perforina/genética , Perforina/metabolismo , Alinhamento de Sequência , Streptococcus pneumoniae/genética , Estreptolisinas/genética
4.
Small ; 16(46): e2003793, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33103323

RESUMO

The generation of effective and safe nanoagents for biological applications requires their physicochemical characteristics to be tunable, and their cellular interactions to be well characterized. Here, the controlled synthesis is developed for preparing high-aspect ratio gold nanotubes (AuNTs) with tailorable wall thickness, microstructure, composition, and optical characteristics. The modulation of optical properties generates AuNTs with strong near infrared absorption. Surface modification enhances dispersibility of AuNTs in aqueous media and results in low cytotoxicity. The uptake and trafficking of these AuNTs by primary mesothelioma cells demonstrate their accumulation in a perinuclear distribution where they are confined initially in membrane-bound vesicles from which they ultimately escape to the cytosol. This represents the first study of the cellular interactions of high-aspect ratio 1D metal nanomaterials and will facilitate the rational design of plasmonic nanoconstructs as cytosolic nanoagents for potential diagnosis and therapeutic applications.


Assuntos
Mesotelioma , Nanoestruturas , Nanotubos , Citosol , Ouro , Humanos , Mesotelioma/tratamento farmacológico
5.
J Cell Sci ; 130(10): 1845-1855, 2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28389579

RESUMO

Serial block face scanning electron microscopy (SBF-SEM) is a powerful method to analyze cells in 3D. Here, working at the resolution limit of the method, we describe a correlative light-SBF-SEM workflow to resolve microtubules of the mitotic spindle in human cells. We present four examples of uses for this workflow that are not practical by light microscopy and/or transmission electron microscopy. First, distinguishing closely associated microtubules within K-fibers; second, resolving bridging fibers in the mitotic spindle; third, visualizing membranes in mitotic cells, relative to the spindle apparatus; and fourth, volumetric analysis of kinetochores. Our workflow also includes new computational tools for exploring the spatial arrangement of microtubules within the mitotic spindle. We use these tools to show that microtubule order in mitotic spindles is sensitive to the level of TACC3 on the spindle.


Assuntos
Processamento de Imagem Assistida por Computador , Microscopia Eletrônica de Varredura/métodos , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Células HeLa , Humanos , Imageamento Tridimensional , Cinetocoros/metabolismo , Cinetocoros/ultraestrutura , Modelos Biológicos , Modelos Moleculares , Fuso Acromático/ultraestrutura
6.
Biochem Soc Trans ; 47(5): 1209-1222, 2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31506331

RESUMO

Due to cell-cycle dysregulation, many cancer cells contain more than the normal compliment of centrosomes, a state referred to as centrosome amplification (CA). CA can drive oncogenic phenotypes and indeed can cause cancer in flies and mammals. However, cells have to actively manage CA, often by centrosome clustering, in order to divide. Thus, CA is also an Achilles' Heel of cancer cells. In recent years, there have been many important studies identifying proteins required for the management of CA and it has been demonstrated that disruption of some of these proteins can cause cancer-specific inhibition of cell growth. For certain targets therapeutically relevant interventions are being investigated, for example, small molecule inhibitors, although none are yet in clinical trials. As the field is now poised to move towards clinically relevant interventions, it is opportune to summarise the key work in targeting CA thus far, with particular emphasis on recent developments where small molecule or other strategies have been proposed. We also highlight the relatively unexplored paradigm of reversing CA, and thus its oncogenic effects, for therapeutic gain.


Assuntos
Centrossomo , Neoplasias/genética , Animais , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Oncogenes , Proteínas/metabolismo
7.
Biochem Soc Trans ; 46(5): 1325-1332, 2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30287508

RESUMO

RAS proteins are small GTPases that regulate signalling networks that control cellular proliferation and survival. They are frequently mutated in cancer and a commonly occurring group of developmental disorders called RASopathies. We discuss recent findings describing how RAS isoforms and different activating mutations differentially contribute to normal and disease-associated biology and the mechanisms that have been proposed to underpin this.


Assuntos
Neoplasias/metabolismo , Transdução de Sinais , Proteínas ras/genética , Proteínas ras/metabolismo , Animais , Membrana Celular/metabolismo , Proliferação de Células , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/metabolismo , Humanos , Mutação , Neoplasias/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica
8.
Histochem Cell Biol ; 148(1): 3-12, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28283744

RESUMO

Due to the physical and physiological properties of the blood-brain barrier (BBB), the transport of neurotherapeutics from blood to brain is still a pharmaceutical challenge. We previously conducted a series of experiments to explore the potential of the anti-transferrin receptor 8D3 monoclonal antibody (mAb) to transport neurotherapeutics across the BBB. In that study, gold nanoparticles (AuNPs) were coated with the 8D3 antibody and administered intravenously to mice. Transmission electron microscopy was used and a two-dimensional (2D) image analysis was performed to detect the AuNPs in the brain capillary endothelial cells (BCECs) and brain parenchyma. In the present work, we determined that serial block-face scanning electron microscopy (SBF-SEM) is a useful tool to study the transcytosis of these AuNPs across the BBB in three dimensions and we, therefore, applied it to gain more knowledge of their transcellular trafficking. The resulting 3D reconstructions provided additional information on the endocytic vesicles containing AuNPs and the endosomal processing that occurs inside BCECs. The passage from 2D to 3D analysis reinforced the trafficking model proposed in the 2D study, and revealed that the vesicles containing AuNPs are significantly larger and more complex than described in our 2D study. We also discuss tradeoffs of using this technique for our application, and conclude that together with other volume electron microscopy imaging techniques, SBF-SEM is a powerful approach that is worth of considering for studies of drug transport across the BBB.


Assuntos
Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/ultraestrutura , Ouro/farmacocinética , Nanopartículas Metálicas/análise , Microscopia Eletrônica de Varredura , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/farmacocinética , Ouro/administração & dosagem , Injeções Intravenosas , Masculino , Nanopartículas Metálicas/administração & dosagem , Camundongos , Camundongos Endogâmicos ICR
9.
Biochem Soc Trans ; 45(5): 1125-1136, 2017 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-28900014

RESUMO

Post-translational modification of proteins by ubiquitylation is increasingly recognised as a highly complex code that contributes to the regulation of diverse cellular processes. In humans, a family of almost 100 deubiquitylase enzymes (DUBs) are assigned to six subfamilies and many of these DUBs can remove ubiquitin from proteins to reverse signals. Roles for individual DUBs have been delineated within specific cellular processes, including many that are dysregulated in diseases, particularly cancer. As potentially druggable enzymes, disease-associated DUBs are of increasing interest as pharmaceutical targets. The biology, structure and regulation of DUBs have been extensively reviewed elsewhere, so here we focus specifically on roles of DUBs in regulating cell cycle processes in mammalian cells. Over a quarter of all DUBs, representing four different families, have been shown to play roles either in the unidirectional progression of the cell cycle through specific checkpoints, or in the DNA damage response and repair pathways. We catalogue these roles and discuss specific examples. Centrosomes are the major microtubule nucleating centres within a cell and play a key role in forming the bipolar mitotic spindle required to accurately divide genetic material between daughter cells during cell division. To enable this mitotic role, centrosomes undergo a complex replication cycle that is intimately linked to the cell division cycle. Here, we also catalogue and discuss DUBs that have been linked to centrosome replication or function, including centrosome clustering, a mitotic survival strategy unique to cancer cells with supernumerary centrosomes.


Assuntos
Centrossomo/metabolismo , Enzimas Desubiquitinantes/química , Enzimas Desubiquitinantes/metabolismo , Animais , Ciclo Celular , Humanos , Família Multigênica , Neoplasias/enzimologia , Processamento de Proteína Pós-Traducional , Ubiquitinação
10.
FASEB J ; 30(12): 4083-4097, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27601439

RESUMO

α1-Antitrypsin is a serine protease inhibitor produced in the liver that is responsible for the regulation of pulmonary inflammation. The commonest pathogenic gene mutation yields Z-α1-antitrypsin, which has a propensity to self-associate forming polymers that become trapped in inclusions of endoplasmic reticulum (ER). It is unclear whether these inclusions are connected to the main ER network in Z-α1-antitrypsin-expressing cells. Using live cell imaging, we found that despite inclusions containing an immobile matrix of polymeric α1-antitrypsin, small ER resident proteins can diffuse freely within them. Inclusions have many features to suggest they represent fragmented ER, and some are physically separated from the tubular ER network, yet we observed cargo to be transported between them in a cytosol-dependent fashion that is sensitive to N-ethylmaleimide and dependent on Sar1 and sec22B. We conclude that protein recycling occurs between ER inclusions despite their physical separation.-Dickens, J. A., Ordóñez, A., Chambers, J. E., Beckett, A. J., Patel, V., Malzer, E., Dominicus, C. S., Bradley, J., Peden, A. A., Prior, I. A., Lomas, D. A., Marciniak, S. J. The endoplasmic reticulum remains functionally connected by vesicular transport after its fragmentation in cells expressing Z-α1-antitrypsin.


Assuntos
Transporte Biológico/fisiologia , Retículo Endoplasmático/metabolismo , Fígado/metabolismo , alfa 1-Antitripsina/metabolismo , Animais , Transporte Biológico/genética , Células CHO , Células Cultivadas , Cricetulus , Mutação/genética , alfa 1-Antitripsina/genética
11.
Cell Commun Signal ; 14: 5, 2016 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-26861207

RESUMO

BACKGROUND: Growth factors induce a characteristically short-lived Ras activation in cells emerging from quiescence. Extensive work has shown that transient as opposed to sustained Ras activation is critical for the induction of mitogenic programs. Mitogen-induced accumulation of active Ras-GTP results from increased nucleotide exchange driven by the nucleotide exchange factor Sos. In contrast, the mechanism accounting for signal termination and prompt restoration of basal Ras-GTP levels is unclear, but has been inferred to involve feedback inhibition of Sos. Remarkably, how GTP-hydrolase activating proteins (GAPs) participate in controlling the rise and fall of Ras-GTP levels is unknown. RESULTS: Monitoring nucleotide exchange of Ras in permeabilized cells we find, unexpectedly, that the decline of growth factor-induced Ras-GTP levels proceeds in the presence of unabated high nucleotide exchange, pointing to GAP activation as a major mechanism of signal termination. Experiments with non-hydrolysable GTP analogues and mathematical modeling confirmed and rationalized the presence of high GAP activity as Ras-GTP levels decline in a background of high nucleotide exchange. Using pharmacological and genetic approaches we document a raised activity of the neurofibromatosis type I tumor suppressor Ras-GAP neurofibromin and an involvement of Rsk1 and Rsk2 in the down-regulation of Ras-GTP levels. CONCLUSIONS: Our findings show that, in addition to feedback inhibition of Sos, feedback stimulation of the RasGAP neurofibromin enforces termination of the Ras signal in the context of growth-factor signaling. These findings ascribe a precise role to neurofibromin in growth factor-dependent control of Ras activity and illustrate how, by engaging Ras-GAP activity, mitogen-challenged cells play safe to ensure a timely termination of the Ras signal irrespectively of the reigning rate of nucleotide exchange.


Assuntos
Ativação Enzimática , Fator de Crescimento Epidérmico/metabolismo , Neurofibromina 1/metabolismo , Transdução de Sinais , Proteínas ras/metabolismo , Animais , Linhagem Celular , Guanosina Trifosfato/metabolismo , Células HEK293 , Células HeLa , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Fosforilação , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Proteína Son Of Sevenless de Drosófila/metabolismo
12.
Biochem J ; 465(3): 405-12, 2015 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-25370603

RESUMO

The inducers of acute pancreatitis trigger a prolonged increase in the cytosolic Ca(2+) concentration ([Ca(2+)]c), which is responsible for the damage to and eventual death of pancreatic acinar cells. Vacuolization is an important indicator of pancreatic acinar cell damage. Furthermore, activation of trypsinogen occurs in the endocytic vacuoles; therefore the vacuoles can be considered as 'initiating' organelles in the development of the cell injury. In the present study, we investigated the relationship between the formation of endocytic vacuoles and Ca(2+) influx developed in response to the inducers of acute pancreatitis [bile acid taurolithocholic acid 3-sulfate (TLC-S) and supramaximal concentration of cholecystokinin-8 (CCK)]. We found that the inhibitor of STIM (stromal interaction molecule)/Orai channels, GSK-7975A, effectively suppressed both the Ca(2+) influx (stimulated by inducers of pancreatitis) and the formation of endocytic vacuoles. Cell death induced by TLC-S or CCK was also inhibited by GSK-7975A. We documented the formation of endocytic vacuoles in response to store-operated Ca(2+) entry (SOCE) induced by thapsigargin [TG; inhibitor of sarcoplasmic/endoplasmic reticulum (ER) Ca(2+) pumps] and observed strong inhibition of TG-induced vacuole formation by GSK-7975A. Finally, we found that structurally-unrelated inhibitors of calpain suppress formation of endocytic vacuoles, suggesting that this Ca2+-dependent protease is a mediator between Ca(2+) elevation and endocytic vacuole formation.


Assuntos
Células Acinares/metabolismo , Cálcio/metabolismo , Pâncreas/citologia , Pâncreas/metabolismo , Vesículas Transportadoras/metabolismo , Vacúolos/metabolismo , Animais , Células Cultivadas , Camundongos
13.
J Proteome Res ; 14(3): 1535-46, 2015 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-25599653

RESUMO

Oncogenic mutations of Ras at codons 12, 13, or 61, that render the protein constitutively active, are found in ∼ 16% of all cancer cases. Among the three major Ras isoforms, KRAS is the most frequently mutated isoform in cancer. Each Ras isoform and tumor type displays a distinct pattern of codon-specific mutations. In colon cancer, KRAS is typically mutated at codon 12, but a significant fraction of patients have mutations at codon 13. Clinical data suggest different outcomes and responsiveness to treatment between these two groups. To investigate the differential effects upon cell status associated with KRAS mutations we performed a quantitative analysis of the proteome and phosphoproteome of isogenic SW48 colon cancer cell lines in which one allele of the endogenous gene has been edited to harbor specific KRAS mutations (G12V, G12D, or G13D). Each mutation generates a distinct signature, with the most variability seen between G13D and the codon 12 KRAS mutants. One notable example of specific up-regulation in KRAS codon 12 mutant SW48 cells is provided by the short form of the colon cancer stem cell marker doublecortin-like Kinase 1 (DCLK1) that can be reversed by suppression of KRAS.


Assuntos
Neoplasias Colorretais/patologia , Genes ras , Mutação , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Quinases Semelhantes a Duplacortina , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica , Regulação para Cima
14.
EMBO J ; 30(5): 906-19, 2011 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-21297582

RESUMO

Kinetochore fibres (K-fibres) of the spindle apparatus move chromosomes during mitosis. These fibres are discrete bundles of parallel microtubules (MTs) that are crosslinked by inter-MT 'bridges' that are thought to improve fibre stability during chromosomal movement. The identity of these bridges is unknown. Clathrin is a multimeric protein that has been shown to stabilise K-fibres during early mitosis by a mechanism independent of its role in membrane trafficking. In this study, we show that clathrin at the mitotic spindle is in a transforming acidic colied-coil protein 3 (TACC3)/colonic, hepatic tumour overexpressed gene (ch-TOG)/clathrin complex. The complex is anchored to the spindle by TACC3 and ch-TOG. Ultrastructural analysis of clathrin-depleted K-fibres revealed a selective loss of a population of short inter-MT bridges and a general loss of MTs. A similar loss of short inter-MT bridges was observed in TACC3-depleted K-fibres. Finally, immunogold labelling confirmed that inter-MT bridges in K-fibres contain clathrin. Our results suggest that the TACC3/ch-TOG/clathrin complex is an inter-MT bridge that stabilises K-fibres by physical crosslinking and by reducing rates of MT catastrophe.


Assuntos
Clatrina/metabolismo , Cinetocoros/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Aurora Quinases , Clatrina/genética , Células HeLa , Humanos , Immunoblotting , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/genética , Mitose , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fuso Acromático/genética
15.
J Cell Sci ; 126(Pt 9): 2102-13, 2013 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-23532825

RESUMO

Microtubule-associated proteins of the mitotic spindle are thought to be important for the initial assembly and the maintenance of spindle structure and function. However, distinguishing assembly and maintenance roles for a given protein is difficult. Most experimental methods for protein inactivation are slow and therefore affect both assembly and maintenance. Here, we have used 'knocksideways' to rapidly (∼5 minutes) and specifically remove TACC3-ch-TOG-clathrin non-motor complexes from kinetochore fibers (K-fibers). This method allows the complex to be inactivated at defined stages of mitosis. Removal of TACC3-ch-TOG-clathrin after nuclear envelope breakdown caused severe delays in chromosome alignment. Inactivation at metaphase, following a normal prometaphase, significantly delayed progression to anaphase. In these cells, K-fiber tension was reduced and the spindle checkpoint was not satisfied. Surprisingly, there was no significant loss of K-fiber microtubules, even after prolonged removal. TACC3-ch-TOG-clathrin removal during metaphase also resulted in a decrease in spindle length and significant alteration in kinetochore dynamics. Our results indicate that TACC3-ch-TOG-clathrin complexes are important for the maintenance of spindle structure and function as well as for initial spindle assembly.


Assuntos
Cromossomos Humanos/metabolismo , Cinetocoros/metabolismo , Metáfase/fisiologia , Microtúbulos/metabolismo , Complexos Multiproteicos/metabolismo , Fuso Acromático/metabolismo , Cromossomos Humanos/genética , Clatrina/genética , Clatrina/metabolismo , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/genética , Complexos Multiproteicos/genética , Fuso Acromático/genética
16.
PLoS Biol ; 10(7): e1001361, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22815649

RESUMO

The heparan sulfate (HS) chains of proteoglycans are a key regulatory component of the extracellular matrices of animal cells, including the pericellular matrix around the plasma membrane. In these matrices they regulate transport, gradient formation, and effector functions of over 400 proteins central to cell communication. HS from different matrices differs in its selectivity for its protein partners. However, there has been no direct test of how HS in the matrix regulates the transport of its partner proteins. We address this issue by single molecule imaging and tracking in fibroblast pericellular matrix of fibroblast growth factor 2 (FGF2), stoichiometrically labelled with small gold nanoparticles. Transmission electron microscopy and photothermal heterodyne imaging (PHI) show that the spatial distribution of the HS-binding sites for FGF2 in the pericellular matrix is heterogeneous over length scales ranging from 22 nm to several µm. Tracking of individual FGF2 by PHI in the pericellular matrix of living cells demonstrates that they undergo five distinct types of motion. They spend much of their time in confined motion (∼110 nm diameter), but they are not trapped and can escape by simple diffusion, which may be slow, fast, or directed. These substantial translocations (µm) cover distances far greater than the length of a single HS chain. Similar molecular motion persists in fixed cells, where the movement of membrane PGs is impeded. We conclude that FGF2 moves within the pericellular matrix by translocating from one HS-binding site to another. The binding sites on HS chains form non-random, heterogeneous networks. These promote FGF2 confinement or substantial translocation depending on their spatial organisation. We propose that this spatial organisation, coupled to the relative selectivity and the availability of HS-binding sites, determines the transport of FGF2 in matrices. Similar mechanisms are likely to underpin the movement of many other HS-binding effectors.


Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Heparitina Sulfato/metabolismo , Sítios de Ligação , Fibroblastos/metabolismo , Microscopia Eletrônica de Transmissão , Nanopartículas , Fosforilação , Transporte Proteico
17.
Semin Cell Dev Biol ; 23(2): 145-53, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21924373

RESUMO

Ras proteins are proto-oncogenes that are frequently mutated in human cancers. Three closely related isoforms, HRAS, KRAS and NRAS, are expressed in all cells and have overlapping but distinctive functions. Recent work has revealed how differences between the Ras isoforms in their trafficking, localization and protein-membrane orientation enable signalling specificity to be determined. We review the various strategies used to characterize compartmentalized Ras localization and signalling. Localization is an important contextual modifier of signalling networks and insights from the Ras system are of widespread relevance for researchers interested in signalling initiated from membranes.


Assuntos
Compartimento Celular , Transdução de Sinais , Proteínas ras/química , Sequência de Aminoácidos , Animais , Membrana Celular/química , Retículo Endoplasmático , Endossomos/química , Ativação Enzimática , Evolução Molecular , Complexo de Golgi/química , Humanos , Dados de Sequência Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas ras/genética
18.
Biochem Soc Trans ; 42(4): 742-6, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25109951

RESUMO

RAS proteins are key signalling hubs that are oncogenically mutated in 30% of all cancer cases. Three genes encode almost identical isoforms that are ubiquitously expressed, but are not functionally redundant. The network responses associated with each isoform and individual oncogenic mutations remain to be fully characterized. In the present article, we review recent data defining the differences between the RAS isoforms and their most commonly mutated codons and discuss the underlying mechanisms.


Assuntos
Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Códon/genética , Humanos , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Isoformas de Proteínas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética
19.
Biochem J ; 454(2): 323-32, 2013 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-23758196

RESUMO

Ras GTPases undergo post-translational modifications that govern their subcellular trafficking and localization. In particular, palmitoylation of the Golgi tags N-Ras and H-Ras for exocytotic transport and residency at the PM (plasma membrane). Following depalmitoylation, PM-Ras redistributes to all subcellular membranes causing an accumulation of palmitate-free Ras at endomembranes, including the Golgi and endoplasmic reticulum. Palmitoylation is unanimously regarded as a critical modification at the crossroads of Ras activity and trafficking control, but its precise relevance to native wild-type Ras function in growth factor signalling is unknown. We show in the present study by use of palmitoylation-deficient N-Ras mutants and via the analysis of palmitate content of agonist-activated GTP-loaded N-Ras that only palmitoylated N-Ras becomes activated by agonists. In line with an essential role of palmitoylation in Ras activation, dominant-negative RasS17N loses its blocking potency if rendered devoid of palmitoylation. Live-cell Ras-GTP imaging shows that N-Ras activation proceeds only at the PM, consistent with activated N-Ras-GTP being palmitoylated. Finally, palmitoylation-deficient N-Ras does not sustain EGF (epidermal growth factor) or serum-elicited mitogenic signalling, confirming that palmitoylation is essential for signal transduction by N-Ras. These findings document that N-Ras activation proceeds at the PM and suggest that depalmitoylation, by removing Ras from the PM, may contribute to the shutdown of Ras signalling.


Assuntos
Membrana Celular/metabolismo , Regulação para Baixo , Fator de Crescimento Epidérmico/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana/metabolismo , Ácido Palmítico/metabolismo , Transdução de Sinais , Proteínas ras/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Embrião de Mamíferos/citologia , Ativação Enzimática , GTP Fosfo-Hidrolases/genética , Humanos , Lipoilação , Proteínas de Membrana/agonistas , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas Mutantes/agonistas , Proteínas Mutantes/metabolismo , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/metabolismo , Proteínas ras/genética
20.
J Cell Biol ; 223(11)2024 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-39186086

RESUMO

Chromosome compaction is a key feature of mitosis and critical for accurate chromosome segregation. However, a precise quantitative analysis of chromosome geometry during mitotic progression is lacking. Here, we use volume electron microscopy to map, with nanometer precision, chromosomes from prometaphase through telophase in human RPE1 cells. During prometaphase, chromosomes acquire a smoother surface, their arms shorten, and the primary centromeric constriction is formed. The chromatin is progressively compacted, ultimately reaching a remarkable nucleosome concentration of over 750 µM in late prometaphase that remains relatively constant during metaphase and early anaphase. Surprisingly, chromosomes then increase their volume in late anaphase prior to deposition of the nuclear envelope. The plateau of total chromosome volume from late prometaphase through early anaphase described here is consistent with proposals that the final stages of chromatin condensation in mitosis involve a limit density, such as might be expected for a process involving phase separation.


Assuntos
Anáfase , Nucleossomos , Prometáfase , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Nucleossomos/genética , Humanos , Linhagem Celular , Cromossomos Humanos/metabolismo , Cromossomos Humanos/genética , Cromatina/metabolismo , Cromatina/genética , Mitose , Centrômero/metabolismo , Centrômero/ultraestrutura , Centrômero/genética
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