共查询到20条相似文献,搜索用时 31 毫秒
1.
Quantification of invasive, intracellular bacteria is critical in many areas of cellular microbiology and immunology. We describe a novel and fast approach to determine invasion of bacterial pathogens in adherent cell types such as epithelial cells or fibroblasts based on flow cytometry. Using the CEACAM-mediated uptake of Opa-expressing Neisseria gonorrhoeae as a well-characterized model of bacterial invasion, we demonstrate that the flow cytometry-based method yields results comparable to a standard antibiotic protection assay. Furthermore, the quantification of intracellular bacteria by the novel approach is not biased by intracellular killing of the microbes and correctly discriminates between cell-associated extracellular and bona fide intracellular bacteria. As flow cytometry-based quantification is also applicable to other pathogen-host interactions such as the integrin-mediated internalization of Staphylococcus aureus, this approach provides a fast and convenient alternative for the quantification of bacterial uptake and should be particularly useful in elucidating the molecular mechanisms of pathogen-triggered host cell invasion. 相似文献
2.
Robustness is one of the principles of design inherent to biological systems. Cellular robustness can be measured as limits of intracellular parameters such as gene expression levels. We have recently developed an experimental approach coined as genetic Tug-Of-War (gTOW), which we used to perform robustness analysis in yeast. Using gTOW, we were able to measure the upper limit of expression of gene targets. In this review, we first elaborate on how the gTOW method compares to current mathematical simulation models prevalently used in the determination of robustness. We then explain the experimental principles underlying gTOW and its associated tools, and we provide concrete examples of robustness analysis using gTOW, i.e. cell cycle and HOG pathway gene expression analysis. Finally, we list a series of Q&As related to the experimental utilization of gTOW and we describe the potential impact of gTOW and its relevance to the understanding of biological systems. 相似文献
3.
Applications of high-throughput RNA interference screens to problems in cell and developmental biology 总被引:4,自引:0,他引:4
RNA interference (RNAi) in tissue culture cells has emerged as an excellent methodology for identifying gene functions systematically and in an unbiased manner. Here, we describe how RNAi high-throughput screening (HTS) in Drosophila cells are currently being performed and emphasize the strengths and weaknesses of the approach. Further, to demonstrate the versatility of the technology, we provide examples of the various applications of the method to problems in signal transduction and cell and developmental biology. Finally, we discuss emerging technological advances that will extend RNAi-based screening methods. 相似文献
4.
Electron tomography (ET) is an indispensable high-resolution tool for three dimensional (3D) imaging in cell biology. When applied to immuno-labeled cells, ET can provide essential insights in both the cellular architecture and the dynamics. Current protocols for 3D immuno-labeling of intracellular antigens include permeabilization steps that cause random, extensive cell membrane disruption. This permeabilization results in a poor cell ultrastructure, limiting the usefulness of the specimens for high-resolution studies. Here we describe a novel method, based on a well-controlled permeabilization by targeted laser cell perforation, that allows for the 3D immuno-localization of cytoplasmic antigens in cultured cells. The approach is unique since it is applicable to both chemically and cryo-fixed cells and leads to a superior ultrastructural preservation for electron microscopy and tomography. 相似文献
5.
Stem cell self-renewal versus differentiation fate decisions are difficult to characterize and analyze due to multiple competing rate processes occurring simultaneously among heterogeneous cell subpopulations. To address this challenge, we describe a mathematical model for cell population dynamics that allows flow cytometry measurement of population distributions of molecular markers to be deconvoluted in terms of subpopulation-specific rate parameters distinguishing commitment to differentiation, proliferation of differentiated cells, and proliferation of undifferentiated cells (i.e., self-renewal). We validate this model-based parameter determination by means of dedicated, independent cell-tracking studies. Our approach facilitates interpretation of relationships underlying effects of external cues on cell responses in differentiating cultures via intracellular signals. 相似文献
6.
Once damaged, cardiac tissue does not readily repair and is therefore a primary target of regenerative therapies. One regenerative approach is the development of scaffolds that functionally mimic the cardiac extracellular matrix (ECM) to deliver stem cells or cardiac precursor populations to the heart. Technological advances in micro/nanotechnology, stem cell biology, biomaterials and tissue decellularization have propelled this promising approach forward. Surprisingly, technological advances in optical imaging methods have not been fully utilized in the field of cardiac regeneration. Here, we describe and provide examples to demonstrate how advanced imaging techniques could revolutionize how ECM-mimicking cardiac tissues are informed and evaluated. 相似文献
7.
Costantino S McQuinn CG Kennedy TE Wiseman PW 《Journal of biochemical and biophysical methods》2008,70(6):1192-1195
We present a straightforward method to create spatial gradients of substrate bound protein for live cell studies using only mechanical parts. Protein concentration gradients on a micron scale can be fabricated in several minutes for a relatively low cost using a method that is generally applicable to any protein and substrate combination. We describe the details of the device construction, and provide examples of mammalian cells grown on substrates patterned with protein concentration gradients using this technique. 相似文献
8.
May C. Morris 《Cell biochemistry and biophysics》2010,56(1):19-37
With the escalation of drug discovery programmes, it has become essential to visualize and monitor biological activities in
healthy and pathological cells, with high spatial and temporal resolution. To this aim, the development of probes and sensors,
which can report on the levels and activities of specific intracellular targets, has become essential. Together with the discovery
of the Green Fluorescent Protein (GFP), and the development of GFP-based reporters, recent advances in the synthesis of small
molecule fluorescent probes, and the explosion of fluorescence-based imaging technologies, the biosensor field has witnessed
a dramatic expansion of fluorescence-based reporters which can be applied to complex biological samples, living cells and
tissues to probe protein/protein interactions, conformational changes and posttranslational modifications. Here, we review
recent developments in the field of fluorescent biosensor technology. We describe different varieties and categories of fluorescent
biosensors together with an overview of the technologies commonly employed to image biosensors in cellulo and in vivo. We
discuss issues and strategies related to the choice of synthetic fluorescent probes, labelling, quenching, caging and intracellular
delivery of biosensors. Finally, we provide examples of some well-characterized genetically encoded FRET reporter systems,
peptide and protein biosensors and describe biosensor applications in a wide variety of fields. 相似文献
9.
Overlaying differential changes in gene expression on protein interaction networks has proven to be a useful approach to interpreting the cell's dynamic response to a changing environment. Despite successes in finding active subnetworks in the context of a single species, the idea of overlaying lists of differentially expressed genes on networks has not yet been extended to support the analysis of multiple species' interaction networks. To address this problem, we designed a scalable, cross-species network search algorithm, neXus (Network-cross(X)-species-Search), that discovers conserved, active subnetworks based on parallel differential expression studies in multiple species. Our approach leverages functional linkage networks, which provide more comprehensive coverage of functional relationships than physical interaction networks by combining heterogeneous types of genomic data. We applied our cross-species approach to identify conserved modules that are differentially active in stem cells relative to differentiated cells based on parallel gene expression studies and functional linkage networks from mouse and human. We find hundreds of conserved active subnetworks enriched for stem cell-associated functions such as cell cycle, DNA repair, and chromatin modification processes. Using a variation of this approach, we also find a number of species-specific networks, which likely reflect mechanisms of stem cell function that have diverged between mouse and human. We assess the statistical significance of the subnetworks by comparing them with subnetworks discovered on random permutations of the differential expression data. We also describe several case examples that illustrate the utility of comparative analysis of active subnetworks. 相似文献
10.
Despite their biological and biotechnological interest, pluripotent embryonic stem cell lines (ES cells) have been isolated from cultured embryos only in a very limited number of mammalian species. Here we review the main molecular mechanisms that have been shown in mouse or primates to regulate the maintenance of pluripotency in vitro. We describe the main signaling pathways that participate in the self-renewal of ES cells and provide an outlook on the epigenetic associated mechanisms. We also propose a practical approach to stem cell differentiation that examines the relationships between the genotype of embryos and their culture conditions and consider nuclear reprogramming as a valuable approach in ES cell derivation in farm animals. 相似文献
11.
Pederson T 《European journal of histochemistry : EJH》2004,48(1):57-64
For more than 60 years, RNA has been detectable in fixed cells and tissues by relatively specific staining methods. More recently, it has become possible to study RNA in unfixed, live cells. This review article describes how the intracellular dynamics and localization of RNA in vivo can be studied by microinjection of fluorescent RNA into cells- an approach we have termed Fluorescent RNA Cytochemistry. Depending on the particular RNA species under investigation, Fluorescent RNA Cytochemistry can operate as a "stain" to reveal intracellular sites at which a given RNA resides, or as a "tracer" to allow movements of a dynamically translocating RNA to be followed in the living cell. Several examples of Fluorescent RNA Cytochemistry are presented, collectively illustrating the range of applicability this approach offers in the toolbox of gene expression, studied as in vivo cell biology. 相似文献
12.
Protein phase transitions are particularly amenable for cell signalling as these highly cooperative processes allow cells to make binary decisions in response to relatively small intracellular changes. The different processes of condensate formation and the distinct material properties of the resulting condensates provide a dictionary to modulate a range of decisions on cell fate. We argue that, on the one hand, the reversibility of liquid demixing offers a chance to arrest cell growth under specific circumstances. On the other hand, the transition to amyloids is better suited for terminal decisions such as those leading to apoptosis and necrosis. Here, we review recent examples of both scenarios, highlighting how mutations in signalling proteins affect the formation of biomolecular condensates with drastic effects on cell survival. 相似文献
13.
14.
植物细胞胞内生物碱跨膜传递模型研究(Ⅰ)-中性生物碱饱和特性模型 总被引:1,自引:1,他引:0
针对制约植物细胞生物碱释放的生物碱跨膜传递这一物理过程,引入有载体参与的主动运输过程来描述植物细胞生物碱的跨膜传递过程,将Michaelis-Menten酶促反应动力学公式应用于载体转运,建立了植物细胞中产物释放的饱和特性模型。将模型在特定参数下进行积分,对已有的实验现象进行数值模拟,并与实验结果比较表明,所建模型能很好地描述低Pka值生物碱的各种跨膜传递现象,并能同时呈现生物碱跨膜传递的线性特性和饱和特性,从而为长期对立的生物碱跨膜传递的简单扩散观点和主动运输观点的统一提供了理论依据 相似文献
15.
Changes in intracellular temperatures reflect the activity of the cell. Thus, the tool to measure intracellular temperatures could provide valuable information about cellular status. We previously reported a method to analyze the intracellular temperature distribution using a fluorescent polymeric thermometer (FPT) in combination with fluorescence lifetime imaging microscopy (FLIM). Intracellular delivery of the FPT used in the previous study required microinjection. We now report a novel FPT that is cell permeable and highly photostable, and we describe the application of this FPT to the imaging of intracellular temperature distributions in various types of mammalian cell lines. This cell-permeable FPT displayed a temperature resolution of 0.05°C to 0.54°C within the range from 28°C to 38°C in HeLa cell extracts. Using our optimized protocol, this cell-permeable FPT spontaneously diffused into HeLa cells within 10 min of incubation and exhibited minimal toxicity over several hours of observation. FLIM analysis confirmed a temperature difference between the nucleus and the cytoplasm and heat production near the mitochondria, which were also detected previously using the microinjected FPT. We also showed that this cell-permeable FPT protocol can be applied to other mammalian cell lines, COS7 and NIH/3T3 cells. Thus, this cell-permeable FPT represents a promising tool to study cellular states and functions with respect to temperature. 相似文献
16.
Risco C Sanmartín-Conesa E Tzeng WP Frey TK Seybold V de Groot RJ 《Structure (London, England : 1993)》2012,20(5):759-766
More than any other methodology, transmission electron microscopy (TEM) has contributed to our understanding of the architecture and organization of cells. With current detection limits approaching atomic resolution, it will ultimately become possible to ultrastructurally image intracellular macromolecular assemblies in situ. Presently, however, methods to unambiguously identify proteins within the crowded environment of the cell's interior are lagging behind. We describe an approach, metal-tagging TEM (METTEM), that allows detection of intracellular proteins in mammalian cells with high specificity, exceptional sensitivity, and at molecular scale resolution. In live cells treated with gold salts, proteins bearing a small metal-binding tag will form 1-nm gold nanoclusters, readily detectable in electron micrographs. The applicability and strength of METTEM is demonstrated by a study of Rubella virus replicase and capsid proteins, which revealed virus-induced cell structures not seen before. 相似文献
17.
Abrahim I. Orabi Kamaldeen A. Muili Dong Wang Shunqian Jin George Perides Sohail Z. Husain 《Journal of visualized experiments : JoVE》2013,(77)
The pancreatic acinar cell is the main parenchymal cell of the exocrine pancreas and plays a primary role in the secretion of pancreatic enzymes into the pancreatic duct. It is also the site for the initiation of pancreatitis. Here we describe how acinar cells are isolated from whole pancreas tissue and intracellular calcium signals are measured. In addition, we describe the techniques of transfecting these cells with adenoviral constructs, and subsequently measuring the leakage of lactate dehydrogenase, a marker of cell injury, during conditions that induce acinar cell injury in vitro. These techniques provide a powerful tool to characterize acinar cell physiology and pathology. 相似文献
18.
Intracellular oxygen-sensitive phosphorescent probes based on cell-penetrating peptides 总被引:2,自引:0,他引:2
Ruslan I. Dmitriev Gelii V. Ponomarev Dmitri B. Papkovsky 《Analytical biochemistry》2010,398(1):24-2518
Probing of molecular oxygen in mammalian cells is important for the analysis of mitochondrial function, metabolic responses, and energetic status of the cells. We describe a new panel of intracellular O2-sensitive probes based on phosphorescent porphyrin dyes conjugated to cell-penetrating peptides. The probes comprising the uncharged derivatives of Pt(II)-coproporphyrin I covalently linked to positively charged TAT-derived peptides are shown to effectively load live mammalian cells without any transfection reagents. The probes work well with all cell types tested, show similar subcellular localization, and produce characteristic responses to cell stimulation with mitochondrial uncouplers and inhibitors. They provide a simple and versatile tool for O2 monitoring in live cells and in tissue, and an alternative to the existing O2 probes which require facilitated transport into the cell. 相似文献
19.