Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 87
Filtrar
Filtros adicionais











Intervalo de ano
2.
Cell Rep ; 24(4): 973-986.e8, 2018 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-30044992

RESUMO

Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and release of extracellular vesicles (EVs), which enable cell-to-cell communication in the nervous system essential for development and adult function. We recently showed human loss-of-function (LOF) mutations in ESCRT-III member CHMP1A cause autosomal recessive microcephaly with pontocerebellar hypoplasia, but its mechanism was unclear. Here, we show Chmp1a is required for progenitor proliferation in mouse cortex and cerebellum and progenitor maintenance in human cerebral organoids. In Chmp1a null mice, this defect is associated with impaired sonic hedgehog (Shh) secretion and intraluminal vesicle (ILV) formation in multivesicular bodies (MVBs). Furthermore, we show CHMP1A is important for release of an EV subtype that contains AXL, RAB18, and TMED10 (ART) and SHH. Our findings show CHMP1A loss impairs secretion of SHH on ART-EVs, providing molecular mechanistic insights into the role of ESCRT proteins and EVs in the brain.

3.
Elife ; 72018 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-29916365

RESUMO

The inner ear is a fluid-filled closed-epithelial structure whose function requires maintenance of an internal hydrostatic pressure and fluid composition. The endolymphatic sac (ES) is a dead-end epithelial tube connected to the inner ear whose function is unclear. ES defects can cause distended ear tissue, a pathology often seen in hearing and balance disorders. Using live imaging of zebrafish larvae, we reveal that the ES undergoes cycles of slow pressure-driven inflation followed by rapid deflation. Absence of these cycles in lmx1bb mutants leads to distended ear tissue. Using serial-section electron microscopy and adaptive optics lattice light-sheet microscopy, we find a pressure relief valve in the ES comprised of partially separated apical junctions and dynamic overlapping basal lamellae that separate under pressure to release fluid. We propose that this lmx1-dependent pressure relief valve is required to maintain fluid homeostasis in the inner ear and other fluid-filled cavities.

4.
Cell Host Microbe ; 22(5): 688-696.e5, 2017 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-29120745

RESUMO

Arenaviruses cause fatal hemorrhagic disease in humans. Old World arenavirus glycoproteins (GPs) mainly engage α-dystroglycan as a cell-surface receptor, while New World arenaviruses hijack transferrin receptor. However, the Lujo virus (LUJV) GP does not cluster with New or Old World arenaviruses. Using a recombinant vesicular stomatitis virus containing LUJV GP as its sole attachment and fusion protein (VSV-LUJV), we demonstrate that infection is independent of known arenavirus receptor genes. A genome-wide haploid genetic screen identified the transmembrane protein neuropilin 2 (NRP2) and tetraspanin CD63 as factors for LUJV GP-mediated infection. LUJV GP binds the N-terminal domain of NRP2, while CD63 stimulates pH-activated LUJV GP-mediated membrane fusion. Overexpression of NRP2 or its N-terminal domain enhances VSV-LUJV infection, and cells lacking NRP2 are deficient in wild-type LUJV infection. These findings uncover this distinct set of host cell entry factors in LUJV infection and are attractive focus points for therapeutic intervention.


Assuntos
Lujo virus/fisiologia , Neuropilina-2/metabolismo , Tetraspanina 30/metabolismo , Proteínas Virais de Fusão/metabolismo , Proteínas Virais/metabolismo , Internalização do Vírus , Proteínas de Transporte , Linhagem Celular , Interações Hospedeiro-Patógeno/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Lujo virus/genética , Lujo virus/patogenicidade , Domínios e Motivos de Interação entre Proteínas , Receptores de Superfície Celular/metabolismo , Receptores da Transferrina , Proteínas Virais de Fusão/genética , Proteínas Virais/genética
5.
Elife ; 62017 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-29019322

RESUMO

The ESCRT machinery mediates reverse membrane scission. By quantitative fluorescence lattice light-sheet microscopy, we have shown that ESCRT-III subunits polymerize rapidly on yeast endosomes, together with the recruitment of at least two Vps4 hexamers. During their 3-45 s lifetimes, the ESCRT-III assemblies accumulated 75-200 Snf7 and 15-50 Vps24 molecules. Productive budding events required at least two additional Vps4 hexamers. Membrane budding was associated with continuous, stochastic exchange of Vps4 and ESCRT-III components, rather than steady growth of fixed assemblies, and depended on Vps4 ATPase activity. An all-or-none step led to final release of ESCRT-III and Vps4. Tomographic electron microscopy demonstrated that acute disruption of Vps4 recruitment stalled membrane budding. We propose a model in which multiple Vps4 hexamers (four or more) draw together several ESCRT-III filaments. This process induces cargo crowding and inward membrane buckling, followed by constriction of the nascent bud neck and ultimately ILV generation by vesicle fission.


Assuntos
Adenosina Trifosfatases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Tomografia com Microscopia Eletrônica , Microscopia de Fluorescência
6.
Nat Biomed Eng ; 1(11): 878-888, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-31015609

RESUMO

Gene disruption by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) is highly efficient and relies on the error-prone non-homologous end-joining pathway. Conversely, precise gene editing requires homology-directed repair (HDR), which occurs at a lower frequency than non-homologous end-joining in mammalian cells. Here, by testing whether manipulation of DNA repair factors improves HDR efficacy, we show that transient ectopic co-expression of RAD52 and a dominant-negative form of tumour protein p53-binding protein 1 (dn53BP1) synergize to enable efficient HDR using a single-stranded oligonucleotide DNA donor template at multiple loci in human cells, including patient-derived induced pluripotent stem cells. Co-expression of RAD52 and dn53BP1 improves multiplexed HDR-mediated editing, whereas expression of RAD52 alone enhances HDR with Cas9 nickase. Our data show that the frequency of non-homologous end-joining-mediated double-strand break repair in the presence of these two factors is not suppressed and suggest that dn53BP1 competitively antagonizes 53BP1 to augment HDR in combination with RAD52. Importantly, co-expression of RAD52 and dn53BP1 does not alter Cas9 off-target activity. These findings support the use of RAD52 and dn53BP1 co-expression to overcome bottlenecks that limit HDR in precision genome editing.


Assuntos
Sistemas CRISPR-Cas , Reparo do DNA , Edição de Genes/métodos , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Expressão Ectópica do Gene , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Reparo de DNA por Recombinação
7.
Elife ; 52016 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-27564575

RESUMO

How proteins control the biogenesis of cellular lipid droplets (LDs) is poorly understood. Using Drosophila and human cells, we show here that seipin, an ER protein implicated in LD biology, mediates a discrete step in LD formation-the conversion of small, nascent LDs to larger, mature LDs. Seipin forms discrete and dynamic foci in the ER that interact with nascent LDs to enable their growth. In the absence of seipin, numerous small, nascent LDs accumulate near the ER and most often fail to grow. Those that do grow prematurely acquire lipid synthesis enzymes and undergo expansion, eventually leading to the giant LDs characteristic of seipin deficiency. Our studies identify a discrete step of LD formation, namely the conversion of nascent LDs to mature LDs, and define a molecular role for seipin in this process, most likely by acting at ER-LD contact sites to enable lipid transfer to nascent LDs.

8.
Sci Rep ; 5: 15990, 2015 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-26549784

RESUMO

Many viruses have evolved strategies of so-called "superinfection exclusion" to prevent re-infection of a cell that the same virus has already infected. Although Old World arenavirus infection results in down-regulation of its viral receptor and thus superinfection exclusion, whether New World arenaviruses have evolved such a mechanism remains unclear. Here we show that acute infection by the New World Junin virus (JUNV) failed to down-regulate the transferrin receptor and did not induce superinfection exclusion. We observed that Vero cells infected by a first round of JUNV (Candid1 strain) preserve an ability to internalize new incoming JUNV particles that is comparable to that of non-infected cells. Moreover, we developed a dual infection assay with the wild-type Candid1 JUNV and a recombinant JUNV-GFP virus to discriminate between first and second infections at the transcriptional and translational levels. We found that Vero and A549 cells already infected by JUNV were fully competent to transcribe viral RNA from a second round of infection. Furthermore, flow cytometry analysis of viral protein expression indicated that viral translation was normal, regardless of whether cells were previously infected or not. We conclude that in acutely infected cells, Junin virus lacks a superinfection exclusion mechanism.


Assuntos
Febre Hemorrágica Americana/genética , Vírus Junin/genética , Receptores da Transferrina/biossíntese , Proteínas Virais/biossíntese , Animais , Cercopithecus aethiops , Regulação Viral da Expressão Gênica , Febre Hemorrágica Americana/virologia , Humanos , Vírus Junin/patogenicidade , RNA Viral/biossíntese , Superinfecção/genética , Células Vero
9.
Science ; 349(6251): aab3500, 2015 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-26315442

RESUMO

Super-resolution fluorescence microscopy is distinct among nanoscale imaging tools in its ability to image protein dynamics in living cells. Structured illumination microscopy (SIM) stands out in this regard because of its high speed and low illumination intensities, but typically offers only a twofold resolution gain. We extended the resolution of live-cell SIM through two approaches: ultrahigh numerical aperture SIM at 84-nanometer lateral resolution for more than 100 multicolor frames, and nonlinear SIM with patterned activation at 45- to 62-nanometer resolution for approximately 20 to 40 frames. We applied these approaches to image dynamics near the plasma membrane of spatially resolved assemblies of clathrin and caveolin, Rab5a in early endosomes, and α-actinin, often in relationship to cortical actin. In addition, we examined mitochondria, actin, and the Golgi apparatus dynamics in three dimensions.


Assuntos
Citoesqueleto/ultraestrutura , Endocitose , Imagem Tridimensional/métodos , Microscopia de Fluorescência/métodos , Organelas/ultraestrutura , Actinina/análise , Actinas/análise , Animais , Linhagem Celular , Clatrina/análise , Vesículas Revestidas por Clatrina/química , Vesículas Revestidas por Clatrina/ultraestrutura , Invaginações Revestidas da Membrana Celular/química , Invaginações Revestidas da Membrana Celular/ultraestrutura , Citoesqueleto/química , Citoesqueleto/metabolismo , Endossomos/química , Endossomos/ultraestrutura , Complexo de Golgi/ultraestrutura , Processamento de Imagem Assistida por Computador , Imagem Tridimensional/instrumentação , Microscopia de Fluorescência/instrumentação , Mitocôndrias/química , Mitocôndrias/ultraestrutura , Organelas/química , Organelas/metabolismo , Proteínas rab5 de Ligação ao GTP/análise
10.
Nat Biotechnol ; 33(8): 870-6, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26192320

RESUMO

A central hurdle in developing small interfering RNAs (siRNAs) as therapeutics is the inefficiency of their delivery across the plasma and endosomal membranes to the cytosol, where they interact with the RNA interference machinery. With the aim of improving endosomal release, a poorly understood and inefficient process, we studied the uptake and cytosolic release of siRNAs, formulated in lipoplexes or lipid nanoparticles, by live-cell imaging and correlated it with knockdown of a target GFP reporter. siRNA release occurred invariably from maturing endosomes within ~5-15 min of endocytosis. Cytosolic galectins immediately recognized the damaged endosome and targeted it for autophagy. However, inhibiting autophagy did not enhance cytosolic siRNA release. Gene knockdown occurred within a few hours of release and required <2,000 copies of cytosolic siRNAs. The ability to detect cytosolic release of siRNAs and understand how it is regulated will facilitate the development of rational strategies for improving the cytosolic delivery of candidate drugs.


Assuntos
Endossomos/metabolismo , Técnicas de Silenciamento de Genes/métodos , Proteínas Luminescentes/farmacocinética , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacocinética , Células HeLa , Humanos , Lipídeos/química , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Nanopartículas/química , RNA Interferente Pequeno/metabolismo
12.
PLoS Pathog ; 10(9): e1004355, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25211455

RESUMO

Cell entry by non-enveloped viruses requires translocation into the cytosol of a macromolecular complex--for double-strand RNA viruses, a complete subviral particle. We have used live-cell fluorescence imaging to follow rotavirus entry and penetration into the cytosol of its ∼ 700 Šinner capsid particle ("double-layered particle", DLP). We label with distinct fluorescent tags the DLP and each of the two outer-layer proteins and track the fates of each species as the particles bind and enter BSC-1 cells. Virions attach to their glycolipid receptors in the host cell membrane and rapidly become inaccessible to externally added agents; most particles that release their DLP into the cytosol have done so by ∼ 10 minutes, as detected by rapid diffusional motion of the DLP away from residual outer-layer proteins. Electron microscopy shows images of particles at various stages of engulfment into tightly fitting membrane invaginations, consistent with the interpretation that rotavirus particles drive their own uptake. Electron cryotomography of membrane-bound virions also shows closely wrapped membrane. Combined with high resolution structural information about the viral components, these observations suggest a molecular model for membrane disruption and DLP penetration.


Assuntos
Antígenos Virais/metabolismo , Proteínas do Capsídeo/metabolismo , Membrana Celular/química , Rotavirus/química , Vírion , Montagem de Vírus/fisiologia , Internalização do Vírus , Animais , Antígenos Virais/química , Antígenos Virais/genética , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Membrana Celular/metabolismo , Células Cultivadas , Cercopithecus aethiops/virologia , Processamento de Imagem Assistida por Computador , Rim/virologia , Microscopia Eletrônica , Mutação/genética , Rotavirus/fisiologia
13.
J Cell Sci ; 127(Pt 18): 3970-82, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-25074807

RESUMO

After activation by Wnt/ß-Catenin ligands, a multi-protein complex assembles at the plasma membrane as membrane-bound receptors and intracellular signal transducers are clustered into the so-called Lrp6-signalosome [Corrected]. However, the mechanism of signalosome formation and dissolution is yet not clear. Our imaging studies of live zebrafish embryos show that the signalosome is a highly dynamic structure. It is continuously assembled by Dvl2-mediated recruitment of the transducer complex to the activated receptors and partially disassembled by endocytosis. We find that, after internalization, the ligand-receptor complex and the transducer complex take separate routes. The Wnt-Fz-Lrp6 complex follows a Rab-positive endocytic path. However, when still bound to the transducer complex, Dvl2 forms intracellular aggregates. We show that this endocytic process is not only essential for ligand-receptor internalization but also for signaling. The µ2-subunit of the endocytic Clathrin adaptor Ap2 interacts with Dvl2 to maintain its stability during endocytosis. Blockage of Ap2µ2 function leads to Dvl2 degradation, inhibiton of signalosome formation at the plasma membrane and, consequently, reduction of signaling. We conclude that Ap2µ2-mediated endocytosis is important to maintain Wnt/ß-catenin signaling in vertebrates.


Assuntos
Endocitose , Complexos Multiproteicos/metabolismo , Via de Sinalização Wnt , Xenopus/metabolismo , beta Catenina/metabolismo , Complexo 2 de Proteínas Adaptadoras/genética , Complexo 2 de Proteínas Adaptadoras/metabolismo , Subunidades mu do Complexo de Proteínas Adaptadoras/genética , Subunidades mu do Complexo de Proteínas Adaptadoras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Desgrenhadas , Feminino , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Complexos Multiproteicos/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Xenopus/embriologia , Xenopus/genética
14.
Cell ; 157(6): 1309-23, 2014 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-24906149

RESUMO

When killer lymphocytes recognize infected cells, perforin delivers cytotoxic proteases (granzymes) into the target cell to trigger apoptosis. What happens to intracellular bacteria during this process is unclear. Human, but not rodent, cytotoxic granules also contain granulysin, an antimicrobial peptide. Here, we show that granulysin delivers granzymes into bacteria to kill diverse bacterial strains. In Escherichia coli, granzymes cleave electron transport chain complex I and oxidative stress defense proteins, generating reactive oxygen species (ROS) that rapidly kill bacteria. ROS scavengers and bacterial antioxidant protein overexpression inhibit bacterial death. Bacteria overexpressing a GzmB-uncleavable mutant of the complex I subunit nuoF or strains that lack complex I still die, but more slowly, suggesting that granzymes disrupt multiple vital bacterial pathways. Mice expressing transgenic granulysin are better able to clear Listeria monocytogenes. Thus killer cells play an unexpected role in bacterial defense.


Assuntos
Antígenos de Diferenciação de Linfócitos T/metabolismo , Infecções Bacterianas/imunologia , Escherichia coli , Leucócitos Mononucleares/imunologia , Listeria monocytogenes , Staphylococcus aureus , Animais , Granzimas/metabolismo , Células HeLa , Humanos , Leucócitos Mononucleares/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Perforina/genética , Perforina/metabolismo , Espécies Reativas de Oxigênio/metabolismo
15.
Nucleus ; 5(1): 11-4, 2014 Jan-Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24637398

RESUMO

Since it became clear that intervening sequences or introns are spliced out from precursor pre-mRNA molecules in the nucleus before mature mRNAs are exported to the cytoplasm, questions were raised about the timing of splicing. Does splicing start while RNA polymerase II is still transcribing? Is splicing a slow or a fast process? Is timing important to control the splicing reaction? Although our understanding on the mechanism and function of splicing is largely based on data obtained using biochemical and large-scale "omic" approaches, microscopy has been instrumental to address questions related to timing. Experiments done with the electron microscope paved the way to the discovery of splicing and provided unequivocal evidence that splicing can occur co-transcriptionally. More recently, live-cell microscopy introduced a technical breakthrough that allows real-time visualization of splicing dynamics. We discuss here some of the microscopy advances that provided the basis for the current conceptual view of the splicing process and we outline a most recent development that permits direct measurement, in living cells, of the time it takes to synthesize and excise an intron from individual pre-mRNA molecules.


Assuntos
Íntrons , Precursores de RNA/genética , Processamento de RNA/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Genoma , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
16.
Cell Rep ; 4(6): 1144-55, 2013 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-24035393

RESUMO

Removal of introns from pre-messenger RNAs (pre-mRNAs) via splicing provides a versatile means of genetic regulation that is often disrupted in human diseases. To decipher how splicing occurs in real time, we directly examined with single-molecule sensitivity the kinetics of intron excision from pre-mRNA in the nucleus of living human cells. By using two different RNA labeling methods, MS2 and λN, we show that ß-globin introns are transcribed and excised in 20-30 s. Furthermore, we show that replacing the weak polypyrimidine (Py) tract in mouse immunoglobulin µ (IgM) pre-mRNA by a U-rich Py decreases the intron lifetime, thus providing direct evidence that splice-site strength influences splicing kinetics. We also found that RNA polymerase II transcribes at elongation rates ranging between 3 and 6 kb min(-1) and that transcription can be rate limiting for splicing. These results have important implications for a mechanistic understanding of cotranscriptional splicing regulation in the live-cell context.


Assuntos
Íntrons , Processamento de RNA , RNA Mensageiro/genética , Animais , Células HeLa , Humanos , Camundongos , Sítios de Splice de RNA/genética , RNA Mensageiro/metabolismo , Globinas beta/genética
17.
J Virol ; 87(21): 11637-47, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23966407

RESUMO

Rabies virus (RABV) causes a fatal zoonotic encephalitis. Disease symptoms require replication and spread of the virus within neuronal cells; however, in infected animals as well as in cell culture the virus replicates in a broad range of cell types. Here we use a single-cycle RABV and a recombinant vesicular stomatitis virus (rVSV) in which the glycoprotein (G) was replaced with that of RABV (rVSV RABV G) to examine RABV uptake into the African green monkey kidney cell line BS-C-1. Combining biochemical studies and real-time spinning-disk confocal fluorescence microscopy, we show that the predominant entry pathway of RABV particles into BS-C-1 cells is clathrin dependent. Viral particles enter cells in pits with elongated structures and incomplete clathrin coats which depend upon actin to complete the internalization process. By measuring the time of internalization and the abundance of the clathrin adaptor protein AP2, we further show that the pits that internalize RABV particles are similar to those that internalize VSV particles. Pharmacological perturbations of dynamin or of actin polymerization inhibit productive infection, linking our observations on particle uptake with viral infectivity. This work extends to RABV particles the finding that clathrin-mediated endocytosis of rhabdoviruses proceeds through incompletely coated pits which depend upon actin.


Assuntos
Actinas/metabolismo , Clatrina/metabolismo , Endocitose , Células Epiteliais/virologia , Vírus da Raiva/fisiologia , Internalização do Vírus , Animais , Linhagem Celular , Cercopithecus aethiops
18.
Immunity ; 38(6): 1164-75, 2013 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-23770227

RESUMO

Stromal-derived follicular dendritic cells (FDCs) are a major reservoir for antigen that are essential for formation of germinal centers, the site where memory and effector B cells differentiate. A long-standing question is how FDCs retain antigen in its native form for extended periods and how they display it to specific B cells. Here we found that FDCs acquired complement-coated immune complexes (ICs) from noncognate B cells via complement receptors 1 and 2 (CD35 and CD21, respectively) and rapidly internalized them by an actin-dependent pathway. ICs were retained intact within a nondegradative cycling compartment and were displayed periodically on the cell surface where they were accessible to antigen-specific B cells. This would explain how antigens are protected from damage and retained over long periods of time, while remaining accessible for B cells.


Assuntos
Complexo Antígeno-Anticorpo/metabolismo , Antígenos/metabolismo , Linfócitos B/imunologia , Células Dendríticas Foliculares/imunologia , Actinas/metabolismo , Animais , Apresentação do Antígeno , Complexo Antígeno-Anticorpo/imunologia , Antígenos/imunologia , Células Cultivadas , Endocitose/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Receptores de Complemento 3b/metabolismo , Receptores de Complemento 3d/metabolismo
19.
J Cell Biol ; 201(3): 449-65, 2013 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-23629967

RESUMO

Basic mechanisms by which cellular barriers sense and respond to integrity disruptions remain poorly understood. Despite its tenuous structure and constitutive exposure to disruptive strains, the vascular endothelium exhibits robust barrier function. We show that in response to micrometer-scale disruptions induced by transmigrating leukocytes, endothelial cells generate unique ventral lamellipodia that propagate via integrins toward and across these "micro-wounds" to close them. This novel actin remodeling activity progressively healed multiple micro-wounds in succession and changed direction during this process. Mechanical probe-induced micro-wounding of both endothelia and epithelia suggests that ventral lamellipodia formed as a response to force imbalance and specifically loss of isometric tension. Ventral lamellipodia were enriched in the Rac1 effectors cortactin, IQGAP, and p47Phox and exhibited localized production of hydrogen peroxide. Together with Apr2/3, these were functionally required for effective micro-wound healing. We propose that barrier disruptions are detected as local release of isometric tension/force unloading, which is directly coupled to reactive oxygen species-dependent self-restorative actin remodeling dynamics.


Assuntos
Células Endoteliais da Veia Umbilical Humana/fisiologia , Pseudópodes/fisiologia , Migração Transendotelial e Transepitelial , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Fenômenos Biomecânicos , Adesão Celular , Células Cultivadas , Técnicas de Cocultura , Cortactina/metabolismo , Humanos , Linfócitos/fisiologia , Microscopia de Fluorescência , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico , Imagem com Lapso de Tempo , Cicatrização , Proteínas rac1 de Ligação ao GTP/metabolismo
20.
Mol Biol Cell ; 24(8): 1196-207, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23427267

RESUMO

Polarized epithelial cells that line the digestive, respiratory, and genitourinary tracts form a barrier that many viruses must breach to infect their hosts. Current understanding of cell entry by mammalian reovirus (MRV) virions and infectious subvirion particles (ISVPs), generated from MRV virions by extracellular proteolysis in the digestive tract, are mostly derived from in vitro studies with nonpolarized cells. Recent live-cell imaging advances allow us for the first time to visualize events at the apical surface of polarized cells. In this study, we used spinning-disk confocal fluorescence microscopy with high temporal and spatial resolution to follow the uptake and trafficking dynamics of single MRV virions and ISVPs at the apical surface of live polarized Madin-Darby canine kidney cells. Both types of particles were internalized by clathrin-mediated endocytosis, but virions and ISVPs exhibited strikingly different trafficking after uptake. While virions reached early and late endosomes, ISVPs did not and instead escaped the endocytic pathway from an earlier location. This study highlights the broad advantages of using live-cell imaging combined with single-particle tracking for identifying key steps in cell entry by viruses.


Assuntos
Orthoreovirus de Mamíferos/fisiologia , Internalização do Vírus , Animais , Transporte Biológico , Linhagem Celular , Polaridade Celular , Vesículas Revestidas por Clatrina/virologia , Invaginações Revestidas da Membrana Celular/virologia , Cães , Endocitose , Endossomos/virologia , Interações Hospedeiro-Patógeno , Cinética , Microscopia de Fluorescência , Análise de Célula Única , Vírion/fisiologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA