RESUMO
The formation of dynamic protein filaments contributes to various biological functions by clustering individual molecules together and enhancing their binding to ligands. We report such a propensity for the BTB domains of certain proteins from the ZBTB family, a large eukaryotic transcription factor family implicated in differentiation and cancer. Working with Xenopus laevis and human proteins, we solved the crystal structures of filaments formed by dimers of the BTB domains of ZBTB8A and ZBTB18 and demonstrated concentration-dependent higher-order assemblies of these dimers in solution. In cells, the BTB-domain filamentation supports clustering of full-length human ZBTB8A and ZBTB18 into dynamic nuclear foci and contributes to the ZBTB18-mediated repression of a reporter gene. The BTB domains of up to 21 human ZBTB family members and two related proteins, NACC1 and NACC2, are predicted to behave in a similar manner. Our results suggest that filamentation is a more common feature of transcription factors than is currently appreciated.
Assuntos
Domínio BTB-POZ , Fatores de Transcrição , Proteínas de Xenopus , Animais , Humanos , Núcleo Celular/metabolismo , Núcleo Celular/genética , Cristalografia por Raios X , Células HEK293 , Modelos Moleculares , Ligação Proteica , Multimerização Proteica , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/química , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Xenopus laevis , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/químicaRESUMO
Poly(ADP-ribose) polymerase 1 (PARP1) has emerged as a central target for cancer therapies due to the ability of PARP inhibitors to specifically kill tumors deficient for DNA repair by homologous recombination. Upon DNA damage, PARP1 quickly binds to DNA breaks and triggers ADP-ribosylation signaling. ADP-ribosylation is important for the recruitment of various factors to sites of damage, as well as for the timely dissociation of PARP1 from DNA breaks. Indeed, PARP1 becomes trapped at DNA breaks in the presence of PARP inhibitors, a mechanism underlying the cytotoxitiy of these inhibitors. Therefore, any cellular process influencing trapping is thought to impact PARP inhibitor efficiency, potentially leading to acquired resistance in patients treated with these drugs. There are numerous ADP-ribosylation targets after DNA damage, including PARP1 itself as well as histones. While recent findings reported that the automodification of PARP1 promotes its release from the DNA lesions, the potential impact of other ADP-ribosylated proteins on this process remains unknown. Here, we demonstrate that histone ADP-ribosylation is also crucial for the timely dissipation of PARP1 from the lesions, thus contributing to cellular resistance to PARP inhibitors. Considering the crosstalk between ADP-ribosylation and other histone marks, our findings open interesting perspectives for the development of more efficient PARP inhibitor-driven cancer therapies.
Assuntos
ADP-Ribosilação , Histonas , Poli(ADP-Ribose) Polimerase-1 , Inibidores de Poli(ADP-Ribose) Polimerases , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Histonas/metabolismo , Dano ao DNA , Resistencia a Medicamentos Antineoplásicos/genética , Linhagem Celular Tumoral , Poli(ADP-Ribose) Polimerases/metabolismo , Poli(ADP-Ribose) Polimerases/genéticaRESUMO
ADP-ribosylation signaling orchestrates the recruitment of various repair actors and chromatin remodeling processes promoting access to lesions during the early stages of the DNA damage response. The chromatin remodeler complex ACF, composed of the ATPase subunit SMARCA5/SNF2H and the cofactor ACF1/BAZ1A, is among the factors that accumulate at DNA lesions in an ADP-ribosylation dependent manner. In this work, we show that each subunit of the ACF complex accumulates to DNA breaks independently from its partner. Furthermore, we demonstrate that the recruitment of SMARCA5 and ACF1 to sites of damage is not due to direct binding to the ADP-ribose moieties but due to facilitated DNA binding at relaxed ADP-ribosylated chromatin. Therefore, our work provides new insights regarding the mechanisms underlying the timely accumulation of ACF1 and SMARCA5 to DNA lesions, where they contribute to efficient DNA damage resolution.
Assuntos
Cromatina , Dano ao DNA , DNA/metabolismo , Adenosina Trifosfatases/metabolismo , Reparo do DNA , ADP-RibosilaçãoRESUMO
Poly(ADP-ribose) polymerase 1 (PARP1) activity is regulated by its co-factor histone poly(ADP-ribosylation) factor 1 (HPF1). The complex formed by HPF1 and PARP1 catalyzes ADP-ribosylation of serine residues of proteins near DNA breaks, mainly PARP1 and histones. However, the effect of HPF1 on DNA repair regulated by PARP1 remains unclear. Here, we show that HPF1 controls prolonged histone ADP-ribosylation in the vicinity of the DNA breaks by regulating both the number and length of ADP-ribose chains. Furthermore, we demonstrate that HPF1-dependent histone ADP-ribosylation triggers the rapid unfolding of chromatin, facilitating access to DNA at sites of damage. This process promotes the assembly of both the homologous recombination and non-homologous end joining repair machineries. Altogether, our data highlight the key roles played by the PARP1/HPF1 complex in regulating ADP-ribosylation signaling as well as the conformation of damaged chromatin at early stages of the DNA damage response.
Assuntos
Cromatina , Histonas , Histonas/metabolismo , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerase-1/genética , ADP-Ribosilação , Dano ao DNA , Reparo do DNA , DNA/metabolismoRESUMO
The DNA-glycosylase OGG1 oversees the detection and clearance of the 7,8-dihydro-8-oxoguanine (8-oxoG), which is the most frequent form of oxidized base in the genome. This lesion is deeply buried within the double-helix and its detection requires careful inspection of the bases by OGG1 via a mechanism that remains only partially understood. By analyzing OGG1 dynamics in the nucleus of living human cells, we demonstrate that the glycosylase constantly samples the DNA by rapidly alternating between diffusion within the nucleoplasm and short transits on the DNA. This sampling process, that we find to be tightly regulated by the conserved residue G245, is crucial for the rapid recruitment of OGG1 at oxidative lesions induced by laser micro-irradiation. Furthermore, we show that residues Y203, N149 and N150, while being all involved in early stages of 8-oxoG probing by OGG1 based on previous structural data, differentially regulate the sampling of the DNA and recruitment to oxidative lesions.
Assuntos
DNA Glicosilases , Humanos , Núcleo Celular/genética , Núcleo Celular/metabolismo , DNA/química , DNA Glicosilases/metabolismo , Reparo do DNARESUMO
TET (ten-eleven translocation) enzymes initiate active cytosine demethylation via the oxidation of 5-methylcytosine. TET1 is composed of a C-terminal domain, which bears the catalytic activity of the enzyme, and a N-terminal region that is less well characterized except for the CXXC domain responsible for the targeting to CpG islands. While cytosine demethylation induced by TET1 promotes transcription, this protein also interacts with chromatin-regulating factors that rather silence this process, the coordination between these two opposite functions of TET1 being unclear. In the present work, we uncover a new function of the N-terminal part of the TET1 protein in the regulation of the chromatin architecture. This domain of the protein promotes the establishment of a compact chromatin architecture displaying reduced exchange rate of core histones and partial dissociation of the histone linker. This chromatin reorganization process, which does not rely on the CXXC domain, is associated with a global shutdown of transcription and an increase in heterochromatin-associated histone epigenetic marks. Based on these findings, we propose that the dense chromatin organization generated by the N-terminal domain of TET1 could contribute to restraining the transcription enhancement induced by the DNA demethylation activity of this enzyme.
Assuntos
Cromatina , Metilação de DNA , 5-Metilcitosina/metabolismo , Cromatina/genética , Citosina/metabolismo , Histonas/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismoRESUMO
Poly(ADP-ribose) polymerase (PARP) inhibitors are used in the treatment of BRCA-deficient cancers, with treatments currently extending toward other homologous recombination defective tumors. In a genome-wide CRISPR knockout screen with olaparib, we identify ALC1 (Amplified in Liver Cancer 1)-a cancer-relevant poly(ADP-ribose)-regulated chromatin remodeling enzyme-as a key modulator of sensitivity to PARP inhibitor. We found that ALC1 can remove inactive PARP1 indirectly through binding to PARylated chromatin. Consequently, ALC1 deficiency enhances trapping of inhibited PARP1, which then impairs the binding of both nonhomologous end-joining and homologous recombination repair factors to DNA lesions. We also establish that ALC1 overexpression, a common feature in multiple tumor types, reduces the sensitivity of BRCA-deficient cells to PARP inhibitors. Together, we conclude that ALC1-dependent PARP1 mobilization is a key step underlying PARP inhibitor resistance.
Assuntos
Cromatina , DNA Helicases , Proteínas de Ligação a DNA , Inibidores de Poli(ADP-Ribose) Polimerases , Linhagem Celular Tumoral , Cromatina/genética , Montagem e Desmontagem da Cromatina , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
The addition of poly(ADP-ribose) (PAR) chains along the chromatin fiber due to PARP1 activity regulates the recruitment of multiple factors to sites of DNA damage. In this manuscript, we investigated how, besides direct binding to PAR, early chromatin unfolding events controlled by PAR signaling contribute to recruitment to DNA lesions. We observed that different DNA-binding, but not histone-binding, domains accumulate at damaged chromatin in a PAR-dependent manner, and that this recruitment correlates with their affinity for DNA. Our findings indicate that this recruitment is promoted by early PAR-dependent chromatin remodeling rather than direct interaction with PAR. Moreover, recruitment is not the consequence of reduced molecular crowding at unfolded damaged chromatin but instead originates from facilitated binding to more exposed DNA. These findings are further substantiated by the observation that PAR-dependent chromatin remodeling at DNA lesions underlies increased DNAse hypersensitivity. Finally, the relevance of this new mode of PAR-dependent recruitment to DNA lesions is demonstrated by the observation that reducing the affinity for DNA of both CHD4 and HP1α, two proteins shown to be involved in the DNA-damage response, strongly impairs their recruitment to DNA lesions.
Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Cromatina/metabolismo , Dano ao DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Poli Adenosina Difosfato Ribose/fisiologia , Sítios de Ligação/genética , Células Cultivadas , Cromatina/química , Homólogo 5 da Proteína Cromobox , Humanos , Conformação de Ácido Nucleico , Poli Adenosina Difosfato Ribose/metabolismo , Ligação ProteicaRESUMO
Genetically encoded Förster's Resonance Energy Transfer (FRET) biosensors are indispensable tools to sense the spatiotemporal dynamics of signal transduction pathways. Investigating the crosstalk between different signaling pathways is becoming increasingly important to follow cell development and fate programs. To this end, FRET biosensors must be optimized to monitor multiple biochemical activities simultaneously and in single cells. In addition, their sensitivity must be increased to follow their activation even when the abundance of the biosensor is low. We describe here the development of a second generation of Aurora kinase A/AURKA biosensors. First, we adapt the original AURKA biosensor-GFP-AURKA-mCherry-to multiplex FRET by using dark acceptors as ShadowG or ShadowY. Then, we use the novel superYFP acceptor protein to measure FRET by 2-color Fluorescence Cross-Correlation Spectroscopy, in cytosolic regions where the abundance of AURKA is extremely low and undetectable with the original AURKA biosensor. These results pave the way to the use of FRET biosensors to follow AURKA activation in conjunction with substrate-based activity biosensors. In addition, they open up the possibility of tracking the activation of small pools of AURKA and its interaction with novel substrates, which would otherwise remain undetectable with classical biochemical approaches.
Assuntos
Aurora Quinase A/análise , Aurora Quinase A/genética , Técnicas Biossensoriais , Transferência Ressonante de Energia de Fluorescência , Mitose/genética , Aurora Quinase A/metabolismo , HumanosRESUMO
One of the first events to occur upon DNA damage is the local opening of the compact chromatin architecture, facilitating access of repair proteins to DNA lesions. This early relaxation is triggered by poly(ADP-ribosyl)ation by PARP1 in addition to ATP-dependent chromatin remodeling. CHD4 recruits to DNA breaks in a PAR-dependent manner, although it lacks any recognizable PAR-binding domain, and has the ability to relax chromatin structure. However, its role in chromatin relaxation at the site of DNA damage has not been explored. Using a live cell fluorescence three-hybrid assay, we demonstrate that the recruitment of CHD4 to DNA damage, while being poly(ADP-ribosyl)ation-dependent, is not through binding poly(ADP-ribose). Additionally, we show that CHD3 is recruited to DNA breaks in the same manner as CHD4 and that both CHD3 and CHD4 play active roles in chromatin remodeling at DNA breaks. Together, our findings reveal a two-step mechanism for DNA damage induced chromatin relaxation in which PARP1 and the PAR-binding remodeler activities of Alc1/CHD1L induce an initial chromatin relaxation phase that promotes the subsequent recruitment of CHD3 and CHD4 via binding to DNA for further chromatin remodeling at DNA breaks.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Quebras de DNA , DNA Helicases/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Linhagem Celular , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/fisiologia , Transdução de SinaisRESUMO
Monitoring of different signalling enzymes in a single assay using multiplex biosensing provides a multidimensional workspace to elucidate biological processes, signalling pathway crosstalk, and determine precise sequence of events at the single living cell level. In this study, we interrogate the complexity in cAMP/PKA-MAPK/ERK1&2 crosstalk by using multi-parameter biosensing experiments to correlate biochemical activities simultaneously in time and space. Using a single excitation wavelength dual colour FLIM method we are able to detect fluorescence lifetime images of two donors to simultaneously measure PKA and ERK1&2 kinase activities in the same cellular localization by using FRET biosensors. To this end, we excite two FRET donors mTFP1 and LSSmOrange with a 440 nm wavelength and we alleviate spectral bleed-through associated limitations with the very dim-fluorescent acceptor ShadowG for mTFP1 and the red-shifted mKate2 for LSSmOrange. The simultaneous recording of PKA and ERK1&2 kinase activities reveals concomitant EGF-mediated activations of both kinases in HeLa cells. Under these conditions the subsequent Forskolin-induced cAMP release reverses the transient increase of EGF-mediated ERK1&2 kinase activity while reinforcing PKA activation. Here we propose a validated methodology for multiparametric kinase biosensing in living cells using FRET-FLIM.
Assuntos
Técnicas Biossensoriais/métodos , Proteínas Quinases Dependentes de AMP Cíclico/análise , Técnicas Citológicas/métodos , Proteína Quinase 1 Ativada por Mitógeno/análise , Proteína Quinase 3 Ativada por Mitógeno/análise , Imagem Óptica/métodos , AMP Cíclico/metabolismo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/metabolismo , Células HeLa , Humanos , Transdução de SinaisRESUMO
Chromatin relaxation is one of the earliest cellular responses to DNA damage. However, what determines these structural changes, including their ATP requirement, is not well understood. Using live-cell imaging and laser microirradiation to induce DNA lesions, we show that the local chromatin relaxation at DNA damage sites is regulated by PARP1 enzymatic activity. We also report that H1 is mobilized at DNA damage sites, but, since this mobilization is largely independent of poly(ADP-ribosyl)ation, it cannot solely explain the chromatin relaxation. Finally, we demonstrate the involvement of Alc1, a poly(ADP-ribose)- and ATP-dependent remodeler, in the chromatin-relaxation process. Deletion of Alc1 impairs chromatin relaxation after DNA damage, while its overexpression strongly enhances relaxation. Altogether our results identify Alc1 as an important player in the fast kinetics of the NAD+- and ATP-dependent chromatin relaxation upon DNA damage in vivo.
Assuntos
DNA Helicases/metabolismo , DNA Helicases/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Poli Adenosina Difosfato Ribose/metabolismo , Técnicas de Cultura de Células , Cromatina/fisiologia , Montagem e Desmontagem da Cromatina/fisiologia , DNA , Dano ao DNA , Reparo do DNA/fisiologia , Histonas/metabolismo , Humanos , Nucleossomos , Imagem Óptica , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli Adenosina Difosfato Ribose/fisiologia , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
DNA replication is essential to maintain genome integrity in S phase of the cell division cycle. Accumulation of stalled replication forks is a major source of genetic instability, and likely constitutes a key driver of tumorigenesis. The mechanisms of regulation of replication fork progression have therefore been extensively investigated, in particular with DNA combing, an optical mapping technique that allows the stretching of single molecules and the mapping of active region for DNA synthesis by fluorescence microscopy. DNA linearization in nanochannels has been successfully used to probe genomic information patterns along single chromosomes, and has been proposed to be a competitive alternative to DNA combing. Yet this conjecture remains to be confirmed experimentally. Here, two complementary techniques are established to detect the genomic distribution of tracks of newly synthesized DNA in human cells by optical mapping in nanochannels. Their respective advantages and limitations are compared, and applied them to detect deregulations of the replication program induced by the antitumor drug hydroxyurea. The developments here thus broaden the field of applications accessible to nanofluidic technologies, and can be used in the future as part for molecular diagnostics in the context of high throughput cancer drug screening.
Assuntos
Replicação do DNA , Nanopartículas/química , Imagem Óptica/métodos , Difusão , Humanos , Microscopia de Força Atômica , Nanopartículas/ultraestrutura , Espectrometria por Raios X , Compostos de Vanádio/químicaRESUMO
In contrast to cytoplasmic organelles, which are usually separated from the rest of the cell by phospholipid membranes, nuclear compartments are readily accessible to diffusing proteins and must rely on different mechanisms to maintain their integrity. Specific interactions between scaffolding proteins are known to have important roles for the formation and maintenance of nuclear structures. General physical mechanisms such as molecular crowding, phase separation or colloidal behavior have also been suggested, but their physiological significance remains uncertain. For macromolecular crowding, a role in the maintenance of nucleoli and promyelocytic leukemia (PML) nuclear bodies has been shown. Here, we tested whether a modulation of the compaction state of chromatin, which directly influences the local crowding state, has an impact on the formation and maintenance of densely packed heterochromatin. By osmotic perturbations, we could modify the packing state of chromatin in a controlled manner and show that chromatin compaction, which is associated with increased crowding conditions, is not, per se, sufficient to initiate the formation of new bona fide heterochromatin structures nor is it necessary to maintain already established heterochromatin domains. In consequence, if an increase in crowding induced by chromatin compaction maybe an early step in heterochromatin formation, specific protein-protein interactions are nevertheless required to make heterochromatin long lasting and independent of the crowding state.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/química , Cromatina/metabolismo , Heterocromatina/metabolismo , Animais , Eucromatina/química , Eucromatina/metabolismo , Heterocromatina/química , Histonas/análise , Histonas/genética , Histonas/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Células NIH 3T3 , Pressão Osmótica , Fotodegradação , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Espectrometria de FluorescênciaRESUMO
Despite our rapidly growing knowledge about the human genome, we do not know all of the genes required for some of the most basic functions of life. To start to fill this gap we developed a high-throughput phenotypic screening platform combining potent gene silencing by RNA interference, time-lapse microscopy and computational image processing. We carried out a genome-wide phenotypic profiling of each of the approximately 21,000 human protein-coding genes by two-day live imaging of fluorescently labelled chromosomes. Phenotypes were scored quantitatively by computational image processing, which allowed us to identify hundreds of human genes involved in diverse biological functions including cell division, migration and survival. As part of the Mitocheck consortium, this study provides an in-depth analysis of cell division phenotypes and makes the entire high-content data set available as a resource to the community.
Assuntos
Divisão Celular/genética , Genoma Humano/genética , Microscopia de Fluorescência/métodos , Fenótipo , Animais , Movimento Celular/genética , Sobrevivência Celular/genética , Cor , Técnicas de Silenciamento de Genes , Genes/genética , Células HeLa , Humanos , Cinética , Camundongos , Mitose/genética , Interferência de RNA , Reprodutibilidade dos Testes , Fuso Acromático/genética , Fuso Acromático/metabolismo , Fatores de TempoRESUMO
OBJECTIVES: To identify independent risk factors for endometritis and urinary tract infection (UTI) after vaginal delivery, and to monitor changes in nosocomial infection rates and derive benchmarks for prevention. DESIGN: Prospective study. METHODS: We analyzed routine surveillance data for all vaginal deliveries between January 1997 and December 2003 at 66 maternity units participating in the Mater Sud-Est surveillance network. Adjusted odds ratios for risk of endometritis or UTI were obtained using a logistic regression model. RESULTS: The overall incidence rates were 0.5% for endometritis and 0.3% for UTI. There was a significant decrease in the incidence and risk of endometritis but not of UTI during the 7-year period. Significant risk factors for endometritis were fever during labor, parity of 1, and instrumental delivery and/or manual removal of the placenta. Significant risk factors for UTI were urinary infection on admission, premature rupture of membranes (more than 12 hours before admission), blood loss of more than 800 mL, parity of 1, instrumental delivery, and receipt of more than 5 vaginal digital examinations. Each maternity unit received a poster showing graphs of the number of expected and observed cases of UTI and endometritis associated with vaginal deliveries, which enabled each maternity unit to determine their rank within the network and to initiate prevention programs. CONCLUSIONS: Although routine surveillance means additional work for maternity units, our results demonstrate the usefulness of regular targeted monitoring of risk factors and of the most common nosocomial infections in obstetrics. Most of the information needed for monitoring is already present in the patients' records.
Assuntos
Infecção Hospitalar/epidemiologia , Parto Obstétrico/efeitos adversos , Parto Obstétrico/métodos , Endometrite/epidemiologia , Infecções Urinárias/epidemiologia , Adulto , Infecção Hospitalar/etiologia , Parto Obstétrico/estatística & dados numéricos , Endometrite/etiologia , Feminino , França/epidemiologia , Humanos , Incidência , Modelos Logísticos , Vigilância da População/métodos , Gravidez , Fatores de Risco , Infecções Urinárias/etiologiaRESUMO
OBJECTIVE: To evaluate whether the adjusted rates of surgical site infection (SSI) and urinary tract infection (UTI) after cesarean delivery decrease in maternity units that perform active healthcare-associated infection surveillance. DESIGN: Trend analysis by means of multiple logistic regression. SETTING: A total of 80 maternity units participating in the Mater Sud-Est surveillance network. PATIENTS: A total of 37,074 cesarean deliveries were included in the surveillance from January 1, 1997, through December 31, 2003. METHODS: We used a logistic regression model to estimate risk-adjusted post-cesarean delivery infection odds ratios. The variables included were the maternity units' annual rate of operative procedures, the level of dispensed neonatal care, the year of delivery, maternal risk factors, and the characteristics of cesarean delivery. The trend of risk-adjusted odds ratios for SSI and UTI during the study period was studied by linear regression. RESULTS: The crude rates of SSI and UTI after cesarean delivery were 1.5% (571 of 37,074 patients) and 1.8% (685 of 37,074 patients), respectively. During the study period, the decrease in SSI and UTI adjusted odds ratios was statistically significant (R=-0.823 [P=.023] and R=-0.906 [P=.005], respectively). CONCLUSION: Reductions of 48% in the SSI rate and 52% in the UTI rate were observed in the maternity units. These unbiased trends could be related to progress in preventive practices as a result of the increased dissemination of national standards and a collaborative surveillance with benchmarking of rates.
Assuntos
Cesárea/efeitos adversos , Infecção da Ferida Cirúrgica/epidemiologia , Infecções Urinárias/epidemiologia , Adulto , Infecção Hospitalar/complicações , Infecção Hospitalar/epidemiologia , Feminino , França/epidemiologia , Unidades Hospitalares , Humanos , Modelos Logísticos , Gravidez , Medição de Risco/métodos , Fatores de Risco , Vigilância de Evento Sentinela , Infecção da Ferida Cirúrgica/etiologia , Infecções Urinárias/etiologiaRESUMO
Myosin Va (MyoVa) is a prime candidate for controlling actin-based organelle motion in neurons and neuroendocrine cells. Its function in secretory granule (SG) trafficking was investigated in enterochromaffin cells by wide-field and total internal reflection fluorescence microscopy. The distribution of endogenous MyoVa partially overlapped with SGs and microtubules. Impairing MyoVa function by means of a truncated construct (MyoVa tail) or RNA interference prevented the formation of SG-rich regions at the cell periphery and reduced SG density in the subplasmalemmal region. Individual SG trajectories were tracked to analyze SG mobility. A wide distribution of their diffusion coefficient, D(xy), was observed. Almost immobile SGs (D(xy) < 5 x 10(-4) microm2 x s(-1)) were considered as docked at the plasma membrane based on two properties: (1) SGs that undergo exocytosis have a D(xy) below this threshold value for at least 2 s before fusion; (2) a negative autocorrelation of the vertical motion was found in subtrajectories with a D(xy) below the threshold. Using this criterion of docking, we found that the main effect of MyoVa inhibition was to reduce the number of docked granules, leading to reduced secretory responses. Surprisingly, this reduction was not attributable to a decreased transport of SGs toward release sites. In contrast, MyoVa silencing reduced the occurrence of long-lasting, but not short-lasting, docking periods. We thus propose that, despite its known motor activity, MyoVa directly mediates stable attachment of SGs at the plasma membrane.
Assuntos
Membrana Celular/fisiologia , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Vesículas Secretórias/fisiologia , Células Cultivadas , Humanos , Vesículas Transportadoras/fisiologiaRESUMO
BON cells are human carcinoid cells that secrete serotonin (5-HT) and various peptides. Secretion of [(3)H]5-HT by cell cultures was investigated. Acetylcholine (Ach) stimulated secretion through a somatostatin-sensitive muscarinic pathway, whereas isoproterenol was inefficient. [(3)H]5-HT secretion also was induced by Ca(2+) in the presence of the ionophore A-23187 or after digitonin permeabilization. These two processes were insensitive to stomatostatin. Ba(2+) induced an efficient somatostatin-sensitive [(3)H]5-HT secretory response. Secretion also was analyzed at the single-cell level, using carbon fiber amperometry and evanescent-field fluorescence microscopy, after labeling the secretory vesicles by transfection of the cells with a NPY-GFP construct. Both techniques revealed slow kinetics of secretory responses, suggesting that ready-to-fuse vesicles do not accumulate in these cells. Single secretory vesicles were imaged either in resting conditions or after addition of Ca(2+) ions to digitonin-permeabilized cells. The three-dimensional movements of the vesicles before exocytosis were analyzed. The mean velocity of vesicles that released their content was lower than that of silent ones. Even in the case of mobile vesicles, exocytosis often was preceded by a period of arrest lasting at least 15 seconds, consistent with a docking/priming step.