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1.
PLoS Pathog ; 17(2): e1009304, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33544760

RESUMEN

S. epidermidis is a substantial component of the human skin microbiota, but also one of the major causes of nosocomial infection in the context of implanted medical devices. We here aimed to advance the understanding of S. epidermidis genotypes and phenotypes conducive to infection establishment. Furthermore, we investigate the adaptation of individual clonal lines to the infection lifestyle based on the detailed analysis of individual S. epidermidis populations of 23 patients suffering from prosthetic joint infection. Analysis of invasive and colonizing S. epidermidis provided evidence that invasive S. epidermidis are characterized by infection-supporting phenotypes (e.g. increased biofilm formation, growth in nutrient poor media and antibiotic resistance), as well as specific genetic traits. The discriminating gene loci were almost exclusively assigned to the mobilome. Here, in addition to IS256 and SCCmec, chromosomally integrated phages was identified for the first time. These phenotypic and genotypic features were more likely present in isolates belonging to sequence type (ST) 2. By comparing seven patient-matched nasal and invasive S. epidermidis isolates belonging to identical genetic lineages, infection-associated phenotypic and genotypic changes were documented. Besides increased biofilm production, the invasive isolates were characterized by better growth in nutrient-poor media and reduced hemolysis. By examining several colonies grown in parallel from each infection, evidence for genetic within-host population heterogeneity was obtained. Importantly, subpopulations carrying IS insertions in agrC, mutations in the acetate kinase (AckA) and deletions in the SCCmec element emerged in several infections. In summary, these results shed light on the multifactorial processes of infection adaptation and demonstrate how S. epidermidis is able to flexibly repurpose and edit factors important for colonization to facilitate survival in hostile infection environments.

2.
J Extracell Vesicles ; 9(1): 1809065, 2020 Aug 27.
Artículo en Inglés | MEDLINE | ID: mdl-32944194

RESUMEN

Extracellular vesicles (EVs) are important means of intercellular communication and a potent tool for regenerative therapy. In ischaemic stroke, transient blockage of a brain artery leads to a lack of glucose and oxygen in the affected brain tissue, provoking neuronal death by necrosis in the core of the ischaemic region. The fate of neurons in the surrounding penumbra region depends on the stimuli, including EVs, received during the following hours. A detailed characterization of such stimuli is crucial not only for understanding stroke pathophysiology but also for new therapeutic interventions. In the present study, we characterize the EVs in mouse brain under physiological conditions and 24 h after induction of transient ischaemia in mice. We show that, in steady-state conditions, microglia are the main source of small EVs (sEVs), whereas after ischaemia the main sEV population originates from astrocytes. Brain sEVs presented high amounts of the prion protein (PrP), which were further increased after stroke. Moreover, EVs were enriched in a proteolytically truncated PrP fragment (PrP-C1). Because of similarities between PrP-C1 and certain viral surface proteins, we studied the cellular uptake of brain-derived sEVs from mice lacking (PrP-KO) or expressing PrP (WT). We show that PrP-KO-sEVs are taken up significantly faster and more efficiently than WT-EVs by primary neurons. Furthermore, microglia and astrocytes engulf PrP-KO-sEVs more readily than WT-sEVs. Our results provide novel information on the relative contribution of brain cell types to the sEV pool in murine brain and indicate that increased release of sEVs by astrocytes together with elevated levels of PrP in sEVs may play a role in intercellular communication at early stages after stroke. In addition, amounts of PrP (and probably PrP-C1) in brain sEVs seem to contribute to regulating their cellular uptake.

3.
Adv Biosyst ; 4(2): e1900162, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-32293134

RESUMEN

The capture of circulating tumor cells (CTCs) is still a challenging application for microfluidic chips, as these cells are rare and hidden in a huge background of blood cells. Here, different microfluidic ceiling designs in regard to their capture efficiency for CTCs in model experiments and more realistic conditions of blood samples spiked with a clinically relevant amount of tumor cells are evaluated. An optimized design for the capture platform that allows highly efficient recovery of CTCs from size-based pre-enriched samples under realistic conditions is obtained. Furthermore, the viability of captured tumor cells as well as single cell recovery for downstream genomic analysis is demonstrated. Additionally, the authors' findings underline the importance of evaluating rational design rules for microfluidic devices based on theoretical models by application-specific experiments.

4.
RNA Biol ; 17(4): 425-440, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-31986967

RESUMEN

The use of disease-specific signatures of microRNAs (miRNAs) in exosomes has become promising for clinical applications, either as biomarkers or direct therapeutic targets. However, a new approach for exosome enrichment and quantification of miRNAs is urgently needed for its clinical application, since the commercial techniques have shortcomings in quantity and quality. To overcome these deficiencies, we developed a new method for purification of exosomes with subsequent miRNA extraction, followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR), and compared our assays with commercial techniques. For the establishment of these methods, numerous reagents, parameters, and combinations thereof were examined. Our new technique for exosome extraction is based on a mannuronate-guluronate polymer (MGP) which avoids co-precipitating plasma proteins. Quality, concentration and biological activity of the isolated exosomes were examined by Western blot, Nanoparticle Tracking Analysis (NTA), and confocal microscopy. A combination of chaotropic and non-chaotropic salts was used to extract miRNAs from plasma, serum, and exosomes, allowing the exclusion of hazardous components, such as phenol/chloroform. The performance of the miRNAs extraction was verified by RT-qPCR. The chemistry and TaqMan probe were also optimized for RT-qPCR. Sensitivity, efficiency, and linearity of RT-qPCR were tested on serial dilutions of synthetic miR-16 and miR-142. Our established procedure covers all steps of miRNA analyses, and measures the levels of either cell-free and exosomal miRNAs in plasma, serum and other body fluids with high performance.

5.
Bone ; 130: 115062, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31678489

RESUMEN

Although inactivating mutations of PLS3, encoding the actin-bundling protein plastin-3, have been identified to cause X-linked osteoporosis, the cellular and molecular influence of PLS3 on bone remodeling is poorly defined. Moreover, although a previous study has demonstrated moderate osteopenia in 12 week-old Pls3-deficient mice based on µCT scanning, there is no reported analysis of such a model on the basis of undecalcified histology and bone-specific histomorphometry. To fill this knowledge gap we applied a deep phenotyping approach and studied Pls3-deficient mice at different ages. Surprisingly, we did not detect significant differences between wildtype and Pls3-deficient littermates with respect to trabecular bone mass, and the same was the case for all histomorphometric parameters determined at 12 weeks of age. Remarkably however, the cortical thickness in both, tibia and femur, was significantly reduced in Pls3-deficient mice in all age groups. We additionally studied the ex vivo behavior of Pls3-deficient primary osteoblasts, which displayed moderately impaired mineralization capacity. Of note, while most osteoblastogenesis markers were not differentially expressed between wildtype and Pls3-deficient cultures, the expression of Sfrp4 was significantly reduced in the latter, a potentially relevant finding, since Sfrp4 inactivation, in mice and humans, specifically causes cortical thinning. We finally addressed the question, if Pls3-deficiency would impair the osteoanabolic influence of parathyroid hormone (PTH). For this purpose we applied daily injection of PTH into wildtype and Pls3-deficient mice and found a similar response regardless of the genotype. Taken together, our data reveal that Pls3-deficiency in mice only recapitulates the cortical bone phenotype of individuals with X-linked osteoporosis by negatively affecting the early stage of cortical bone acquisition.

6.
Nat Commun ; 10(1): 5448, 2019 11 29.
Artículo en Inglés | MEDLINE | ID: mdl-31784514

RESUMEN

Amphisomes are organelles of the autophagy pathway that result from the fusion of autophagosomes with late endosomes. While biogenesis of autophagosomes and late endosomes occurs continuously at axon terminals, non-degradative roles of autophagy at boutons are barely described. Here, we show that in neurons BDNF/TrkB traffick in amphisomes that signal locally at presynaptic boutons during retrograde transport to the soma. This is orchestrated by the Rap GTPase-activating (RapGAP) protein SIPA1L2, which connects TrkB amphisomes to a dynein motor. The autophagosomal protein LC3 regulates RapGAP activity of SIPA1L2 and controls retrograde trafficking and local signaling of TrkB. Following induction of presynaptic plasticity, amphisomes dissociate from dynein at boutons enabling local signaling and promoting transmitter release. Accordingly, sipa1l2 knockout mice show impaired BDNF-dependent presynaptic plasticity. Taken together, the data suggest that in hippocampal neurons, TrkB-signaling endosomes are in fact amphisomes that during retrograde transport have local signaling capacity in the context of presynaptic plasticity.


Asunto(s)
Autofagosomas/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Endosomas/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Plasticidad Neuronal/genética , Neuronas/metabolismo , Terminales Presinápticos/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Transporte Axonal , Axones/metabolismo , Dineínas/metabolismo , Proteínas Activadoras de GTPasa/genética , Hipocampo , Ratones , Ratones Noqueados , Transporte de Proteínas
7.
Front Mol Neurosci ; 12: 224, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31616248

RESUMEN

The endocannabinoid system (ECS) consists particularly of cannabinoid receptors 1 and 2 (CB1 and CB2), their endogenous ligands, and enzymes that synthesize and degrade their ligands. It acts in a variety of organs and disease states ranging from cancer progression over neuropathic pain to neurodegeneration. Protein components engaged in the signaling, trafficking, and homeostasis machinery of the G-protein coupled CB2, are however largely unknown. It is therefore important to identify further interaction partners to better understand CB2 receptor functions in physiology and pathophysiology. For this purpose, we used an affinity purification and mass spectrometry-based proteomics approach of Strep-HA-CB2 receptor in HEK293 cells. After subtraction of background interactions and protein frequency library assessment we could identify 83 proteins that were classified by the identification of minimally 2 unique peptides as highly probable interactors. A functional protein association network analysis obtained an interaction network with a significant enrichment of proteins functionally involved in protein metabolic process, in endoplasmic reticulum, response to stress but also in lipid metabolism and membrane organization. The network especially contains proteins involved in biosynthesis and trafficking like calnexin, Sec61A, tubulin chains TUBA1C and TUBB2B, TMED2, and TMED10. Six proteins that were only expressed in stable CB2 expressing cells were DHC24, DHRS7, GGT7, HECD3, KIAA2013, and PLS1. To exemplify the validity of our approach, we chose a candidate having a relatively low number of edges in the network to increase the likelihood of a direct protein interaction with CB2 and focused on the scaffold/phagosomal protein p62/SQSTM1. Indeed, we independently confirmed the interaction by co-immunoprecipitation and immunocytochemical colocalization studies. 3D reconstruction of confocal images furthermore showed CB2 localization in close proximity to p62 positive vesicles at the cell membrane. In summary, we provide a comprehensive repository of the CB2 interactome in HEK293 cells identified by a systematic unbiased approach, which can be used in future experiments to decipher the signaling and trafficking complex of this cannabinoid receptor. Future studies will have to analyze the exact mechanism of the p62-CB2 interaction as well as its putative role in disease pathophysiology.

8.
J Am Soc Nephrol ; 30(5): 824-839, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30971456

RESUMEN

BACKGROUND: About 3%-5% of adults with membranous nephropathy have autoantibodies directed against thrombospondin type 1 domain-containing 7A (THSD7A), a podocyte-expressed transmembrane protein. However, the temporal and spatial expression of THSD7A and its biologic function for podocytes are unknown, information that is needed to understand the effects of THSD7A autoantibodies in this disease. METHODS: Using a variety of microscopic techniques, we analyzed THSD7A localization in postnatal, adult, and autoantibody-injected mice as well as in human podocytes. We also analyzed THSD7A function in human podocytes using confocal microscopy; Western blotting; and adhesion and migration assays. RESULTS: We found that THSD7A expression begins on glomerular vascularization with slit diaphragm formation in development. THSD7A localizes to the basal aspect of foot processes, closely following the meanders of the slit diaphragm in human and mice. Autoantibodies binding to THSD7A localize to the slit diaphragm. In human podocytes, THSD7A expression is accentuated at filopodia and thin arborized protrusions, an expression pattern associated with decreased membrane activity of cytoskeletal regulators. We also found that, phenotypically, THSD7A expression in human podocytes is associated not only with increases in cell size, enhanced adhesion, and reduced detachment from collagen type IV-coated plates but also, with decreased ability to migrate. CONCLUSIONS: Our findings suggest that THSD7A functions as a foot process protein involved in the stabilization of the slit diaphragm of mature podocytes and that autoantibodies to THSD7A, on the basis of their localization, might structurally and functionally alter the slit diaphragm's permeability to protein.


Asunto(s)
Antígenos de Superficie/genética , Glomerulonefritis Membranosa/genética , Glomérulos Renales/metabolismo , Proteínas de la Membrana/metabolismo , Trombospondinas/inmunología , Animales , Antígenos de Superficie/inmunología , Autoanticuerpos/inmunología , Western Blotting , Células Cultivadas , Regulación de la Expresión Génica , Tasa de Filtración Glomerular , Glomerulonefritis Membranosa/fisiopatología , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Podocitos/inmunología , Proteinuria/metabolismo , Sensibilidad y Especificidad , Trombospondinas/metabolismo
9.
J Vis Exp ; (143)2019 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-30735154

RESUMEN

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.


Asunto(s)
Células Cultivadas/ultraestructura , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Fluorescente/métodos
10.
PLoS Pathog ; 14(12): e1007527, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30586431

RESUMEN

Type III secretion systems (T3SSs) are essential virulence factors of numerous bacterial pathogens. Upon host cell contact the T3SS machinery-also named injectisome-assembles a pore complex/translocon within host cell membranes that serves as an entry gate for the bacterial effectors. Whether and how translocons are physically connected to injectisome needles, whether their phenotype is related to the level of effector translocation and which target cell factors trigger their formation have remained unclear. We employed the superresolution fluorescence microscopy techniques Stimulated Emission Depletion (STED) and Structured Illumination Microscopy (SIM) as well as immunogold electron microscopy to visualize Y. enterocolitica translocons during infection of different target cell types. Thereby we were able to resolve translocon and needle complex proteins within the same injectisomes and demonstrate that these fully assembled injectisomes are generated in a prevacuole, a PI(4,5)P2 enriched host cell compartment inaccessible to large extracellular proteins like antibodies. Furthermore, the operable translocons were produced by the yersiniae to a much larger degree in macrophages (up to 25% of bacteria) than in HeLa cells (2% of bacteria). However, when the Rho GTPase Rac1 was activated in the HeLa cells, uptake of the yersiniae into the prevacuole, translocon formation and effector translocation were strongly enhanced reaching the same levels as in macrophages. Our findings indicate that operable T3SS translocons can be visualized as part of fully assembled injectisomes with superresolution fluorescence microscopy techniques. By using this technology, we provide novel information about the spatiotemporal organization of T3SS translocons and their regulation by host cell factors.


Asunto(s)
Sistemas de Secreción Tipo III , Yersiniosis/transmisión , Yersinia enterocolitica/patogenicidad , Humanos , Microscopía Fluorescente
11.
J Microsc ; 269(3): 282-290, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-28960301

RESUMEN

Understanding the cellular processes that occur between the cytosol and the plasma membrane is an important task for biological research. Till now, however, it was not possible to combine fast and high-resolution imaging of both the isolated plasma membrane and the surrounding intracellular volume. Here, we demonstrate the combination of fast high-resolution spinning disk (SD) and total internal reflection fluorescence (TIRF) microscopy for specific imaging of the plasma membrane. A customised SD-TIRF microscope was used with specific design of the light paths that allowed, for the first time, live SD-TIRF experiments at high acquisition rates. A series of experiments is shown to demonstrate the feasibility and performance of our setup.


Asunto(s)
Membrana Celular/ultraestructura , Citoplasma/ultraestructura , Microscopía Intravital/métodos , Microscopía Fluorescente/métodos , Células Cultivadas , Humanos , Microscopía Intravital/instrumentación , Microscopía Fluorescente/instrumentación
12.
J Neurosci ; 38(1): 137-148, 2018 01 03.
Artículo en Inglés | MEDLINE | ID: mdl-29138282

RESUMEN

Reelin controls neuronal migration and layer formation. Previous studies in reeler mice deficient in Reelin focused on the result of the developmental process in fixed tissue sections. It has remained unclear whether Reelin affects the migratory process, migration directionality, or migrating neurons guided by the radial glial scaffold. Moreover, Reelin has been regarded as an attractive signal because newly generated neurons migrate toward the Reelin-containing marginal zone. Conversely, Reelin might be a stop signal because migrating neurons in reeler, but not in wild-type mice, invade the marginal zone. Here, we monitored the migration of newly generated proopiomelanocortin-EGFP-expressing dentate granule cells in slice cultures from reeler, reeler-like mutants and wild-type mice of either sex using real-time microscopy. We discovered that not the actual migratory process and migratory speed, but migration directionality of the granule cells is controlled by Reelin. While wild-type granule cells migrated toward the marginal zone of the dentate gyrus, neurons in cultures from reeler and reeler-like mutants migrated randomly in all directions as revealed by vector analyses of migratory trajectories. Moreover, live imaging of granule cells in reeler slices cocultured to wild-type dentate gyrus showed that the reeler neurons changed their directions and migrated toward the Reelin-containing marginal zone of the wild-type culture, thus forming a compact granule cell layer. In contrast, directed migration was not observed when Reelin was ubiquitously present in the medium of reeler slices. These results indicate that topographically administered Reelin controls the formation of a granule cell layer.SIGNIFICANCE STATEMENT Neuronal migration and the various factors controlling its onset, speed, directionality, and arrest are poorly understood. Slice cultures offer a unique model to study the migration of individual neurons in an almost natural environment. In the present study, we took advantage of the expression of proopiomelanocortin-EGFP by newly generated, migrating granule cells to analyze their migratory trajectories in hippocampal slice cultures from wild-type mice and mutants deficient in Reelin signaling. We show that the compartmentalized presence of Reelin is essential for the directionality, but not the actual migratory process or speed, of migrating granule cells leading to their characteristic lamination in the dentate gyrus.


Asunto(s)
Moléculas de Adhesión Celular Neuronal/fisiología , Movimiento Celular/fisiología , Giro Dentado/citología , Proteínas de la Matriz Extracelular/fisiología , Proteínas del Tejido Nervioso/fisiología , Serina Endopeptidasas/fisiología , Animales , Movimiento Celular/genética , Células Cultivadas , Corteza Cerebral/citología , Gránulos Citoplasmáticos/fisiología , Células Ependimogliales , Femenino , Cinética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes Neurológicos , Mutación , Neuronas/fisiología , Proopiomelanocortina/genética , Proopiomelanocortina/metabolismo
13.
Development ; 143(6): 1029-40, 2016 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-26893343

RESUMEN

In reeler mutant mice, which are deficient in reelin (Reln), the lamination of the cerebral cortex is disrupted. Reelin signaling induces phosphorylation of LIM kinase 1, which phosphorylates the actin-depolymerizing protein cofilin in migrating neurons. Conditional cofilin mutants show neuronal migration defects. Thus, both reelin and cofilin are indispensable during cortical development. To analyze the effects of cofilin phosphorylation on neuronal migration we used in utero electroporation to transfect E14.5 wild-type cortical neurons with pCAG-EGFP plasmids encoding either a nonphosphorylatable form of cofilin 1 (cofilin(S3A)), a pseudophosphorylated form (cofilin(S3E)) or wild-type cofilin 1 (cofilin(WT)). Wild-type controls and reeler neurons were transfected with pCAG-EGFP. Real-time microscopy and histological analyses revealed that overexpression of cofilin(WT) and both phosphomutants induced migration defects and morphological abnormalities of cortical neurons. Of note, reeler neurons and cofilin(S3A)- and cofilin(S3E)-transfected neurons showed aberrant backward migration towards the ventricular zone. Overexpression of cofilin(S3E), the pseudophosphorylated form, partially rescued the migration defect of reeler neurons, as did overexpression of Limk1. Collectively, the results indicate that reelin and cofilin cooperate in controlling cytoskeletal dynamics during neuronal migration.


Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Movimiento Celular , Forma de la Célula , Corteza Cerebral/citología , Cofilina 1/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Serina Endopeptidasas/metabolismo , Animales , Recuento de Células , Electroporación , Embrión de Mamíferos/citología , Femenino , Ratones Endogámicos C57BL , Neuronas/metabolismo , Transfección
14.
Oncotarget ; 6(21): 18577-89, 2015 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-26124177

RESUMEN

Drosophila homologue of Diaphanous 1 (DIAPH1) regulates actin polymerization and microtubule (MT) stabilization upon stimulation with lysophosphatidic acid (LPA). Recently, we showed strongly reduced lung metastasis of DIAPH1-depleted colon cancer cells but we found accumulations of DIAPH1-depleted cells in bone marrow. Here, we analyzed possible organ- or tissue-specific metastasis of DIAPH1-depleted HCT-116 cells. Our data confirmed that depletion of DIAPH1 strongly inhibited lung metastasis and revealed that, in contrast to control cells, DIAPH1-depleted cells did not form metastases in further organs. Detailed mechanistic analysis on cells that were not stimulated with LPA to activate the cytoskeleton-modulating activity of DIAPH1, revealed that even under basal conditions DIAPH1 was essential for cellular adhesion to collagen. In non-stimulated cells DIAPH1 did not control actin dynamics but, interestingly, was essential for stabilization of microtubules (MTs). Additionally, DIAPH1 controlled directed vesicle trafficking and with this, local clustering of the adhesion protein integrin-ß1 at the plasma membrane. Therefore, we conclude that under non-stimulating conditions DIAPH1 controls cellular adhesion by stabilizing MTs required for local clustering of integrin-ß1 at the plasma membrane. Thus, blockade of DIAPH1-tubulin interaction may be a promising approach to inhibit one of the earliest steps in the metastatic cascade of colon cancer.


Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neoplasias del Colon/metabolismo , Microtúbulos/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Western Blotting , Adhesión Celular/efectos de los fármacos , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Forminas , Células HCT116 , Células HEK293 , Humanos , Integrina beta1/metabolismo , Lisofosfolípidos/farmacología , Ratones SCID , Microscopía Fluorescente , Microtúbulos/efectos de los fármacos , Metástasis de la Neoplasia , Interferencia de ARN , Imagen de Lapso de Tiempo , Trasplante Heterólogo
15.
Tissue Cell ; 47(3): 266-72, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25890870

RESUMEN

The vascular endothelium as well as subendothelium are objects of many researches as it is directly involved in a multiplicity of physiological and pathological settings. Detailed study of endothelial function became feasible with the development of techniques to culture endothelial cells (EC) in vitro. Limitations of this approach have become apparent with the realization that cell culture dedifferentiate with time and do not exhibit properties of intact tissue. Here we describe the development of a novel ex vivo tissue model to study cell-vascular wall interactions by using isolated mouse aorta patches. Validation of this model was performed by demonstrating cell attachment and changes in cell shape typical for cell spreading during adhesion. A major advantage of this model is that cell-endothelium interaction and its molecular backgrounds can now be studied more feasibly on an intact and native tissue.


Asunto(s)
Aorta/fisiología , Adhesión Celular/fisiología , Desdiferenciación Celular/fisiología , Endotelio Vascular/citología , Animales , Aorta/citología , Comunicación Celular/fisiología , Técnicas de Cultivo de Célula , Forma de la Célula/fisiología , Endotelio Vascular/fisiología , Ratones
16.
Anal Bioanal Chem ; 407(14): 4029-34, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25855152

RESUMEN

In this paper, we explain in detail the wavelength dependence of the elastic scattering pattern of individual, optically isolated gold nanorods by using confocal microscopy in combination with higher order laser modes, i.e., radially/azimuthally polarized laser modes. We demonstrate that the spectral dependence of the scattering pattern is mostly caused by the relative strength of the gold nanorods' plasmonic modes at different wavelengths. Since the gold nanorods' plasmonic modes are determined by the particles' geometrical parameter, e.g., size and aspect ratio, as well as the refractive index of the surrounding medium, we show that the spectral dependence of the scattering pattern is a simple, not invasive way to determine, e.g., the gold nanorod aspect ratio or physical variation of the local environment. Thus, a further development of spectral imaging of gold nanorods can lead to the employment of this technique in biomedical assays involving also living samples.


Asunto(s)
Técnicas Biosensibles/métodos , Oro/química , Nanopartículas del Metal/química , Análisis Espectral/métodos , Microscopía Confocal/métodos , Coloración y Etiquetado
17.
Cytoskeleton (Hoboken) ; 72(2): 93-100, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25620569

RESUMEN

Inositol-1,4,5-trisphosphate-3-kinase-A (ITPKA) has been considered as an actin bundling protein because its N-terminal actin binding domain (ABD) induces formation of linear actin bundles. Since in many cancer cell lines ITPKA is essential for formation of lamellipodia, which consist of cross-linked actin filaments, here we analyzed if full length-ITPKA may induce formation of more complex actin structures. Indeed, we found that incubation of F-actin with ITPKA resulted in formation of dense, branched actin networks. Based on our result that ITPKA does not exhibit an additional C-terminal ABD, we exclude that ITPKA cross-links actin filaments by simultaneous F-actin binding with two different ABDs. Instead, stimulated-emission-depletion-microscopy and measurement of InsP3 Kinase activity give evidence that that N-terminal ABD-homodimers of ITPKA bind to F-actin while the monomeric C-termini insert between adjacent actin filaments. Thereby, they prevent formation of thick actin bundles but induce formation of thin branched actin structures. Interestingly, when embedded in this dense actin network, InsP3 Kinase activity is doubled and the product of InsP3 Kinase activity, Ins(1,3,4,5)P4 , inhibits spontaneous actin polymerization which may reflect a local negative feedback regulation of InsP3 Kinase activity. In conclusion, we demonstrate that not only the ABD of ITPKA modulates actin dynamics but reveal that the InsP3 Kinase domain substantially contributes to this process.


Asunto(s)
Actinas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Dominio Catalítico , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo
18.
Chem Soc Rev ; 43(4): 1263-86, 2014 Feb 21.
Artículo en Inglés | MEDLINE | ID: mdl-24365864

RESUMEN

While single-molecule fluorescence from emitters with high quantum efficiencies such as organic dye molecules can easily be detected by modern apparatus, many less efficient emission processes such as Raman scattering and metal luminescence require dramatic enhancement to exceed the single-particle detection limit. This enhancement can be achieved using resonant optical systems such as plasmonic particles or nanoantennas, the study of which has led to substantial progress in understanding the interaction of quantum emitters with their electromagnetic environment. This review is focused on the advances in measurement techniques and potential applications enabled by a deeper understanding of fundamental optical interaction processes occurring between single quantum systems on the nanoscale. While the affected phenomena are numerous, including molecular fluorescence and also exciton luminescence and Raman scattering, the interaction itself can often be described from a unified point of view. Starting from a single underlying model, this work elucidates the dramatic enhancement potential of plasmonic tips and nanoparticles and also the more deterministic influence of a Fabry-Pérot microresonator. With the extensive knowledge of the radiative behavior of a quantum system, insight can be gained into nonradiative factors as well, such as energy transfer phenomena or spatial and chemical configurations in single molecules.

19.
Histochem Cell Biol ; 141(4): 407-21, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24292845

RESUMEN

Merkel cells, the neurosecretory cells of skin, are essential for light-touch responses and may probably fulfill additional functions. Whether these cells derive from an epidermal or a neural lineage has been a matter of dispute for a long time. In mice, recent studies have clearly demonstrated an epidermal origin of Merkel cells. Given the differences in Merkel cell distribution between human and murine skin, it is, however, unclear whether the same holds true for human Merkel cells. We therefore attempted to gain insight into the human Merkel cell lineage by co-immunodetection of the Merkel cell marker protein cytokeratin 20 (CK20) with various proteins known to be expressed either in epidermal or in neural stem cells of the skin. Neither Sox10 nor Pax3, both established markers of the neural crest lineage, exhibited any cell co-labeling with CK20. By contrast, ß1 integrin, known to be enriched in epidermal stem cells, was found in nearly 70 % of interfollicular epidermal and 25 % of follicular Merkel cells. Moreover, LRIG1, also enriched in epidermal stem cells, displayed significant co-immunolabeling with CK20 as well (approximately 20 % in the interfollicular epidermis and 7 % in the hair follicle, respectively). Further epidermal markers were detected in sporadic Merkel cells. Cells co-expressing CK20 with epidermal markers may represent a transitory state between stem cells and differentiated cells. ß1 integrin is probably also synthesized by a large subset of mature Merkel cells. Summarizing, our data suggest that human Merkel cells may originate from epidermal rather than neural progenitors.


Asunto(s)
Linaje de la Célula , Células Epidérmicas , Células de Merkel/citología , Epidermis/química , Epidermis/metabolismo , Humanos , Inmunohistoquímica , Integrina beta1/análisis , Integrina beta1/metabolismo , Queratina-20/análisis , Queratina-20/metabolismo , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/metabolismo , Células de Merkel/química , Células de Merkel/metabolismo , Microscopía Confocal , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box/análisis , Factores de Transcripción Paired Box/metabolismo , Factores de Transcripción SOXE/análisis , Factores de Transcripción SOXE/metabolismo
20.
EMBO Mol Med ; 5(12): 1871-86, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24127423

RESUMEN

Mucolipidosis type II (MLII) is a severe multi-systemic genetic disorder caused by missorting of lysosomal proteins and the subsequent lysosomal storage of undegraded macromolecules. Although affected children develop disabling skeletal abnormalities, their pathogenesis is not understood. Here we report that MLII knock-in mice, recapitulating the human storage disease, are runted with accompanying growth plate widening, low trabecular bone mass and cortical porosity. Intralysosomal deficiency of numerous acid hydrolases results in accumulation of storage material in chondrocytes and osteoblasts, and impaired bone formation. In osteoclasts, no morphological or functional abnormalities are detected whereas osteoclastogenesis is dramatically increased in MLII mice. The high number of osteoclasts in MLII is associated with enhanced osteoblastic expression of the pro-osteoclastogenic cytokine interleukin-6, and pharmacological inhibition of bone resorption prevented the osteoporotic phenotype of MLII mice. Our findings show that progressive bone loss in MLII is due to the presence of dysfunctional osteoblasts combined with excessive osteoclastogenesis. They further underscore the importance of a deep skeletal phenotyping approach for other lysosomal diseases in which bone loss is a prominent feature.


Asunto(s)
Desarrollo Óseo , Mucolipidosis/patología , Osteoclastos/metabolismo , Animales , Conservadores de la Densidad Ósea/farmacología , Desarrollo Óseo/genética , Huesos/efectos de los fármacos , Huesos/metabolismo , Huesos/patología , Células Cultivadas , Preescolar , Condrocitos/citología , Condrocitos/metabolismo , Condrocitos/patología , Difosfonatos/farmacología , Modelos Animales de Enfermedad , Femenino , Humanos , Interleucina-6/metabolismo , Ratones , Ratones Endogámicos C57BL , Mucolipidosis/diagnóstico por imagen , Mucolipidosis/genética , Osteoclastos/citología , Osteoclastos/patología , Osteogénesis , Ligando RANK/metabolismo , Radiografía , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo
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