RESUMO
Understanding how the nuclear pore complex (NPC) is assembled is of fundamental importance to grasp the mechanisms behind its essential function and understand its role during the evolution of eukaryotes1-4. There are at least two NPC assembly pathways-one during the exit from mitosis and one during nuclear growth in interphase-but we currently lack a quantitative map of these events. Here we use fluorescence correlation spectroscopy calibrated live imaging of endogenously fluorescently tagged nucleoporins to map the changes in the composition and stoichiometry of seven major modules of the human NPC during its assembly in single dividing cells. This systematic quantitative map reveals that the two assembly pathways have distinct molecular mechanisms, in which the order of addition of two large structural components, the central ring complex and nuclear filaments are inverted. The dynamic stoichiometry data was integrated to create a spatiotemporal model of the NPC assembly pathway and predict the structures of postmitotic NPC assembly intermediates.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear , Humanos , Interfase , Mitose , Poro Nuclear/química , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Espectrometria de FluorescênciaRESUMO
Mitotic bookmarking transcription factors remain bound to chromosomes during mitosis and were proposed to regulate phenotypic maintenance of stem and progenitor cells at the mitosis-to-G1 (M-G1) transition. However, mitotic bookmarking remains largely unexplored in most stem cell types, and its functional relevance for cell fate decisions remains unclear. Here we screened for mitotic chromosome binding within the pluripotency network of embryonic stem (ES) cells and show that SOX2 and OCT4 remain bound to mitotic chromatin through their respective DNA-binding domains. Dynamic characterization using photobleaching-based methods and single-molecule imaging revealed quantitatively similar specific DNA interactions, but different nonspecific DNA interactions, of SOX2 and OCT4 with mitotic chromatin. Using ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) to assess the genome-wide distribution of SOX2 on mitotic chromatin, we demonstrate the bookmarking activity of SOX2 on a small set of genes. Finally, we investigated the function of SOX2 mitotic bookmarking in cell fate decisions and show that its absence at the M-G1 transition impairs pluripotency maintenance and abrogates its ability to induce neuroectodermal differentiation but does not affect reprogramming efficiency toward induced pluripotent stem cells. Our study demonstrates the mitotic bookmarking property of SOX2 and reveals its functional importance in pluripotency maintenance and ES cell differentiation.
Assuntos
Diferenciação Celular/genética , Mitose/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Animais , Reprogramação Celular/genética , Cromatina/metabolismo , Células-Tronco Embrionárias , Fase G1 , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Células NIH 3T3 , Placa Neural/citologia , Placa Neural/fisiologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Ligação ProteicaRESUMO
Transcription factors (TFs) regulate gene expression in both prokaryotes and eukaryotes by recognizing and binding to specific DNA promoter sequences. In higher eukaryotes, it remains unclear how the duration of TF binding to DNA relates to downstream transcriptional output. Here, we address this question for the transcriptional activator NF-κB (p65), by live-cell single molecule imaging of TF-DNA binding kinetics and genome-wide quantification of p65-mediated transcription. We used mutants of p65, perturbing either the DNA binding domain (DBD) or the protein-protein transactivation domain (TAD). We found that p65-DNA binding time was predominantly determined by its DBD and directly correlated with its transcriptional output as long as the TAD is intact. Surprisingly, mutation or deletion of the TAD did not modify p65-DNA binding stability, suggesting that the p65 TAD generally contributes neither to the assembly of an "enhanceosome," nor to the active removal of p65 from putative specific binding sites. However, TAD removal did reduce p65-mediated transcriptional activation, indicating that protein-protein interactions act to translate the long-lived p65-DNA binding into productive transcription.
Assuntos
NF-kappa B/genética , Fator de Transcrição RelA/genética , Fatores de Transcrição/genética , Ativação Transcricional/genética , Proteínas de Ligação a DNA/genética , Expressão Gênica/genética , Genoma Humano/genética , Células HeLa , Humanos , Cinética , Proteínas Mutantes/química , Proteínas Mutantes/genética , NF-kappa B/química , Domínios e Motivos de Interação entre Proteínas/genética , Imagem Individual de Molécula , Fator de Transcrição RelA/química , Fatores de Transcrição/químicaRESUMO
Cytoplasmic flows are an ubiquitous feature of biological systems, in particular in large cells, such as oocytes and eggs in early animal development. Here we show that cytoplasmic flows in starfish oocytes, which can be imaged well with transmission light microscopy, are fully determined by the cortical dynamics during surface contraction waves. We first show that the dynamics of the oocyte surface is highly symmetric around the animal-vegetal axis. We then mathematically solve the Stokes equation for flows inside a deforming sphere using the measured surface displacements as boundary conditions. Our theoretical predictions agree very well with the intracellular flows quantified by particle image velocimetry, proving that during this stage the starfish cytoplasm behaves as a simple Newtonian fluid on the micrometer scale. We calculate the pressure field inside the oocyte and find that its gradient is too small as to explain polar body extrusion, in contrast to earlier suggestions. Myosin II inhibition by blebbistatin confirms this conclusion, because it diminishes cell shape changes and hydrodynamic flow, but does not abolish polar body formation.
Assuntos
Citoplasma/fisiologia , Oócitos/citologia , Estrelas-do-Mar/fisiologia , Actinas/química , Algoritmos , Animais , Citoplasma/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/química , Imageamento Tridimensional , Modelos Teóricos , Miosina Tipo II/metabolismo , Distribuição Normal , Corpos Polares , Rotação , Água do Mar , Propriedades de SuperfícieRESUMO
The neurotrophin receptor TrkA (also known as NTRK1) is known to be crucially involved in several physio-pathological processes. However, a clear description of the early steps of ligand-induced TrkA responses at the cell plasma membrane is missing. We have exploited single particle tracking and TIRF microscopy to study TrkA membrane lateral mobility and changes of oligomerization state upon binding of diverse TrkA agonists (NGF, NGF R100E HSANV mutant, proNGF and NT-3). We show that, in the absence of ligands, most of the TrkA receptors are fast moving monomers characterized by an average diffusion coefficient of 0.47 µm(2)/second; about 20% of TrkA molecules move at least an order of magnitude slower and around 4% are almost immobile within regions of about 0.6 µm diameter. Ligand binding results in increased slow and/or immobile populations over the fast one, slowing down of non-immobile trajectories and reduction of confinement areas, observations that are consistent with the formation of receptor dimeric and oligomeric states. We demonstrate that the extent of TrkA lateral mobility modification is strictly ligand dependent and that each ligand promotes distinct trajectory patterns of TrkA receptors at the cell membrane (ligand 'fingerprinting' effect). This ligand signature of receptor dynamics results from a differential combination of receptor-binding affinity, intracellular effectors recruited in the signalling platforms and formation of signalling and/or recycling endosome precursors. Thus, our data uncover a close correlation between the initial receptor membrane dynamics triggered upon binding and the specific biological outcomes induced by different ligands for the same receptor.
Assuntos
Receptor trkA/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Humanos , Ligantes , Fosforilação , Ligação Proteica , Receptor trkA/química , Transdução de SinaisRESUMO
OBJECTIVE: Vascular calcification is highly correlated with cardiovascular disease morbidity and mortality. Osteoprotegerin (OPG) is a secreted decoy receptor for receptor activator of NF-κB ligand (RANKL). Inactivation of OPG in apolipoprotein E-deficient (ApoE-/-) mice increases lesion size and calcification. The mechanism(s) by which OPG is atheroprotective and anticalcific have not been entirely determined. We investigated whether OPG-deficient vascular smooth muscle cells (VSMCs) are more susceptible to mineralization and whether RANKL mediates this process. RESULTS: Lesion-free aortas from 12-week-old ApoE-/-OPG-/- mice had spotty calcification, an appearance of osteochondrogenic factors and a decrease of smooth muscle markers when compared to ApoE-/-OPG+/+ aortas. In osteogenic conditions, VSMCs isolated from ApoE-/-OPG-/- (KO-VSMC) mice deposited more calcium than VSMCs isolated from ApoE-/-OPG+/+ (WT-VSMC) mice. Gene expression and biochemical analysis indicated accelerated osteochondrogenic differentiation. Ablation of RANKL signaling in KO-VSMCs rescued the accelerated calcification. While WT-VSMCs did not respond to RANKL treatment, KO-VSMCs responded with enhanced calcification and the upregulation of osteochondrogenic genes. RANKL strongly induced interleukin 6 (IL-6), which partially mediated RANKL-dependent calcification and gene expression in KO-VSMCs. CONCLUSIONS: OPG inhibits vascular calcification by regulating the procalcific effects of RANKL on VSMCs and is thus a possible target for therapeutic intervention.
Assuntos
Apolipoproteínas E/deficiência , Interleucina-6/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Osteoprotegerina/deficiência , Ligante RANK/metabolismo , Transdução de Sinais , Calcificação Vascular/metabolismo , Animais , Apolipoproteínas E/genética , Diferenciação Celular , Células Cultivadas , Condrogênese , Genótipo , Camundongos , Camundongos Knockout , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Osteogênese , Osteoprotegerina/genética , Fenótipo , Ligante RANK/genética , Interferência de RNA , Transdução Genética , Calcificação Vascular/genética , Calcificação Vascular/patologia , Calcificação Vascular/prevenção & controleRESUMO
BACKGROUND: Vascular calcification is highly correlated with cardiovascular disease (CVD) morbidity and mortality, and it is associated with inflammation. Receptor activator of NF-ĸB ligand (RANKL) inhibition in vivo has been shown to reduce vascular calcification in a mouse model of atherosclerosis. Therefore, we tested the hypothesis that RANKL regulates smooth muscle cell (SMC) calcification by modulating macrophage production of pro-calcific cytokines. METHODS: We used a bone marrow-derived macrophage (BMDM)/SMC co-culture system and examined the effects of RANKL on BMDM activation and SMC matrix calcification. RESULTS: Treatment with RANKL alone did not stimulate SMC calcification induced by elevated phosphate. BMDMs differentiated with macrophage colony-stimulating factor and placed in co-culture with SMCs increased phosphate-induced SMC calcification. RANKL added to the BMDM/SMC co-cultures further enhanced SMC calcification. Treatment of BMDMs with RANKL resulted in increased expression of IL-6 and TNF-α. Thus, increased expression of these pro-calcific cytokines in macrophages may mediate RANKL-induced SMC calcification in a paracrine fashion. Addition of neutralizing IL-6 and TNF-α antibodies together with RANKL treatment significantly reduced the RANKL induction of SMC calcification. CONCLUSION: RANKL activation of pro-inflammatory and pro-calcific pathways in macrophages may contribute to vascular calcification and inflammation.
Assuntos
Calcinose/etiologia , Interleucina-6/metabolismo , Macrófagos/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Fosfatos/farmacologia , Ligante RANK/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Calcinose/complicações , Técnicas de Cocultura , Citocinas , Interleucina-6/farmacologia , Macrófagos/metabolismo , Camundongos , Miócitos de Músculo Liso/metabolismo , Osteoclastos/citologia , Osteoprotegerina/biossíntese , Ligante RANK/biossíntese , Receptor Ativador de Fator Nuclear kappa-B/biossíntese , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
OBJECTIVE: Glutamate-cysteine ligase (GCL) is the rate-limiting step in glutathione synthesis. The enzyme is a heterodimer composed of a catalytic subunit, GCLC, and a modifier subunit, GCLM. We generated apolipoprotein E (apoE)-/- mice deficient in GCLM (apoE-/-/Gclm-/-) and transgenic mice that overexpress GCLC specifically in macrophages (apoE-/-/Gclc-Tg) to test the hypothesis that significantly altering the availability of glutathione has a measurable impact on both the initiation and progression of atherosclerosis. METHODS AND RESULTS: Atherosclerotic plaque size and composition were measured in the innominate artery in chow-fed male and female mice at 20, 30, 40, and 50 weeks of age and in the aortic sinus at 40 and 50 weeks of age. The apoE-/-/Gclm-/- mice more rapidly developed complex lesions, whereas the apoE-/-/Gclc-Tg mice had reduced lesion development compared with the littermate apoE-/- control mice. Transplantation of bone marrow from the apoE-/-/Gclm-/- and apoE-/-/Gclc-Tg mice into apoE-/- mice with established lesions also stimulated or inhibited further lesion development at 30 weeks posttransplant. CONCLUSION: Gain and loss of function in the capacity to synthesize glutathione especially in macrophages has reciprocal effects on the initiation and progression of atherosclerosis at multiple sites in apoE-/- mice.
Assuntos
Apolipoproteínas E/deficiência , Aterosclerose/metabolismo , Aterosclerose/patologia , Glutationa/metabolismo , Animais , Apolipoproteínas E/genética , Tronco Braquiocefálico/metabolismo , Tronco Braquiocefálico/patologia , Modelos Animais de Doenças , Progressão da Doença , Feminino , Glutamato-Cisteína Ligase/deficiência , Glutamato-Cisteína Ligase/genética , Lipídeos/sangue , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Seio Aórtico/metabolismo , Seio Aórtico/patologiaRESUMO
In eukaryotes, transcription is a discontinuous process with mRNA being generated in bursts, after the binding of transcription factors (TFs) to regulatory elements on the genome. Live-cell single-molecule microscopy has highlighted that transcriptional bursting can be controlled by tuning TF/DNA binding kinetics. Yet the timescales of these two processes seem disconnected with TF/DNA interactions typically lasting orders of magnitude shorter than transcriptional bursts. To test models that could reconcile these discrepancies, reliable measurements of TF binding kinetics are needed, also accounting for the current limitations in performing these single-molecule measurements at specific regulatory elements. Here, we review the recent studies linking TF binding kinetics to transcriptional bursting and outline some current and future challenges that need to be addressed to provide a microscopic description of transcriptional regulation kinetics.
Assuntos
Fatores de Transcrição , Transcrição Gênica , Sítios de Ligação , Regulação da Expressão Gênica , Cinética , Ligação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The cryopreservation of hematopoietic cells using dimethyl sulfoxide (DMSO) and serum is a common procedure used in transplantation. However, DMSO has clinical and biological side effects due to its toxicity, and serum introduces variation and safety risks. Inspired by natural antifreeze proteins, a novel class of ice-interactive cryoprotectants was developed. The corresponding DMSO-, protein-, and serum-free cryopreservation media candidates were screened through a series of biological assays using human cell lines, peripheral blood cells, and bone marrow cells. XT-Thrive-A and XT-Thrive-B were identified as lead candidates to rival cryopreservation with 10% DMSO in serum based on post-thaw cell survival and short-term proliferation assays. The effectiveness of the novel cryopreservation media in freezing hematopoietic stem cells from human whole bone marrow was assessed by extreme limiting dilution analysis in immunodeficient mice. Stem cell frequencies were measured 12 weeks after transplant based on bone marrow engraftment of erythroid, myeloid, B-lymphoid, and CD34+ progenitors measured by flow cytometry. The recovered numbers of cryopreserved stem cells were similar among XT-Thrive A, XT-Thrive B, and DMSO with serum groups. These findings show that cryoprotectants developed through biomimicry of natural antifreeze proteins offers a substitute for DMSO-based media for the cryopreservation of hematopoietic stem cells.
Assuntos
Criopreservação , Dimetil Sulfóxido , Animais , Antígenos CD34/análise , Sobrevivência Celular , Criopreservação/métodos , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Células-Tronco Hematopoéticas , Humanos , CamundongosRESUMO
Nuclear pore complexes (NPCs) are large macromolecular machines that mediate the traffic between the nucleus and the cytoplasm. In vertebrates, each NPC consists of â¼1000 proteins, termed nucleoporins, and has a mass of more than 100 MDa. While a pseudo-atomic static model of the central scaffold of the NPC has recently been assembled by integrating data from isolated proteins and complexes, many structural components still remain elusive due to the enormous size and flexibility of the NPC. Here, we explored the power of three-dimensional (3D) superresolution microscopy combined with computational classification and averaging to explore the 3D structure of the NPC in single human cells. We show that this approach can build the first integrated 3D structural map containing both central as well as peripheral NPC subunits with molecular specificity and nanoscale resolution. Our unbiased classification of more than 10,000 individual NPCs indicates that the nuclear ring and the nuclear basket can adopt different conformations. Our approach opens up the exciting possibility to relate different structural states of the NPC to function in situ.
Assuntos
Microscopia de Fluorescência/métodos , Complexo de Proteínas Formadoras de Poros Nucleares/ultraestrutura , Poro Nuclear/ultraestrutura , Animais , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Poro Nuclear/metabolismo , Poro Nuclear/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismoRESUMO
We address the contribution of kinase domain structure and catalytic activity to membrane trafficking of TrkA receptor tyrosine kinase. We conduct a systematic comparison between TrkA-wt, an ATP-binding defective mutant (TrkA-K544N) and other mutants displaying separate functional impairments of phosphorylation, ubiquitination, or recruitment of intracellular partners. We find that only K544N mutation endows TrkA with restricted membrane mobility and a substantial increase of cell surface pool already in the absence of ligand stimulation. This mutation is predicted to drive a structural destabilization of the αC helix in the N-lobe by molecular dynamics simulations, and enhances interactions with elements of the actin cytoskeleton. On the other hand, a different TrkA membrane immobilization is selectively observed after NGF stimulation, requires both phosphorylation and ubiquitination to occur, and is most probably related to the signaling abilities displayed by the wt but not mutated receptors. In conclusion, our results allow to distinguish two different TrkA membrane immobilization modes and demonstrate that not all kinase-inactive mutants display identical membrane trafficking.
Assuntos
Receptor trkA/metabolismo , Citoesqueleto de Actina/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Humanos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Fator de Crescimento Neural/farmacologia , Fosforilação/efeitos dos fármacos , Conformação Proteica em alfa-Hélice , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Transporte Proteico , Receptor trkA/química , Receptor trkA/genética , Ubiquitinação/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/química , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
We present here the analysis of experimental Stark effect measurements made using photofragment quantum beat spectroscopy on the |4,0(-)>, |5,0(-)>, |8,0(+)> and |4,0(-)>|2> vibrational states of H(2)O [ Callegari , A. ; et al. Science 2002 , 297 , 993.]. To link the measured Stark coefficients with the dipole surface, we analyze our results using a coupled anharmonic oscillator model, which takes into account the local-mode nature of higly excited OH stretching vibrations in water, and the tunneling between the two equivalent bonds. The large inertial frame tilt associated with the local-mode bond stretching results in a complex interaction between rotational-, vibrational-, and tunneling-motion, all of which become deeply entangled in the Stark coefficients. A perturbational approach makes it possible to analyze the problem at increasingly higher levels of approximation and to disentangle the different contributions, according to the different time scales involved. This simple model reproduces most experimental values to within a few percent, even for these highly vibrationally excited levels, and gives valuable insight into the complex rotational and vibrational motions that link the dipole moment surface with the Stark coefficients.
RESUMO
Efficacy of adult (bone marrow, BM) versus fetal (amniotic fluid, AF) mesenchymal stem cells (MSCs) to replenish damaged rat heart tissues with new cardiovascular cells has not yet been established. We investigated on the differentiation potential of these two rat MSC populations in vitro and in a model of acute necrotizing injury (ANI) induced by cryoinjury. Isolated BM-MSCs and AF-MSCs were characterized by flow cytometry and cytocentrifugation and their potential for osteogenic, adipogenic, and cardiovascular differentiation assayed in vitro using specific induction media. The left anterior ventricular wall of syngeneic Fisher 344 (n = 48) and athymic nude (rNu) rats (n = 6) was subjected to a limited, nontransmural epicardial ANI in the approximately one third of wall thickness without significant hemodynamic effects. The time window for in situ stem cell transplantation was established at day 7 postinjury. Fluorochrome (CMTMR)-labeled BM-MSCs (2 x 10(6)) or AF-MSCs (2 x 10(6)) were injected in syngeneic animals (n = 26) around the myocardial lesion via echocardiographic guidance. Reliability of CMTMR cell tracking in this context was ascertained by transplanting genetically labeled BM-MSCs or AF-MSCs, expressing the green fluorescent protein (GFP), in rNu rats with ANI. Comparison between the two methods of cell tracking 30 days after cell transplantation gave slightly different values (1420,58 +/- 129,65 cells/mm2 for CMTMR labeling and 1613.18 +/- 643.84 cells/mm2 for genetic labeling; p = NS). One day after transplantation about one half CMTMR-labeled AF-MSCs engrafted to the injured heart (778.61 +/- 156.28 cells/mm2) in comparison with BM-MSCs (1434.50 +/- 173.80 cells/mm2, p < 0.01). Conversely, 30 days after cell transplantation survived MSCs were similar: 1275.26 +/- 74.51/mm2 (AF-MSCs) versus 1420.58 +/- 129.65/mm2 for BM-MSCs (p = NS). Apparent survival gain of AF-MSCs between the two time periods was motivated by the cell proliferation rate calculated at day 30, which was lower for BM-MSCs (6.79 +/- 0.48) than AF-MSCs (10.83 +/- 3.50; p < 0.01), in the face of a similar apoptotic index (4.68 +/- 0.20 for BM-MSCs and 4.16 +/- 0.58 for AF-MSCs; p = NS). These cells were also studied for their expression of markers specific for endothelial cells (ECs), smooth muscle cells (SMCs), and cardiomyocytes (CMs) using von Willebrand factor (vWf), smooth muscle (SM) alpha-actin, and cardiac troponin T, respectively. Grafted BM-MSCs or AF-MSCs were found as single cell/small cell clusters or incorporated in the wall of microvessels. A larger number of ECs (227.27 +/- 18.91 vs. 150.36 +/- 24.08 cells/mm2, p < 0.01) and CMs (417.91 +/- 100.95 vs. 237.43 +/- 79.99 cells/mm2, p < 0.01) originated from AF-MSCs than from BM-MSCs. Almost no SMCs were seen with AF-MSCs, in comparison to BM-MSCs (98.03 +/- 40.84 cells/mm2), in concordance with lacking of arterioles, which, instead, were well expressed with BM-MSCs (71.30 +/- 55.66 blood vessels/mm2). The number of structurally organized capillaries was slightly different with the two MSCs (122.49 +/- 17.37/mm2 for AF-MSCs vs. 148.69 +/- 54.41/mm2 for BM-MSCs; p = NS). Collectively, these results suggest that, in the presence of the same postinjury microenvironment, the two MSC populations from different sources are able to activate distinct differentiation programs that potentially can bring about a myocardial-capillary or myocardial-capillary-arteriole reconstitution.
Assuntos
Células-Tronco Adultas/transplante , Temperatura Baixa/efeitos adversos , Células-Tronco Fetais/transplante , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Miocárdio/patologia , Células-Tronco Adultas/citologia , Células-Tronco Adultas/fisiologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Ecocardiografia , Células-Tronco Fetais/citologia , Células-Tronco Fetais/fisiologia , Coração/anatomia & histologia , Coração/fisiologia , Hemodinâmica , Humanos , Imunofenotipagem , Masculino , Células-Tronco Mesenquimais/citologia , Necrose , Osteogênese/fisiologia , Ratos , Ratos Endogâmicos F344 , Ratos NusRESUMO
We have developed a simple and accurate method for calibrating the amplitude of vibration of quartz tuning fork sensors commonly used in atomic force- and near field optical-microscopy. Unlike interferometric methods, which require a complex optical setup, the method we present requires only a simple measurement of the electro-mechanical properties of the tuning-fork oscillator and can be performed in a matter of minutes without disturbing the experimental setup. Comparison with interferometric methods shows that an accuracy of better than few percent can be routinely achieved.
RESUMO
Capture of each and every chromosome by spindle microtubules is essential to prevent chromosome loss and aneuploidy. In somatic cells, astral microtubules search and capture chromosomes forming lateral attachments to kinetochores. However, this mechanism alone is insufficient in large oocytes. We have previously shown that a contractile F-actin network is additionally required to collect chromosomes scattered in the 70-µm starfish oocyte nucleus. How this F-actin-driven mechanism is coordinated with microtubule capture remained unknown. Here, we show that after nuclear envelope breakdown Arp2/3-nucleated F-actin "patches" form around chromosomes in a Ran-GTP-dependent manner, and we propose that these structures sterically block kinetochore-microtubule attachments. Once F-actin-driven chromosome transport is complete, coordinated disassembly of F-actin patches allows synchronous capture by microtubules. Our observations indicate that this coordination is necessary because early capture of chromosomes by microtubules would interfere with F-actin-driven transport leading to chromosome loss and formation of aneuploid eggs.
Assuntos
Actinas/metabolismo , Cromossomos/metabolismo , Meiose , Microtúbulos/metabolismo , Oócitos/metabolismo , Estrelas-do-Mar/citologia , Actinas/análise , Animais , Cinetocoros/metabolismo , Cinetocoros/fisiologia , Oócitos/ultraestrutura , Fuso Acromático/metabolismo , Fuso Acromático/fisiologia , Estrelas-do-Mar/metabolismo , Estrelas-do-Mar/ultraestruturaRESUMO
The potential of collagen scaffolds for promoting angiogenesis/arteriogenesis was studied in vivo by implantation on healthy or cryoinjured left ventricles of rats up to 60 days post-injury. Blood vessels content and extra-vascular cell infiltration were evaluated within the collagen scaffold, the cryoinjured areas, and the "border zones" of the myocardium facing the cryoinjured zones. The collagen cardiac patches were almost completely absorbed in 60 days and became populated by new arterioles and capillaries in both intact and cryoinjured heart (arterioles in cryoinjured vs. intact zones were about 2,3-fold higher; capillaries in cryoinjured vs. intact zones were 1.7-fold higher). Collagen cardiac patches exerted a "trophic" effect on the organizing granulation tissue that emerged from the wound-healing process, increasing vessel density of 2.7-fold for arterioles and 4-fold for capillaries. Interstitial cells in collagen cardiac patches rarely (<1%) expressed cardiogenic stem cells markers such as Sca-1- or MDR1, whereas markers of neural crest cells GFAP(+)/nestin(+) cells ranged from 3/30% to 30/70% in collagen cardiac patches placed on intact vs. cryoinjured heart, respectively. Myofibroblasts and cardiomyocytes (CM) were absent but macrophages populated the collagen scaffolds even after 60 days from implantation. Western blotting of collagen cardiac patches after implantation on intact/cryoinjured hearts confirmed that markers of endothelial and smooth muscle cells, but not of CM, were expressed. The porous collagen scaffold was able to elicit a powerful angiogenetic and arteriogenetic response in the intact and cryoinjured hearts, representing an ideal tool for therapeutic angio-arteriogenesis and a potentially useful substrate for stem cell seeding.
Assuntos
Colágeno/farmacologia , Traumatismos Cardíacos , Neovascularização Patológica/induzido quimicamente , Neovascularização Patológica/patologia , Animais , Colágeno/ultraestrutura , Microscopia Eletrônica de Varredura , Fenótipo , Ratos , Ratos WistarRESUMO
Amidoferrocenyl-functionalised single wall carbon nanotubes (Fc-SWNT) are efficient exoreceptors for the redox recognition of H2PO4-.
RESUMO
There is a wide interest in studying the membrane mobility of Nerve Growth Factor (NGF) tropomyosin receptor kinase A (TrkA) at the single molecule level, in order to elucidate its diverse signaling responses related to different receptor functions. Here we present an experimental strategy based on the acyl carrier protein (ACP) tag in order to study the dynamics of the high-affinity NGF receptor TrkA in the membrane of PC12nnr5 cells. We present a single-particle tracking (SPT) study using highly photostable semiconductor quantum dots (Qdots) conjugated to ACP-tagged TrkA receptors. We demonstrate that ACP-TrkA shows biochemical and biological properties identical to those of its unmodified counterpart and that single receptor molecules in living cells display distinct diffusive regimes and a highly heterogeneous dynamics.
Assuntos
Proteína de Transporte de Acila/metabolismo , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Técnicas de Sonda Molecular , Neoplasias da Próstata/metabolismo , Pontos Quânticos , Receptor trkA/metabolismo , Animais , Linhagem Celular Tumoral , Masculino , Células PC12 , Ratos , Coloração e RotulagemRESUMO
PURPOSE: Wound healing of the cryo-injured bladder can bring about organ remodeling because of incomplete reconstitution of depleted smooth muscle cells. Stem cell transplantation could be beneficial to improve smooth muscle cell regeneration and/or modulate the remodeling process. The repair of bladder injury using adult-type stem cells would be useful for adult urological patients but unsuited for neonatal patients, in whom major benefits are likely to derive from fetal-type stem cells. MATERIALS AND METHODS: The smooth muscle cell differentiation potential of fetal-type vs adult-type stem cells was evaluated by injecting green fluorescent protein labeled mesenchymal stem cells from rat amniotic fluid or bone marrow, respectively, in cryo-injured rat bladder walls. RESULTS: At 30 days after transplantation only a few fetal-type or adult-type mesenchymal stem cells gave rise to enteric or vascular smooth muscle cells, whereas most mesenchymal stem cells appeared incapable of specific differentiation. In vitro co-culture experiments of smooth muscle cells with fetal-type or adult-type mesenchymal stem cells selectively labeled with distinct fluorochromes showed the presence of hybrid cells, suggesting that some mesenchymal stem cells can undergo cell fusion. Surprisingly the major effect of rat bone marrow or amniotic fluid mesenchymal stem cell transplantation seemed to be preventing cryo-injury induced hypertrophy of surviving smooth muscle cells. CONCLUSIONS: In this model stem cell transplantation has a limited effect on smooth muscle cell regeneration. Instead it can regulate post-injury bladder remodeling, possibly via a paracrine mechanism.