RESUMO
Comparisons and linkage between multiple imaging scales are essential for neural circuit connectomics. Here, we report 20 new recombinant rabies virus (RV) vectors that we have developed for multi-scale and multi-modal neural circuit mapping tools. Our new RV tools for mesoscale imaging express a range of improved fluorescent proteins. Further refinements target specific neuronal subcellular locations of interest. We demonstrate the discovery power of these new tools including the detection of detailed microstructural changes of rabies-labeled neurons in aging and Alzheimer's disease mouse models, live imaging of neuronal activities using calcium indicators, and automated measurement of infected neurons. RVs that encode GFP and ferritin as electron microscopy (EM) and fluorescence microscopy reporters are used for dual EM and mesoscale imaging. These new viral variants significantly expand the scale and power of rabies virus-mediated neural labeling and circuit mapping across multiple imaging scales in health and disease.
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The technique of colour EM that was recently developed enabled localisation of specific macromolecules/proteins of interest by the targeted deposition of diaminobenzidine (DAB) conjugated to lanthanide chelates. By acquiring lanthanide elemental maps by energy-filtered transmission electron microscopy (EFTEM) and overlaying them in pseudo-colour over the conventional greyscale TEM image, a colour EM image is generated. This provides a powerful tool for visualising subcellular component/s, by the ability to clearly distinguish them from the general staining of the endogenous cellular material. Previously, the lanthanide elemental maps were acquired at the high-loss M4,5 edge (excitation of 3d electrons), where the characteristic signal is extremely low and required considerably long exposures. In this paper, we explore the possibility of acquiring the elemental maps of lanthanides at their N4,5 edge (excitation of 4d electrons), which occurring at a much lower energy-loss regime, thereby contains significantly greater total characteristic signal owing to the higher inelastic scattering cross-sections at the N4,5 edge. Acquiring EFTEM lanthanide elemental maps at the N4,5 edge instead of the M4,5 edge, provides â¼4× increase in signal-to-noise and â¼2× increase in resolution. However, the interpretation of the lanthanide maps acquired at the N4,5 edge by the traditional 3-window method, is complicated due to the broad shape of the edge profile and the lower signal-above-background ratio. Most of these problems can be circumvented by the acquisition of elemental maps with the more sophisticated technique of EFTEM Spectrum Imaging (EFTEM SI). Here, we also report the chemical synthesis of novel second-generation DAB lanthanide metal chelate conjugates that contain 2 lanthanide ions per DAB molecule in comparison with 0.5 lanthanide ion per DAB in the first generation. Thereby, fourfold more Ln3+ per oxidised DAB would be deposited providing significant amplification of signal. This paper applies the colour EM technique at the intermediate-loss energy-loss regime to three different cellular targets, namely using mitochondrial matrix-directed APEX2, histone H2B-Nucleosome and EdU-DNA. All the examples shown in the paper are single colour EM images only.
Assuntos
Elementos da Série dos Lantanídeos , Microscopia Eletrônica de Transmissão por Filtração de Energia , Diagnóstico por Imagem , Elétrons , Coloração e RotulagemRESUMO
Synthesizing, localizing, and stabilizing new protein copies at synapses are crucial factors in maintaining the synaptic changes required for storing long-term memories. PKMζ recently emerged as a molecule putatively responsible for maintaining encoded memories over time because its presence correlates with late LTP and because its inhibition disrupts LTP in vitro and long-term memory storage in vivo. Here we investigated PKMζ stability in rat neurons to better understand its role during information encoding and storage. We used TimeSTAMP reporters to track the synthesis and degradation of PKMζ as well as a related atypical PKC, PKCλ. These reporters revealed that both PKMζ and PKCλ were upregulated after chemical LTP induction; however, these new PKMζ copies exhibited more rapid turnover than basally produced PKMζ, particularly in dendritic spines. In contrast to PKMζ, new PKCλ copies exhibited elevated stability. Stable information storage over long periods of time is more challenging the shorter the metabolic lifetime of the candidate molecules.
Assuntos
Espinhas Dendríticas/metabolismo , Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Proteólise , Sinapses/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Espinhas Dendríticas/fisiologia , Estabilidade Enzimática , Células HEK293 , Humanos , Isoenzimas/biossíntese , Isoenzimas/genética , Potenciação de Longa Duração , Dados de Sequência Molecular , Proteína Quinase C/genética , Ratos , Ratos Sprague-Dawley , Sinapses/fisiologia , Regulação para CimaRESUMO
Energy filtered transmission electron microscopy techniques are regularly used to build elemental maps of spatially distributed nanoparticles in materials and biological specimens. When working with thick biological sections, electron energy loss spectroscopy techniques involving core-loss electrons often require exposures exceeding several minutes to provide sufficient signal to noise. Image quality with these long exposures is often compromised by specimen drift, which results in blurring and reduced resolution. To mitigate drift artifacts, a series of short exposure images can be acquired, aligned, and merged to form a single image. For samples where the target elements have extremely low signal yields, the use of charge coupled device (CCD)-based detectors for this purpose can be problematic. At short acquisition times, the images produced by CCDs can be noisy and may contain fixed pattern artifacts that impact subsequent correlative alignment. Here we report on the use of direct electron detection devices (DDD's) to increase the signal to noise as compared with CCD's. A 3× improvement in signal is reported with a DDD versus a comparably formatted CCD, with equivalent dose on each detector. With the fast rolling-readout design of the DDD, the duty cycle provides a major benefit, as there is no dead time between successive frames.
Assuntos
Astrócitos/ultraestrutura , Células Epiteliais/ultraestrutura , Microscopia Eletrônica de Transmissão por Filtração de Energia/instrumentação , Microscopia Eletrônica de Transmissão por Filtração de Energia/métodos , Razão Sinal-Ruído , Coloração e Rotulagem/métodos , Animais , Encéfalo/patologia , Células HeLa , Humanos , Camundongos Endogâmicos C57BLRESUMO
We introduce Fe-TAML, a small molecule-based peroxidase as a versatile new member of the correlated fluorescence and electron microscopy toolkit. The utility of the probe is demonstrated by high resolution imaging of newly synthesized DNA (through biorthogonal labeling), genetically tagged proteins (using HaloTag), and untagged endogenous proteins (via immunostaining). EM visualization in these applications is facilitated by exploiting Fe-TAML's catalytic activity for the deposition of localized osmiophilic precipitates based on polymerized 3,3'-diaminobenzidine. Optimized conditions for synthesizing and implementing Fe-TAML based probes are also described. Overall, Fe-TAML is a new chemical biology tool that can be used to visualize diverse biomolecular species along nanometer and micron scales within cells.
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The mitochondrial inner membrane (IM) serves as the site for ATP production by hosting the oxidative phosphorylation complex machinery most notably on the crista membranes. Disruption of the crista structure has been implicated in a variety of cardiovascular and neurodegenerative diseases. Here, we characterize ChChd3, a previously identified PKA substrate of unknown function (Schauble, S., King, C. C., Darshi, M., Koller, A., Shah, K., and Taylor, S. S. (2007) J. Biol. Chem. 282, 14952-14959), and show that it is essential for maintaining crista integrity and mitochondrial function. In the mitochondria, ChChd3 is a peripheral protein of the IM facing the intermembrane space. RNAi knockdown of ChChd3 in HeLa cells resulted in fragmented mitochondria, reduced OPA1 protein levels and impaired fusion, and clustering of the mitochondria around the nucleus along with reduced growth rate. Both the oxygen consumption and glycolytic rates were severely restricted. Ultrastructural analysis of these cells revealed aberrant mitochondrial IM structures with fragmented and tubular cristae or loss of cristae, and reduced crista membrane. Additionally, the crista junction opening diameter was reduced to 50% suggesting remodeling of cristae in the absence of ChChd3. Analysis of the ChChd3-binding proteins revealed that ChChd3 interacts with the IM proteins mitofilin and OPA1, which regulate crista morphology, and the outer membrane protein Sam50, which regulates import and assembly of ß-barrel proteins on the outer membrane. Knockdown of ChChd3 led to almost complete loss of both mitofilin and Sam50 proteins and alterations in several mitochondrial proteins, suggesting that ChChd3 is a scaffolding protein that stabilizes protein complexes involved in maintaining crista architecture and protein import and is thus essential for maintaining mitochondrial structure and function.
Assuntos
Mitocôndrias Hepáticas/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Mitocôndrias Hepáticas/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismoRESUMO
The C-terminus of the most abundant and best-studied gap-junction protein, connexin43, contains multiple phosphorylation sites and protein-binding domains that are involved in regulation of connexin trafficking and channel gating. It is well-documented that SDS/PAGE of NRK (normal rat kidney) cell lysates reveals at least three connexin43-specific bands (P0, P1 and P2). P1 and P2 are phosphorylated on multiple, unidentified serine residues and are found primarily in gap-junction plaques. In the present study we prepared monoclonal antibodies against a peptide representing the last 23 residues at the C-terminus of connexin43. Immunofluorescence studies showed that one antibody (designated CT1) bound primarily to connexin43 present in the Golgi apparatus, whereas the other antibody (designated IF1) labelled predominately connexin43 present in gap junctions. CT1 immunoprecipitates predominantly the P0 form whereas IF1 recognized all three bands. Peptide mapping, mutational analysis and protein-protein interaction experiments revealed that unphosphorylated Ser364 and/or Ser365 are critical for CT1 binding. The IF1 paratope binds to residues Pro375-Asp379 and requires Pro375 and Pro377. These proline residues are also necessary for ZO-1 interaction. These studies indicate that the conformation of Ser364/Ser365 is important for intracellular localization, whereas the tertiary structure of Pro375-Asp379 is essential in targeting and regulation of gap junctional connexin43.
Assuntos
Anticorpos/imunologia , Conexina 43/química , Junções Comunicantes/química , Complexo de Golgi/química , Animais , Linhagem Celular , Conexina 43/genética , Conexina 43/imunologia , Cães , Eletroforese em Gel de Poliacrilamida , Imunoprecipitação , Microscopia Confocal , Microscopia de Fluorescência , Mutagênese Sítio-Dirigida , Fosforilação , Conformação Proteica , RatosRESUMO
Biological samples are frequently stained with heavy metals in preparation for examining the macro, micro and ultra-structure using X-ray microtomography and electron microscopy. A single X-ray microtomography scan reveals detailed 3D structure based on staining density, yet it lacks both material composition and functional information. Using a commercially available polychromatic X-ray source, energy integrating detectors and a two-scan configuration labelled by their energy- "High" and "Low", we demonstrate how a specific element, here shown with iron, can be detected from a mixture with other heavy metals. With proper selection of scan configuration, achieving strong overlap of source characteristic emission lines and iron K-edge absorption, iron absorption was enhanced enabling K-edge imaging. Specifically, iron images were obtained by scatter plot material analysis, after selecting specific regions within scatter plots generated from the "High" and "Low" scans. Using this method, we identified iron rich regions associated with an iron staining reaction that marks the nodes of Ranvier along nerve axons within mouse spinal roots, also stained with osmium metal commonly used for electron microscopy.
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Axônios/metabolismo , Ferro/análise , Raízes Nervosas Espinhais/diagnóstico por imagem , Microtomografia por Raio-X/instrumentação , Animais , Metais Pesados , Camundongos , Imagens de Fantasmas , Raízes Nervosas Espinhais/metabolismo , Coloração e RotulagemRESUMO
Electron microscopy (EM) remains the primary method for imaging cellular and tissue ultrastructure, although simultaneous localization of multiple specific molecules continues to be a challenge for EM. We present a method for obtaining multicolor EM views of multiple subcellular components. The method uses sequential, localized deposition of different lanthanides by photosensitizers, small-molecule probes, or peroxidases. Detailed view of biological structures is created by overlaying conventional electron micrographs with pseudocolor lanthanide elemental maps derived from distinctive electron energy-loss spectra of each lanthanide deposit via energy-filtered transmission electron microscopy. This results in multicolor EM images analogous to multicolor fluorescence but with the benefit of the full spatial resolution of EM. We illustrate the power of this methodology by visualizing hippocampal astrocytes to show that processes from two astrocytes can share a single synapse. We also show that polyarginine-based cell-penetrating peptides enter the cell via endocytosis, and that newly synthesized PKMζ in cultured neurons preferentially localize to the postsynaptic membrane.
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Elementos da Série dos Lantanídeos/análise , Microscopia Eletrônica de Transmissão por Filtração de Energia/métodos , Animais , Astrócitos/ultraestrutura , Peptídeos Penetradores de Células/análise , Células Cultivadas , Cães , Células HEK293 , Hipocampo/citologia , Humanos , Células Madin Darby de Rim Canino , Masculino , Camundongos Endogâmicos BALB CRESUMO
PURPOSE: Transgenic (Col1a1(r/r)) mice gradually develop elevated intraocular pressure (IOP) with open angles. The present study was undertaken to evaluate optic nerve axonal loss with time in these mice. METHODS: The IOP of transgenic (Col1a1(r/r)) mice and control wild-type (Col1a1(+/+)) mice was measured at 7, 12, 16, 24, 36, and 54 weeks of age using a microneedle method. Transgenic Col1a1(r/r) and control Col1a1(+/+) mice at 24 and 54 weeks of age were randomly selected and their optic nerves were processed conventionally for electron microscopy. Optic nerve cross-sections were collected 300 micro m posterior to the globe. Low (200X) and high (10,000X) magnification images were collected systematically and were masked before analysis. For each nerve, cross-sectional area was measured in low magnification images, and axonal number was counted in high magnification images. RESULTS: Mean IOP of the transgenic Col1a1(r/r) mice was significantly higher than that of the control Col1a1(+/+) mice at 16, 24, 36, and 54 weeks by 21%, 42%, 41%, and 33% respectively (P < 0.05). The mean axonal density and total axonal number in the transgenic Col1a1(r/r) mice at 54 weeks of age (n = 10) was significantly less than those in the control Col1a1(+/+) mice at 24 weeks (n = 5) and 54 weeks (n = 5; P = 0.0081 and P = 0.020, respectively, analysis of variance, P < 0.05 for pair-wise comparisons). The mean axonal density and total axonal number in the transgenic Col1a1(r/r) mice at 54 weeks also were significantly less than in the transgenic Col1a1(r/r) mice at 24 weeks (n = 10). Mean axonal loss between 24 and 54 weeks of age in the transgenic Col1a1(r/r) mice was 28.7%. CONCLUSIONS: Transgenic Col1a1(r/r) mice develop sustained elevation of IOP and progressive optic nerve axon loss. This suggests that these mice may be useful as a mouse model of primary open angle glaucoma as well as for assessing the relationship between collagen type I metabolism and optic nerve axon loss.
Assuntos
Axônios/ultraestrutura , Colágeno Tipo I/genética , Glaucoma de Ângulo Aberto/genética , Mutação , Doenças do Nervo Óptico/genética , Animais , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Modelos Animais de Doenças , Glaucoma de Ângulo Aberto/patologia , Pressão Intraocular/genética , Camundongos , Camundongos Transgênicos , Doenças do Nervo Óptico/patologiaRESUMO
PURPOSE: To assess the relationship between regional variation of axon loss and optic nerve head anatomy in laser-induced experimental glaucoma in the mouse. METHODS: Experimental glaucoma was induced unilaterally in eight NIH Swiss black mice. Intraocular pressure (IOP) was measured for 12 weeks, and the mice were killed. The eyes were enucleated, and both optic nerves were dissected and processed conventionally for electron microscopy. Low- and high-magnification images of the optic nerve cross sections 300 microm posterior to the globe were collected systematically and masked before analysis. For each nerve, cross-sectional area was measured in low-magnification micrographs. Axon number and density were determined in the high-magnification micrographs. Loss of axonal density was compared between the superior and inferior and nasal and temporal areas of the optic nerve cross section. Additional cross-section micrographs were collected at 10- or 20-microm intervals throughout the optic nerve head. RESULTS: In the treated (glaucoma) eyes, mean IOP was 44% higher than that in the control eyes. The optic nerve cross-sectional area, mean axonal density, and total axonal number were significantly less than those in the control eyes (P < 0.01 for each). Axon loss in the superior optic nerve was greater than in the inferior optic nerve in each glaucomatous eye (P = 0.012). The ratio of axonal density in the superior and inferior optic nerve (superior-to-inferior [S/I] ratio) in all treated eyes was <1.0 and significantly lower than that in the control eyes (P = 0.012). The central retinal vessels occupied approximately 20% of the central optic nerve head cross-sectional area, gradually shifted position ventrally as they progressed toward the scleral foramen (the mouse does not have a lamina cribrosa), and exited the inferior retrobulbar optic nerve adjacent to the posterior of the globe. CONCLUSIONS: Ocular hypertension in the mouse eye sufficient to cause optic nerve damage induces preferential loss of superior optic nerve axons. Optic nerve axon loss appeared less among the axons that were near the major optic nerve blood vessels at the scleral foramen. Topographic differences in optic nerve axon loss should be considered when evaluating optic nerve damage in experimental laser-induced glaucoma in the mouse.
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Glaucoma/patologia , Nervo Óptico/patologia , Animais , Axônios/patologia , Vasos Sanguíneos/patologia , Glaucoma/fisiopatologia , Pressão Intraocular , Camundongos , Disco Óptico/irrigação sanguíneaRESUMO
PURPOSE: To evaluate optic nerve damage in mice after laser-induced ocular hypertension. METHODS: Ocular hypertension was induced unilaterally in 13 NIH Black Swiss mice by laser photocoagulation of the limbus. Over the following 12 weeks, intraocular pressure (IOP) was measured at regular intervals by the microneedle method. The optic nerves of these mice and of seven normal untreated mice were then processed conventionally for electron microscopy, and cross sections of the nerve 300 micro m posterior to the globe were collected. Low- and high-magnification images were collected systematically and masked before analysis. For each nerve, cross-sectional area was measured in low-magnification micrographs, and axon and glia numbers were counted in high-magnification micrographs. RESULTS: In normal untreated mice, the average number of axons was 59,597 +/- 3,112 (mean +/- SD). Variation among these measurements was 5.7% +/- 3.9%. After laser treatment, the duration of high IOP ranged from 2 to 12 weeks (6.2 +/- 3.6 weeks, mean +/- SD). The mean IOP in the treated eyes was 1.3 times greater than the mean IOP in the control eyes (P = 0.0012). The maximum IOP in the treated eyes was 1.6 times greater than that observed in the control eyes (P < 0.0001). The optic nerve cross-sectional area, mean axon density, and total number of axons in the treated eyes were significantly less than in the control eyes (28.5% +/- 23.4%, 57.8% +/- 37.8%, and 63.1% +/- 38.1%, respectively; P < 0.005 for each). The decrease in optic nerve cross-sectional area and the positive integral of elevated IOP and duration of IOP elevation correlated significantly with total axon loss (r(2) = 0.79, P < 0.0001 and r(2) = 0.36, P = 0.040, respectively). The number of astrocytes per cross section of optic nerve was significantly greater in the treated eyes than in the control eyes (P = 0.014). CONCLUSIONS: Laser-induced ocular hypertension in mouse eyes can induce optic nerve axon loss that correlates with the magnitude and duration of elevated IOP.
Assuntos
Modelos Animais de Doenças , Hipertensão/complicações , Doenças do Nervo Óptico/etiologia , Animais , Axônios/patologia , Axônios/ultraestrutura , Contagem de Células , Pressão Intraocular , Camundongos , Neuroglia/patologia , Neuroglia/ultraestrutura , Nervo Óptico/ultraestrutura , Doenças do Nervo Óptico/patologiaRESUMO
The MAP kinase kinase kinase TGFß-activated kinase 1 (TAK1) is activated by TLRs, IL-1, TNF, and TGFß and in turn activates IKK-NF-κB and JNK, which regulate cell survival, growth, tumorigenesis, and metabolism. TAK1 signaling also upregulates AMPK activity and autophagy. Here, we investigated TAK1-dependent regulation of autophagy, lipid metabolism, and tumorigenesis in the liver. Fasted mice with hepatocyte-specific deletion of Tak1 exhibited severe hepatosteatosis with increased mTORC1 activity and suppression of autophagy compared with their WT counterparts. TAK1-deficient hepatocytes exhibited suppressed AMPK activity and autophagy in response to starvation or metformin treatment; however, ectopic activation of AMPK restored autophagy in these cells. Peroxisome proliferator-activated receptor α (PPARα) target genes and ß-oxidation, which regulate hepatic lipid degradation, were also suppressed in hepatocytes lacking TAK1. Due to suppression of autophagy and ß-oxidation, a high-fat diet challenge aggravated steatohepatitis in mice with hepatocyte-specific deletion of Tak1. Notably, inhibition of mTORC1 restored autophagy and PPARα target gene expression in TAK1-deficient livers, indicating that TAK1 acts upstream of mTORC1. mTORC1 inhibition also suppressed spontaneous liver fibrosis and hepatocarcinogenesis in animals with hepatocyte-specific deletion of Tak1. These data indicate that TAK1 regulates hepatic lipid metabolism and tumorigenesis via the AMPK/mTORC1 axis, affecting both autophagy and PPARα activity.
Assuntos
Autofagia/fisiologia , Ácidos Graxos/metabolismo , Fígado Gorduroso/prevenção & controle , Neoplasias Hepáticas Experimentais/prevenção & controle , MAP Quinase Quinase Quinases/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Autofagia/efeitos dos fármacos , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/metabolismo , MAP Quinase Quinase Quinases/deficiência , MAP Quinase Quinase Quinases/genética , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/metabolismo , Oxirredução , PPAR alfa/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismoRESUMO
Protein synthesis is highly regulated throughout nervous system development, plasticity and regeneration. However, tracking the distributions of specific new protein species has not been possible in living neurons or at the ultrastructural level. Previously we created TimeSTAMP epitope tags, drug-controlled tags for immunohistochemical detection of specific new proteins synthesized at defined times. Here we extend TimeSTAMP to label new protein copies by fluorescence or photo-oxidation. Live microscopy of a fluorescent TimeSTAMP tag reveals that copies of the synaptic protein PSD95 are synthesized in response to local activation of growth factor and neurotransmitter receptors, and preferentially localize to stimulated synapses in rat neurons. Electron microscopy of a photo-oxidizing TimeSTAMP tag reveals new PSD95 at developing dendritic structures of immature neurons and at synapses in differentiated neurons. These results demonstrate the versatility of the TimeSTAMP approach for visualizing newly synthesized proteins in neurons.
Assuntos
Proteínas de Bactérias/análise , Corantes Fluorescentes , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Luminescentes/análise , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Animais , Animais Recém-Nascidos , Proteínas de Bactérias/ultraestrutura , Células Cultivadas , Proteína 4 Homóloga a Disks-Large , Epitopos/análise , Epitopos/ultraestrutura , Células HEK293 , Humanos , Proteínas Luminescentes/ultraestrutura , Microscopia Eletrônica/métodos , Microscopia de Fluorescência/métodos , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Although RII protein kinase A (PKA) regulatory subunits are constitutively localized to discrete cellular compartments through binding to A-kinase-anchoring proteins (AKAPs), RI subunits are primarily diffuse in the cytoplasm. In this paper, we report a novel AKAP-dependent localization of RIα to distinct organelles, specifically, multivesicular bodies (MVBs). This localization depends on binding to AKAP11, which binds tightly to free RIα or RIα in complex with catalytic subunit (holoenzyme). However, recruitment to MVBs occurs only with the release of PKA catalytic subunit (PKAc). This recruitment is reversed by reassociation with PKAc, and it is disrupted by the presence of AKAP peptides, mutations in the RIα AKAP-binding site, or knockdown of AKAP11. Cyclic adenosine monophosphate binding not only unleashes active PKAc but also leads to the targeting of AKAP11:RIα to MVBs. Therefore, we show that the RIα holoenzyme is part of a signaling complex with AKAP11, in which AKAP11 may direct RIα functionality after disassociation from PKAc. This model defines a new paradigm for PKA signaling.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Corpos Multivesiculares , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Domínio Catalítico , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Isoenzimas/metabolismo , Dados de Sequência Molecular , Mutação , Ligação Proteica , Transporte Proteico , Transdução de SinaisRESUMO
Avian pulmonary capillaries differ from those of mammals in three important ways. The blood-gas barrier is much thinner, it is more uniform in thickness, and the capillaries are far more rigid when their transmural pressure is altered. The thinness of the barrier is surprising because it predisposes the capillaries to stress failure. A possible mechanism for these differences is that avian pulmonary capillaries, unlike mammalian, are supported from the outside by air capillaries, but the details of the support are poorly understood. To clarify this we studied the blood and air capillaries in chicken lung using transmission electron microscopy (EM) and two relatively new techniques that allow 3D visualization: electron tomography and serial block-face scanning EM. These studies show that the pulmonary capillaries are flanked by epithelial bridges composed of two extremely thin epithelial cells with large surface areas. The junctions of the bridges with the capillary walls show thickening of the epithelial cells and an accumulation of extracellular matrix. Collapse of the pulmonary capillaries when the pressure outside them is increased is apparently prevented by the guy wire-like action of the epithelial bridges. The enlarged junctions between the bridges and the walls could provide a mechanism that limits the hoop stress in the capillary walls when the pressure inside them is increased. The support of the pulmonary capillaries may also be explained by an interdependence mechanism whereby the capillaries are linked to a rigid assemblage of air capillaries. These EM studies show the supporting structures in greater detail than has previously been possible, particularly in 3D, and they allow a more complete analysis of the mechanical forces affecting avian pulmonary capillaries.
Assuntos
Barreira Alveolocapilar/ultraestrutura , Capilares/ultraestrutura , Células Epiteliais/ultraestrutura , Imageamento Tridimensional , Junções Intercelulares/ultraestrutura , Microscopia Eletrônica de Transmissão , Animais , Capilares/citologia , Permeabilidade Capilar/fisiologia , Comunicação Celular/fisiologia , Galinhas , Tomografia com Microscopia Eletrônica/métodos , Pulmão/anatomia & histologia , Pulmão/fisiologia , Microscopia Eletrônica de Transmissão/métodos , Circulação Pulmonar/fisiologiaRESUMO
Bioremediation of chromium through the reduction of hexavalent chromium (as the chromate ion, CrO42-) is based on the notion that the product, trivalent chromium (Cr(III)), is less toxic than chromate. In this study, we show that soluble Cr(III), present at pH 6-8 as the Cr3+ ion and/or hydroxyl complexes (henceforth referred to as uncomplexed Cr(III)), can be found transiently in significant concentrations and has a deleterious effect on Shewanella sp. MR-4. However, Cr(lll) complexed to an organic ligand or precipitated as Cr(OH)3(s) has little or no effect on cells. Similarly, during the reduction of Cr(VI) by strain MR-4, complexation of the product, Cr(lll), results in increased cell survival and extended Cr(VI) reduction activity. These results and gene expression data obtained by qRT-PCR (quantitative reverse transcription-PCR) suggestthatthe observed toxic effect of Cr(II) formed during Cr(VI) reduction or added as an uncomplexed species is due to the interference with basic cell activities such as DNA transcription and/or replication. Important implications for the bioremediation of Cr(VI)-contaminated sites emerge from this study: Cr(VI) reduction by Shewanella sp. MR-4 is enhanced and sustained by the presence of compounds able to complex Cr(III) as it is being formed but, in turn, the complexation of Cr(III) precludes its precipitation and immobilization.
Assuntos
Cromo/química , Cromo/toxicidade , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Shewanella/efeitos dos fármacos , Poluentes Químicos da Água , Poluição da Água/prevenção & controle , Biodegradação Ambiental , Microscopia Eletrônica de Transmissão , Oxirredução , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Shewanella/genética , Análise de SobrevidaRESUMO
Microbial reduction is a promising strategy for chromium remediation, but the effects of competing electron acceptors are still poorly understood. We investigated chromate (Cr(VI)) reduction in batch cultures of Shewanella oneidensis MR-1 under aerobic and denitrifying conditions and in the absence of an additional electron acceptor. Growth and Cr(VI) removal patterns suggested a cometabolic reduction; in the absence of nitrate or oxygen, MR-1 reduced Cr(VI), but without any increase in viable cell counts and rates gradually decreased when cells were respiked. Only a small fraction (1.6%) of the electrons from lactate were transferred to Cr(VI). The 48-h transformation capacity (Tc) was 0.78 mg (15 micromoles) Cr(VI) reduced. [mg protein](-1) for high levels of Cr(VI) added as a single spike. For low levels of Cr(VI) added sequentially, Tc increased to 3.33 mg (64 micromoles) Cr(VI) reduced. [mg protein](-1), indicating that it is limited by toxicity at higher concentrations. During denitrification and aerobic growth, MR-1 reduced Cr(VI), with much faster rates under denitrifying conditions. Cr(VI) had no effect on nitrate reduction at 6 microM, was strongly inhibitory at 45 microM, and stopped nitrate reduction above 200 microM. Cr(VI) had no effect on aerobic growth at 60 microM, but severely inhibited growth above 150 microM. A factor that likely plays a role in Cr(VI) toxicity is intracellular reduced chromium. Transmission electron microscopy (TEM) and electron energy loss spectroscopy (EELS) of denitrifying cells exposed to Cr(VI) showed reduced chromium precipitates both extracellularly on the cell surface and, for the first time, as electron-dense round globules inside cells.
Assuntos
Cromo/metabolismo , Shewanella putrefaciens/genética , Shewanella putrefaciens/metabolismo , Biodegradação Ambiental , Carcinógenos Ambientais/metabolismo , Células Cultivadas , Meios de Cultivo Condicionados , Inibidores do Crescimento/biossíntese , Inibidores do Crescimento/metabolismo , Microscopia Eletrônica , Oxirredução , Shewanella putrefaciens/crescimento & desenvolvimento , Solubilidade , Análise EspectralRESUMO
Bcl-2 family proteins regulate the release of proteins like cytochrome c from mitochondria during apoptosis. We used cell-free systems and ultimately a vesicular reconstitution from defined molecules to show that outer membrane permeabilization by Bcl-2 family proteins requires neither the mitochondrial matrix, the inner membrane, nor other proteins. Bid, or its BH3-domain peptide, activated monomeric Bax to produce membrane openings that allowed the passage of very large (2 megadalton) dextran molecules, explaining the translocation of large mitochondrial proteins during apoptosis. This process required cardiolipin and was inhibited by antiapoptotic Bcl-x(L). We conclude that mitochondrial protein release in apoptosis can be mediated by supramolecular openings in the outer mitochondrial membrane, promoted by BH3/Bax/lipid interaction and directly inhibited by Bcl-x(L).
Assuntos
Apoptose , Proteínas de Transporte/metabolismo , Metabolismo dos Lipídeos , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Cardiolipinas/metabolismo , Ácidos Cólicos , Vesículas Citoplasmáticas , Detergentes , Dextranos , Retículo Endoplasmático/metabolismo , Fluoresceína , Humanos , Membranas Intracelulares/metabolismo , Lipossomos/metabolismo , Peptídeos/metabolismo , Permeabilidade , Proteínas Recombinantes de Fusão/metabolismo , Xenopus , Proteína X Associada a bcl-2RESUMO
We describe the technique and application of energy filtering, automated most-probable loss (MPL) tomography to intermediate voltage electron microscopy (IVEM). We show that for thick, selectively stained biological specimens, this method produces a dramatic increase in resolution of the projections and the computed volumes versus standard unfiltered transmission electron microscopy (TEM) methods. This improvement in resolution is attributed to the reduction of chromatic aberration, which results from the large percentage of inelastic electron-scattering events for thick specimens. These improvements are particularly evident at the large tilt angles required to improve tomographic resolution in the z-direction. This method effectively increases the usable thickness of selectively stained samples that can be imaged at a given accelerating voltage by dramatically improving resolution versus unfiltered TEM and increasing signal-to-noise versus zero-loss imaging, thereby expanding the utility of the IVEM to deliver information from within specimens up to 3 microm thick.