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Fluorescent Probes for Subcellular Localization during Osteclast Formation.

ABSTRACT: Labeling of a small bioactive molecule with fluorescent probe has been becoming an essential tool in cell biology to reveal the subcellular distribution and the location of a molecular target. QOA-8a is a novel molecule with potent antiosteoporotic effect in vivo. To investigate the molecular mechanism of QOA-8a, novel fluorescence-tagged chemical probes as bioactive as their parent molecule were designed and synthesized. The fluorescent compound 12 showed a more potent inhibitory activity on RANKL-induced osteoclastogenesis at 2 ?M compared with that of QOA-8a. Microscopy experiments revealed that almost all of probe 12 accumulated in the fusing region, with little in the osteoclast precursors or the mature osteoclasts during osteoclast formation. The result suggests the location of the binding target of QOA-8a, which might greatly narrow down the search field of the target protein(s).

SUBMITTER: Wu J

PROVIDER: S-EPMC4137367 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Fluorescent Probes for Subcellular Localization during Osteclast Formation.

Wu Jing J Shen Qi Q Wang Yue Y Zhao Dan D Peng Chen C Li Jian-Xin JX

ACS medicinal chemistry letters 20140611 8

Labeling of a small bioactive molecule with fluorescent probe has been becoming an essential tool in cell biology to reveal the subcellular distribution and the location of a molecular target. QOA-8a is a novel molecule with potent antiosteoporotic effect in vivo. To investigate the molecular mechanism of QOA-8a, novel fluorescence-tagged chemical probes as bioactive as their parent molecule were designed and synthesized. The fluorescent compound 12 showed a more potent inhibitory activity on RA ...[more]

PMID: 25147613

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Project description:BackgroundTo study the chemical determinants of small molecule transport inside cells, it is crucial to visualize relationships between the chemical structure of small molecules and their associated subcellular distribution patterns. For this purpose, we experimented with cells incubated with a synthetic combinatorial library of fluorescent, membrane-permeant small molecule chemical agents. With an automated high content screening instrument, the intracellular distribution patterns of these chemical agents were microscopically captured in image data sets, and analyzed off-line with machine vision and cheminformatics algorithms. Nevertheless, it remained challenging to interpret correlations linking the structure and properties of chemical agents to their subcellular localization patterns in large numbers of cells, captured across large number of images.ResultsTo address this challenge, we constructed a Multidimensional Online Virtual Image Display (MOVID) visualization platform using off-the-shelf hardware and software components. For analysis, the image data set acquired from cells incubated with a combinatorial library of fluorescent molecular probes was sorted based on quantitative relationships between the chemical structures, physicochemical properties or predicted subcellular distribution patterns. MOVID enabled visual inspection of the sorted, multidimensional image arrays: Using a multipanel desktop liquid crystal display (LCD) and an avatar as a graphical user interface, the resolution of the images was automatically adjusted to the avatar's distance, allowing the viewer to rapidly navigate through high resolution image arrays, zooming in and out of the images to inspect and annotate individual cells exhibiting interesting staining patterns. In this manner, MOVID facilitated visualization and interpretation of quantitative structure-localization relationship studies. MOVID also facilitated direct, intuitive exploration of the relationship between the chemical structures of the probes and their microscopic, subcellular staining patterns.ConclusionMOVID can provide a practical, graphical user interface and computer-assisted image data visualization platform to facilitate bioimage data mining and cheminformatics analysis of high content, phenotypic screening experiments.

Project description:Carbonic anhydrases (CAs) play fundamental roles in several physiological events, and emerging evidence points at their involvement in an array of disorders, including cancer. The expression of CAs in the different cells of teeth is unknown, let alone their expression patterns during odontogenesis. As a first step towards understanding the role of CAs during odontogenesis, we used immunohistochemistry, histochemistry and in situ hybridization to reveal hitherto unknown dynamic distribution patterns of eight CAs in mice. The most salient findings include expression of CAII/Car2 not only in maturation-stage ameloblasts (MA) but also in the papillary layer, dental papilla mesenchyme, odontoblasts and the epithelial rests of Malassez. We uncovered that the latter form lace-like networks around incisors; hitherto these have been known to occur only in molars. All CAs studied were produced by MA, however CAIV, CAIX and CARPXI proteins were distinctly enriched in the ruffled membrane of the ruffled MA but exhibited a homogeneous distribution in smooth-ended MA. While CAIV, CAVI/Car6, CAIX, CARPXI and CAXIV were produced by all odontoblasts, CAIII distribution displayed a striking asymmetry, in that it was virtually confined to odontoblasts in the root of molars and root analog of incisors. Remarkably, from initiation until near completion of odontogenesis and in several other tissues, CAXIII localized mainly in intracellular punctae/vesicles that we show to overlap with LAMP-1- and LAMP-2-positive vesicles, suggesting that CAXIII localizes within lysosomes. We showed that expression of CAs in developing teeth is not confined to cells involved in biomineralization, pointing at their participation in other biological events. Finally, we uncovered novel sites of CA expression, including the developing brain and eye, the olfactory epithelium, melanoblasts, tongue, notochord, nucleus pulposus and sebaceous glands. Our study provides important information for future single or multiple gene targeting strategies aiming at deciphering the function of CAs during odontogenesis.

Project description:BackgroundIntrinsically fluorescent proteins have revolutionized studies in molecular cell biology. The parallel application of these proteins in dual- or multilabeling experiments such as subcellular localization studies requires non-overlapping emission spectra for unambiguous detection of each label. In the red spectral range, almost exclusively DsRed and derivatives thereof are used today. To test the suitability of the red fluorescent protein eqFP611 as an alternative in higher plants, the behavior of this protein was analyzed in terms of expression, subcellular targeting and compatibility with GFP in tobacco.ResultsWhen expressed transiently in tobacco protoplasts, eqFP611 accumulated over night to levels easily detectable by fluorescence microscopy. The native protein was found in the nucleus and in the cytosol and no detrimental effects on cell viability were observed. When fused to N-terminal mitochondrial and peroxisomal targeting sequences, the red fluorescence was located exclusively in the corresponding organelles in transfected protoplasts. Upon co-expression with GFP in the same cells, fluorescence of both eqFP611 and GFP could be easily distinguished, demonstrating the potential of eqFP611 in dual-labeling experiments with GFP. A series of plasmids was constructed for expression of eqFP611 in plants and for simultaneous expression of this fluorescent protein together with GFP. Transgenic tobacco plants constitutively expressing mitochondrially targeted eqFP611 were generated. The red fluorescence was stably transmitted to the following generations, making these plants a convenient source for protoplasts containing an internal marker for mitochondria.ConclusionIn plants, eqFP611 is a suitable fluorescent reporter protein. The unmodified protein can be expressed to levels easily detectable by epifluorescence microscopy without adverse affect on the viability of plant cells. Its subcellular localization can be manipulated by N-terminal signal sequences. eqFP611 and GFP are fully compatible in dual-labeling experiments.

Dataset Information

Fluorescent Probes for Subcellular Localization during Osteclast Formation.

Publications

Fluorescent Probes for Subcellular Localization during Osteclast Formation.

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