Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 28
Filtrar
1.
Cell ; 166(3): 664-678, 2016 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-27397507

RESUMO

Nuclear pore complexes (NPCs) span the nuclear envelope (NE) and mediate nucleocytoplasmic transport. In metazoan oocytes and early embryos, NPCs reside not only within the NE, but also at some endoplasmic reticulum (ER) membrane sheets, termed annulate lamellae (AL). Although a role for AL as NPC storage pools has been discussed, it remains controversial whether and how they contribute to the NPC density at the NE. Here, we show that AL insert into the NE as the ER feeds rapid nuclear expansion in Drosophila blastoderm embryos. We demonstrate that NPCs within AL resemble pore scaffolds that mature only upon insertion into the NE. We delineate a topological model in which NE openings are critical for AL uptake that nevertheless occurs without compromising the permeability barrier of the NE. We finally show that this unanticipated mode of pore insertion is developmentally regulated and operates prior to gastrulation.


Assuntos
Embrião não Mamífero/metabolismo , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Oócitos/metabolismo , Animais , Blastoderma/metabolismo , Blastoderma/ultraestrutura , Drosophila , Embrião não Mamífero/ultraestrutura , Desenvolvimento Embrionário , Retículo Endoplasmático/metabolismo , Gastrulação , Oócitos/ultraestrutura
2.
Cell ; 150(1): 122-35, 2012 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-22770216

RESUMO

Mitosis in metazoa requires nuclear envelope (NE) disassembly and reassembly. NE disassembly is driven by multiple phosphorylation events. Mitotic phosphorylation of the protein BAF reduces its affinity for chromatin and the LEM family of inner nuclear membrane proteins; loss of this BAF-mediated chromatin-NE link contributes to NE disassembly. BAF must reassociate with chromatin and LEM proteins at mitotic exit to reform the NE; however, how its dephosphorylation is regulated is unknown. Here, we show that the C. elegans protein LEM-4L and its human ortholog Lem4 (also called ANKLE2) are both required for BAF dephosphorylation. They act in part by inhibiting BAF's mitotic kinase, VRK-1, in vivo and in vitro. In addition, Lem4/LEM-4L interacts with PP2A and is required for it to dephosphorylate BAF during mitotic exit. By coordinating VRK-1- and PP2A-mediated signaling on BAF, Lem4/LEM-4L controls postmitotic NE formation in a function conserved from worms to humans.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Proteínas de Membrana/metabolismo , Mitose , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Caenorhabditis elegans/enzimologia , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Proteínas de Membrana/química , Mutação , Proteínas Nucleares/química , Proteínas Serina-Treonina Quinases/genética
3.
Allergy ; 79(7): 1893-1907, 2024 07.
Artigo em Inglês | MEDLINE | ID: mdl-38573073

RESUMO

BACKGROUND: Extracellular vesicles (EVs) have been implicated in the pathogenesis of asthma, however, how EVs contribute to immune dysfunction and type 2 airway inflammation remains incompletely understood. We aimed to elucidate roles of airway EVs and their miRNA cargo in the pathogenesis of NSAID-exacerbated respiratory disease (N-ERD), a severe type 2 inflammatory condition. METHODS: EVs were isolated from induced sputum or supernatants of cultured nasal polyp or turbinate tissues of N-ERD patients or healthy controls by size-exclusion chromatography and characterized by particle tracking, electron microscopy and miRNA sequencing. Functional effects of EV miRNAs on gene expression and mediator release by human macrophages or normal human bronchial epithelial cells (NHBEs) were studied by RNA sequencing, LC-MS/MS and multiplex cytokine assays. RESULTS: EVs were highly abundant in secretions from the upper and lower airways of N-ERD patients. N-ERD airway EVs displayed profoundly altered immunostimulatory capacities and miRNA profiles compared to airway EVs of healthy individuals. Airway EVs of N-ERD patients, but not of healthy individuals induced inflammatory cytokine (GM-CSF and IL-8) production by NHBEs. In macrophages, N-ERD airway EVs exhibited an impaired potential to induce cytokine and prostanoid production, while enhancing M2 macrophage activation. Let-7 family miRNAs were highly enriched in sputum EVs from N-ERD patients and mimicked suppressive effects of N-ERD EVs on macrophage activation. CONCLUSION: Aberrant airway EV miRNA profiles may contribute to immune dysfunction and chronic type 2 inflammation in N-ERD. Let-7 family miRNAs represent targets for correcting aberrant macrophage activation and mediator responses in N-ERD.


Assuntos
Anti-Inflamatórios não Esteroides , Vesículas Extracelulares , Macrófagos , MicroRNAs , Humanos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/imunologia , MicroRNAs/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Anti-Inflamatórios não Esteroides/efeitos adversos , Citocinas/metabolismo , Masculino , Feminino , Pessoa de Meia-Idade , Ativação de Macrófagos/imunologia , Ativação de Macrófagos/genética , Adulto
4.
J Microsc ; 294(3): 276-294, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38656474

RESUMO

Modern life science research is a collaborative effort. Few research groups can single-handedly support the necessary equipment, expertise and personnel needed for the ever-expanding portfolio of technologies that are required across multiple disciplines in today's life science endeavours. Thus, research institutes are increasingly setting up scientific core facilities to provide access and specialised support for cutting-edge technologies. Maintaining the momentum needed to carry out leading research while ensuring high-quality daily operations is an ongoing challenge, regardless of the resources allocated to establish such facilities. Here, we outline and discuss the range of activities required to keep things running once a scientific imaging core facility has been established. These include managing a wide range of equipment and users, handling repairs and service contracts, planning for equipment upgrades, renewals, or decommissioning, and continuously upskilling while balancing innovation and consolidation.


Assuntos
Disciplinas das Ciências Biológicas , Disciplinas das Ciências Biológicas/métodos
5.
Appl Environ Microbiol ; 88(7): e0240721, 2022 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-35311510

RESUMO

The phylum "Candidatus Omnitrophica" (candidate division OP3) is ubiquitous in anaerobic habitats but is currently characterized only by draft genomes from metagenomes and single cells. We had visualized cells of the phylotype OP3 LiM in methanogenic cultures on limonene as small epibiotic cells. In this study, we enriched OP3 cells by double density gradient centrifugation and obtained the first closed genome of an apparently clonal OP3 cell population by applying metagenomics and PCR for gap closure. Filaments of acetoclastic Methanosaeta, the largest morphotype in the culture community, contained empty cells, cells devoid of rRNA or of both rRNA and DNA, and dead cells according to transmission electron microscopy (TEM), thin-section TEM, scanning electron microscopy (SEM), catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH), and LIVE/DEAD imaging. OP3 LiM cells were ultramicrobacteria (200 to 300 nm in diameter) and showed two physiological stages in CARD-FISH fluorescence signals: strong signals of OP3 LiM cells attached to Bacteria and to Archaea indicated many rRNA molecules and an active metabolism, whereas free-living OP3 cells had weak signals. Metaproteomics revealed that OP3 LiM lives with highly expressed secreted proteins involved in depolymerization and uptake of macromolecules and an active glycolysis and energy conservation by the utilization of pyruvate via a pyruvate:ferredoxin oxidoreductase and an Rnf complex (ferredoxin:NAD oxidoreductase). Besides sugar fermentation, a nucleotidyl transferase may contribute to energy conservation by phosphorolysis, the phosphate-dependent depolymerization of nucleic acids. Thin-section TEM showed distinctive structures of predation. Our study demonstrated a predatory metabolism for OP3 LiM cells, and therefore, we propose the name "Candidatus Velamenicoccus archaeovorus" gen. nov., sp. nov., for OP3 LiM. IMPORTANCE Epibiotic bacteria are known to live on and off bacterial cells. Here, we describe the ultramicrobacterial anaerobic epibiont OP3 LiM living on Archaea and Bacteria. We detected sick and dead cells of the filamentous archaeon Methanosaeta in slowly growing methanogenic cultures. OP3 LiM lives as a sugar fermenter, likely on polysaccharides from outer membranes, and has the genomic potential to live as a syntroph. The predatory lifestyle of OP3 LiM was supported by its genome, the first closed genome for the phylum "Candidatus Omnitrophica," and by images of cell-to-cell contact with prey cells. We propose naming OP3 LiM "Candidatus Velamenicoccus archaeovorus." Its metabolic versatility explains the ubiquitous presence of "Candidatus Omnitrophica" 3 in anoxic habitats and gives ultramicrobacterial epibionts an important role in the recycling and remineralization of microbial biomass. The removal of polysaccharides from outer membranes by ultramicrobacteria may also influence biological interactions between pro- and eukaryotes.


Assuntos
Ferredoxinas , Ácido Pirúvico , Archaea/metabolismo , Bactérias/genética , Ferredoxinas/metabolismo , Hibridização in Situ Fluorescente , Methanosarcinaceae/metabolismo , Oxirredutases/metabolismo , Filogenia , Ácido Pirúvico/metabolismo , RNA Ribossômico 16S/genética , Açúcares/metabolismo
7.
Appl Environ Microbiol ; 85(19)2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31324630

RESUMO

Large surface-to-volume ratios provide optimal nutrient uptake conditions for small microorganisms in oligotrophic habitats. The surface area can be increased with appendages. Here, we describe chains of interconnecting vesicles protruding from cells of strain Hel3_A1_48, affiliating with Formosa spp. within the Flavobacteriia and originating from coastal free-living bacterioplankton. The chains were up to 10 µm long and had vesicles emanating from the outer membrane with a single membrane and a size of 80 to 100 nm by 50 to 80 nm. Cells extruded membrane tubes in the exponential phase, whereas vesicle chains dominated on cells in the stationary growth phase. This formation is known as pearling, a physical morphogenic process in which membrane tubes protrude from liposomes and transform into chains of interconnected vesicles. Proteomes of whole-cell membranes and of detached vesicles were dominated by outer membrane proteins, including the type IX secretion system and surface-attached peptidases, glycoside hydrolases, and endonucleases. Fluorescein-labeled laminarin stained the cells and the vesicle chains. Thus, the appendages provide binding domains and degradative enzymes on their surfaces and probably storage volume in the vesicle lumen. Both may contribute to the high abundance of these Formosa-affiliated bacteria during laminarin utilization shortly after spring algal blooms.IMPORTANCE Microorganisms produce membrane vesicles. One synthesis pathway seems to be pearling that describes the physical formation of vesicle chains from phospholipid vesicles via extended tubes. Bacteria with vesicle chains had been observed as well as bacteria with tubes, but pearling was so far not observed. Here, we report the observation of, initially, tubes and then vesicle chains during the growth of a flavobacterium, suggesting biopearling of vesicle chains. The flavobacterium is abundant during spring bacterioplankton blooms developing after algal blooms and has a special set of enzymes for laminarin, the major storage polysaccharide of microalgae. We demonstrated with fluorescently labeled laminarin that the vesicle chains bind laminarin or contain laminarin-derived compounds. Proteomic analyses revealed surface-attached degradative enzymes on the outer membrane vesicles. We conclude that the large surface area and the lumen of vesicle chains may contribute to the ecological success of this marine bacterium.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/fisiologia , Flavobacterium/fisiologia , Organismos Aquáticos/fisiologia , Eutrofização , Vesículas Extracelulares/fisiologia , Vesículas Extracelulares/ultraestrutura , Glucanos/metabolismo , Lipossomos , Microscopia Eletrônica , Proteômica
8.
J Cell Sci ; 129(8): 1552-65, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26906412

RESUMO

The mechanism of endoplasmic reticulum (ER) morphogenesis is incompletely understood. ER tubules are shaped by the reticulons (RTNs) and DP1/Yop1p family members, but the mechanism of ER sheet formation is much less clear. Here, we characterize TMEM170A, a human transmembrane protein, which localizes in ER and nuclear envelope membranes. Silencing or overexpressing TMEM170A in HeLa K cells alters ER shape and morphology. Ultrastructural analysis reveals that downregulation of TMEM170A specifically induces tubular ER formation, whereas overexpression of TMEM170A induces ER sheet formation, indicating that TMEM170A is a newly discovered ER-sheet-promoting protein. Additionally, downregulation of TMEM170A alters nuclear shape and size, decreases the density of nuclear pore complexes (NPCs) in the nuclear envelope and causes either a reduction in inner nuclear membrane (INM) proteins or their relocalization to the ER. TMEM170A interacts with RTN4, a member of the reticulon family; simultaneous co-silencing of TMEM170A and RTN4 rescues ER, NPC and nuclear-envelope-related phenotypes, implying that the two proteins have antagonistic effects on ER membrane organization, and nuclear envelope and NPC formation.


Assuntos
Retículo Endoplasmático/fisiologia , Proteínas de Membrana/metabolismo , Proteínas Nogo/metabolismo , Membrana Nuclear/metabolismo , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas Nogo/genética , Poro Nuclear/metabolismo , Transporte Proteico/genética , RNA Interferente Pequeno/genética , Transgenes
9.
J Cell Sci ; 128(22): 4183-95, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26446261

RESUMO

Changes in cell morphology require coordination of plasma membrane turnover and cytoskeleton dynamics, processes that are regulated by Rho GTPases. Here, we describe how a direct interaction between the Rho GTPase Cdc42 and the GTPase-activating protein (GAP) GRAF1 (also known as ARHGAP26), facilitates rapid cell surface turnover at the leading edge. Both Cdc42 and GRAF1 were required for fluid-phase uptake and regulated the generation of transient GRAF1-coated endocytic carriers, which were distinct from clathrin-coated vesicles. GRAF1 was found to transiently assemble at discrete Cdc42-enriched punctae at the plasma membrane, resulting in a corresponding decrease in the microdomain association of Cdc42. However, Cdc42 captured in its active state was, through a GAP-domain-mediated interaction, localised together with GRAF1 on accumulated internal structures derived from the cell surface. Correlative fluorescence and electron tomography microscopy revealed that these structures were clusters of small membrane carriers with defective endosomal processing. We conclude that a transient interaction between Cdc42 and GRAF1 drives endocytic turnover and controls the transition essential for endosomal maturation of plasma membrane internalised by this mechanism.


Assuntos
Actinas/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Endocitose , Proteínas Ativadoras de GTPase/genética , Células HeLa , Humanos , Proteína cdc42 de Ligação ao GTP/genética
10.
EMBO J ; 31(18): 3678-90, 2012 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-22850673

RESUMO

The eukaryotic genome is replicated according to a specific spatio-temporal programme. However, little is known about both its molecular control and biological significance. Here, we identify mouse Rif1 as a key player in the regulation of DNA replication timing. We show that Rif1 deficiency in primary cells results in an unprecedented global alteration of the temporal order of replication. This effect takes place already in the first S-phase after Rif1 deletion and is neither accompanied by alterations in the transcriptional landscape nor by major changes in the biochemical identity of constitutive heterochromatin. In addition, Rif1 deficiency leads to both defective G1/S transition and chromatin re-organization after DNA replication. Together, these data offer a novel insight into the global regulation and biological significance of the replication-timing programme in mammalian cells.


Assuntos
Replicação do DNA , Regulação da Expressão Gênica , Proteínas de Ligação a Telômeros/genética , Alelos , Animais , Ciclo Celular , Feminino , Fase G1 , Genoma , Genótipo , Heterocromatina/química , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal/métodos , Matriz Nuclear/metabolismo , Fase S , Transcrição Gênica
11.
J Cell Sci ; 127(Pt 2): 277-80, 2014 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-24259664

RESUMO

We report the presence of a membranous tubulovesicular network in the planctomycete bacterium Gemmata obscuriglobus. This endomembrane system interacts with membrane coat proteins and is capable of protein internalization and degradation. Taken together, this suggests that the planctomycetal bacterium could illuminate the emergence of complex endomembrane systems.


Assuntos
Compartimento Celular , Planctomycetales/citologia , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Planctomycetales/ultraestrutura
12.
PLoS Biol ; 11(5): e1001565, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23700385

RESUMO

The division of cellular space into functionally distinct membrane-defined compartments has been one of the major transitions in the history of life. Such compartmentalization has been claimed to occur in members of the Planctomycetes, Verrucomicrobiae, and Chlamydiae bacterial superphylum. Here we have investigated the three-dimensional organization of the complex endomembrane system in the planctomycete bacteria Gemmata obscuriglobus. We reveal that the G. obscuriglobus cells are neither compartmentalized nor nucleated as none of the spaces created by the membrane invaginations are closed; instead, they are all interconnected. Thus, the membrane organization of G. obscuriglobus, and most likely all PVC members, is not different from, but an extension of, the "classical" Gram-negative bacterial membrane system. Our results have implications for our definition and understanding of bacterial cell organization, the genesis of complex structure, and the origin of the eukaryotic endomembrane system.


Assuntos
Proteínas de Bactérias/metabolismo , Membranas Intracelulares/ultraestrutura , Planctomycetales/metabolismo , Compartimento Celular , Membranas Intracelulares/metabolismo , Microscopia Eletrônica , Planctomycetales/ultraestrutura
13.
PLoS Pathog ; 8(12): e1003056, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23236278

RESUMO

All positive strand RNA viruses are known to replicate their genomes in close association with intracellular membranes. In case of the hepatitis C virus (HCV), a member of the family Flaviviridae, infected cells contain accumulations of vesicles forming a membranous web (MW) that is thought to be the site of viral RNA replication. However, little is known about the biogenesis and three-dimensional structure of the MW. In this study we used a combination of immunofluorescence- and electron microscopy (EM)-based methods to analyze the membranous structures induced by HCV in infected cells. We found that the MW is derived primarily from the endoplasmic reticulum (ER) and contains markers of rough ER as well as markers of early and late endosomes, COP vesicles, mitochondria and lipid droplets (LDs). The main constituents of the MW are single and double membrane vesicles (DMVs). The latter predominate and the kinetic of their appearance correlates with kinetics of viral RNA replication. DMVs are induced primarily by NS5A whereas NS4B induces single membrane vesicles arguing that MW formation requires the concerted action of several HCV replicase proteins. Three-dimensional reconstructions identify DMVs as protrusions from the ER membrane into the cytosol, frequently connected to the ER membrane via a neck-like structure. In addition, late in infection multi-membrane vesicles become evident, presumably as a result of a stress-induced reaction. Thus, the morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those of the closely related flaviviruses. These results reveal unexpected similarities between HCV and distantly related positive-strand RNA viruses presumably reflecting similarities in cellular pathways exploited by these viruses to establish their membranous replication factories.


Assuntos
Retículo Endoplasmático , Hepacivirus , Hepatite C , Membranas Intracelulares , RNA Viral/biossíntese , Linhagem Celular , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Retículo Endoplasmático/virologia , Hepacivirus/fisiologia , Hepacivirus/ultraestrutura , Hepatite C/metabolismo , Hepatite C/patologia , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Membranas Intracelulares/virologia , Microscopia Eletrônica de Transmissão/métodos , Replicação Viral/fisiologia
14.
Dev Biol ; 365(2): 445-57, 2012 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-22426005

RESUMO

Nuclear pore complexes (NPCs) are large macromolecular structures embedded in the nuclear envelope (NE), where they facilitate exchange of molecules between the cytoplasm and the nucleoplasm. In most cell types, NPCs are evenly distributed around the NE. However, the mechanisms dictating NPC distribution are largely unknown. Here, we used the model organism Caenorhabditis elegans to identify genes that affect NPC distribution during early embryonic divisions. We found that down-regulation of the Sm proteins, which are core components of the spliceosome, but not down-regulation of other splicing factors, led to clustering of NPCs. Down-regulation of Sm proteins also led to incomplete disassembly of NPCs during mitosis, but had no effect on lamina disassembly, suggesting that the defect in NPC disassembly was not due to a general defect in nuclear envelope breakdown. We further found that these mitotic NPC remnants persisted on an ER membrane that juxtaposes the mitotic spindle. At the end of mitosis, the remnant NPCs moved toward the chromatin and the reforming NE, where they ultimately clustered by forming membrane stacks perforated by NPCs. Our results suggest a novel, splicing-independent, role for Sm proteins in NPC disassembly, and point to a possible link between NPC disassembly in mitosis and NPC distribution in the subsequent interphase.


Assuntos
Caenorhabditis elegans/embriologia , Poro Nuclear/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/ultraestrutura , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Regulação para Baixo , Embrião não Mamífero , Mitose , Poro Nuclear/genética , Poro Nuclear/ultraestrutura , Interferência de RNA , Ribonucleoproteínas Nucleares Pequenas/antagonistas & inibidores , Ribonucleoproteínas Nucleares Pequenas/genética , Spliceossomos/genética , Spliceossomos/metabolismo
15.
PLoS Biol ; 8(1): e1000281, 2010 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-20087413

RESUMO

The development of the endomembrane system was a major step in eukaryotic evolution. Membrane coats, which exhibit a unique arrangement of beta-propeller and alpha-helical repeat domains, play key roles in shaping eukaryotic membranes. Such proteins are likely to have been present in the ancestral eukaryote but cannot be detected in prokaryotes using sequence-only searches. We have used a structure-based detection protocol to search all proteomes for proteins with this domain architecture. Apart from the eukaryotes, we identified this protein architecture only in the Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) bacterial superphylum, many members of which share a compartmentalized cell plan. We determined that one such protein is partly localized at the membranes of vesicles formed inside the cells in the planctomycete Gemmata obscuriglobus. Our results demonstrate similarities between bacterial and eukaryotic compartmentalization machinery, suggesting that the bacterial PVC superphylum contributed significantly to eukaryogenesis.


Assuntos
Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Membrana/química , Bactérias/classificação , Bactérias/citologia , Proteínas de Bactérias/fisiologia , Evolução Biológica , Compartimento Celular , Proteínas de Membrana/fisiologia , Filogenia , Estrutura Terciária de Proteína , Proteoma , Análise de Sequência de Proteína
16.
Commun Biol ; 6(1): 488, 2023 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-37147475

RESUMO

Microtubules are key to multiple neuronal functions involving the transport of organelles, however, their relationship to neurotransmitter release is still unresolved. Here, we show that microtubules present in the presynaptic compartment of cholinergic autaptic synapses are dynamic. To investigate how the balance between microtubule growth and shrinkage affects neurotransmission we induced synchronous microtubule depolymerization by photoactivation of the chemical inhibitor SBTub3. The consequence was an increase in spontaneous neurotransmitter release. An analogous effect was obtained by dialyzing the cytosol with Kif18A, a plus-end-directed kinesin with microtubule depolymerizing activity. Kif18A also inhibited the refilling of the readily releasable pool of synaptic vesicles during high frequency stimulation. The action of Kif18A was associated to one order of magnitude increases in the numbers of exo-endocytic pits and endosomes present in the presynaptic terminal. An enhancement of spontaneous neurotransmitter release was also observed when neurons were dialyzed with stathmin-1, a protein with a widespread presence in the nervous system that induces microtubule depolymerization. Taken together, these results support that microtubules restrict spontaneous neurotransmitter release as well as promote the replenishment of the readily releasable pool of synaptic vesicles.


Assuntos
Sinapses , Vesículas Sinápticas , Sinapses/metabolismo , Vesículas Sinápticas/metabolismo , Transmissão Sináptica/fisiologia , Microtúbulos/metabolismo , Neurotransmissores/metabolismo
17.
Theranostics ; 13(15): 5170-5182, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37908732

RESUMO

Rationale: Intrinsic brain tumors, such as gliomas are largely resistant to immunotherapies including immune checkpoint blockade. Adoptive cell therapies (ACT) including chimeric antigen receptor (CAR) or T cell receptor (TCR)-transgenic T cell therapy targeting glioma-associated antigens are an emerging field in glioma immunotherapy. However, imaging techniques for non-invasive monitoring of adoptively transferred T cells homing to the glioma microenvironment are currently lacking. Methods: Ultrasmall iron oxide nanoparticles (NP) can be visualized non-invasively by magnetic resonance imaging (MRI) and dedicated MRI sequences such as T2* mapping. Here, we develop a protocol for efficient ex vivo labeling of murine and human TCR-transgenic and CAR T cells with iron oxide NPs. We assess labeling efficiency and T cell functionality by flow cytometry and transmission electron microscopy (TEM). NP labeled T cells are visualized by MRI at 9.4 T in vivo after adoptive T cell transfer and correlated with 3D models of cleared brains obtained by light sheet microscopy (LSM). Results: NP are incorporated into T cells in subcellular cytoplasmic vesicles with high labeling efficiency without interfering with T cell viability, proliferation and effector function as assessed by cytokine secretion and antigen-specific killing assays in vitro. We further demonstrate that adoptively transferred T cells can be longitudinally monitored intratumorally by high field MRI at 9.4 Tesla in a murine glioma model with high sensitivity. We find that T cell influx and homogenous spatial distribution of T cells within the TME as assessed by T2* imaging predicts tumor response to ACT whereas incomplete T cell coverage results in treatment resistance. Conclusion: This study showcases a rational for monitoring adoptive T cell therapies non-invasively by iron oxide NP in gliomas to track intratumoral T cell influx and ultimately predict treatment outcome.


Assuntos
Glioma , Linfócitos T , Humanos , Animais , Camundongos , Glioma/diagnóstico por imagem , Glioma/terapia , Imunoterapia Adotiva , Receptores de Antígenos de Linfócitos T , Terapia Baseada em Transplante de Células e Tecidos , Microambiente Tumoral
18.
Opt Express ; 20(24): 26778-85, 2012 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-23187532

RESUMO

We have applied Fresnel Coherent Diffractive Imaging (FCDI) to image an intact pollen grain from Convallaria majalis. This approach allows us to resolve internal structures without the requirement to chemically treat or slice the sample into thin sections. Coherent X-ray diffraction data from this pollen grain-composed of a hologram and higher resolution scattering information-was collected at a photon energy of 1820 eV and reconstructed using an iterative algorithm. A comparison with images recorded using transmission electron microscopy demonstrates that, while the resolution of these images is limited by the available flux and mechanical stability, we observed structures internal to the pollen grain-the intine/exine separations and pore dimensions-finer than 60 nm. The potential of this technique for further biological imaging applications is discussed.


Assuntos
Algoritmos , Convallaria/ultraestrutura , Imageamento Tridimensional/métodos , Microscopia Eletrônica de Transmissão/métodos , Pólen/ultraestrutura , Difração de Raios X/métodos , Holografia
19.
Cell Host Microbe ; 28(6): 853-866.e5, 2020 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-33245857

RESUMO

Pathogenesis induced by SARS-CoV-2 is thought to result from both an inflammation-dominated cytokine response and virus-induced cell perturbation causing cell death. Here, we employ an integrative imaging analysis to determine morphological organelle alterations induced in SARS-CoV-2-infected human lung epithelial cells. We report 3D electron microscopy reconstructions of whole cells and subcellular compartments, revealing extensive fragmentation of the Golgi apparatus, alteration of the mitochondrial network and recruitment of peroxisomes to viral replication organelles formed by clusters of double-membrane vesicles (DMVs). These are tethered to the endoplasmic reticulum, providing insights into DMV biogenesis and spatial coordination of SARS-CoV-2 replication. Live cell imaging combined with an infection sensor reveals profound remodeling of cytoskeleton elements. Pharmacological inhibition of their dynamics suppresses SARS-CoV-2 replication. We thus report insights into virus-induced cytopathic effects and provide alongside a comprehensive publicly available repository of 3D datasets of SARS-CoV-2-infected cells for download and smooth online visualization.


Assuntos
COVID-19/genética , Retículo Endoplasmático/ultraestrutura , SARS-CoV-2/ultraestrutura , Compartimentos de Replicação Viral/ultraestrutura , COVID-19/diagnóstico por imagem , COVID-19/patologia , COVID-19/virologia , Morte Celular/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/virologia , Humanos , Microscopia Eletrônica , Pandemias , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Compartimentos de Replicação Viral/metabolismo , Replicação Viral/genética
20.
J Vis Exp ; (139)2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30247481

RESUMO

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.


Assuntos
Células Imobilizadas/metabolismo , Técnicas Histológicas/métodos , Microscopia Eletrônica/métodos , Humanos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA