High-Content Screening

Lederman, Lynne

doi:10.2144/000112505

Cited by 41 publications

(13 citation statements)

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“…Secondly color deconvolution requires reference values that represent the color of a typical kinetoplast and nucleus, in this case these reference values are the average intensity of kinetoplast and nuclear DNA in the MGB and BPI images (see Methods). Our set of tools, written for use in ImageJ [28], for the analysis of trypanosomatid cells and their DNA by color deconvolution can automate these steps.…”

Section: Resultsmentioning

confidence: 99%

“…Automated image analysis was performed in ImageJ [28]. Our tools for automated correction chromatic aberration, color deconvolution and automated micrograph analysis were written in the ImageJ macro language and are described below.…”

Section: Methodsmentioning

confidence: 99%

“…We combined this with image processing by color deconvolution to separate the signal from kinetoplast and nuclear DNA to two separate images thus allowing unambiguous identification of kinetoplasts and nuclei and greatly simplifying quantitation of the kinetoplast and nuclear DNA content of a cell. In addition to developing this staining approach we used it as a basis to develop software tools to demonstrate this capability, in the form of a set of macros for the open source scientific image analysis software ImageJ [28], for high-throughput analysis of trypanosomatid morphology from micrographs.…”

Section: Introductionmentioning

confidence: 99%

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Detailed interrogation of trypanosome cell biology via differential organelle staining and automated image analysis

2012

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BackgroundMany trypanosomatid protozoa are important human or animal pathogens. The well defined morphology and precisely choreographed division of trypanosomatid cells makes morphological analysis a powerful tool for analyzing the effect of mutations, chemical insults and changes between lifecycle stages. High-throughput image analysis of micrographs has the potential to accelerate collection of quantitative morphological data. Trypanosomatid cells have two large DNA-containing organelles, the kinetoplast (mitochondrial DNA) and nucleus, which provide useful markers for morphometric analysis; however they need to be accurately identified and often lie in close proximity. This presents a technical challenge. Accurate identification and quantitation of the DNA content of these organelles is a central requirement of any automated analysis method.ResultsWe have developed a technique based on double staining of the DNA with a minor groove binding (4'', 6-diamidino-2-phenylindole (DAPI)) and a base pair intercalating (propidium iodide (PI) or SYBR green) fluorescent stain and color deconvolution. This allows the identification of kinetoplast and nuclear DNA in the micrograph based on whether the organelle has DNA with a more A-T or G-C rich composition. Following unambiguous identification of the kinetoplasts and nuclei the resulting images are amenable to quantitative automated analysis of kinetoplast and nucleus number and DNA content. On this foundation we have developed a demonstrative analysis tool capable of measuring kinetoplast and nucleus DNA content, size and position and cell body shape, length and width automatically.ConclusionsOur approach to DNA staining and automated quantitative analysis of trypanosomatid morphology accelerated analysis of trypanosomatid protozoa. We have validated this approach using Leishmania mexicana, Crithidia fasciculata and wild-type and mutant Trypanosoma brucei. Automated analysis of T. brucei morphology was of comparable quality to manual analysis while being faster and less susceptible to experimentalist bias. The complete data set from each cell and all analysis parameters used can be recorded ensuring repeatability and allowing complete data archiving and reanalysis.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Detailed interrogation of trypanosome cell biology via differential organelle staining and automated image analysis

2012

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“…CellMask Orange was excited at 561 nm at 2.3% power (100 mW), and emission was monitored in a 570- to 620-nm window. Images (average of 8 scans) were processed using ZEN software (Zeiss), ImageJ (NIH, Bethesda, MD) (96), and Photoshop (Adobe).…”

Section: Methodsmentioning

confidence: 99%

TOR Complex 2-Regulated Protein Kinase Fpk1 Stimulates Endocytosis via Inhibition of Ark1/Prk1-Related Protein Kinase Akl1 in Saccharomyces cerevisiae

Roelants

Leskoske

Pedersen

et al. 2017

Molecular and Cellular Biology

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Depending on the stress, plasma membrane alterations activate or inhibit yeast target of rapamycin (TOR) complex 2, which, in turn, upregulates or downregulates the activity of its essential downstream effector, protein kinase Ypk1. Through phosphorylation of multiple substrates, Ypk1 controls many processes that restore homeostasis. One such substrate is protein kinase Fpk1, which is negatively regulated by Ypk1. Fpk1 phosphorylates and stimulates flippases that translocate aminoglycerophospholipids from the outer to the inner leaflet of the plasma membrane. Fpk1 has additional roles, but other substrates were uncharacterized. We show that Fpk1 phosphorylates and inhibits protein kinase Akl1, related to protein kinases Ark1 and Prk1, which modulate the dynamics of actin patch-mediated endocytosis. Akl1 has two Fpk1 phosphorylation sites (Ark1 and Prk1 have none) and is hypophosphorylated when Fpk1 is absent. Conversely, under conditions that inactivate TORC2-Ypk1 signaling, which alleviates Fpk1 inhibition, Akl1 is hyperphosphorylated. Monitoring phosphorylation of known Akl1 substrates (Sla1 and Ent2) confirmed that Akl1 is hyperactive when not phosphorylated by Fpk1. Fpk1-mediated negative regulation of Akl1 enhances endocytosis, because an Akl1 mutant immune to Fpk1 phosphorylation causes faster dissociation of Sla1 from actin patches, confers elevated resistance to doxorubicin (a toxic compound whose entry requires endocytosis), and impedes Lucifer yellow uptake (a marker of fluid phase endocytosis). Thus, TORC2-Ypk1, by regulating Fpk1-mediated phosphorylation of Akl1, adjusts the rate of endocytosis.

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“…After another 36 h (transfection)/24 h (treatment), medium was removed and slides were fixed in 4% PFA for immunofluorescence staining as described above. Qualitative and quantitative image analysis was done using Image J image analysis software (National Institutes of Health, Bethesda, USA) [45]. 3D surface plots were generated using the 3D surface plot add-in for Image J (Kai Uwe Bartel, HTW Berlin, GER).…”

Section: Methodsmentioning

confidence: 99%

Expression and Y435-phosphorylation of Abelson interactor 1 (Abi1) promotes tumour cell adhesion, extracellular matrix degradation and invasion by colorectal carcinoma cells

et al. 2014

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BackgroundThe Abelson tyrosine kinase (c-Abl) inhibitor STI571 (Glivec®) has been shown to effectively inhibit colorectal cancer cell migration and invasion. The c-Abl substrate abelson interactor 1 (Abi1) is a key regulator of actin reorganization and upregulated in colorectal carcinoma. The specific role of Abi1 in relation to extracellular matrix degradation and effects of targeting Abi1 phosphorylation have not yet been examined. Here, we investigated the role of Abi1 in relation to invasive properties in colorectal cancer.Methods and resultsIn 56 primary human colorectal carcinoma samples, we found overexpression of Abi1 in 39% at the invasive edge of the tumour, associated with an infiltrative phenotype and high-grade tumour cell budding (p = 0.001). To explore the role of Abi1 in vitro, we employed the Abi1 expressing and KRAS-mutated CHD1 model and performed matrix degradation assays that showed Abi1 localization at specific sites of matrix degradation. Moreover, quantification of matrix dissolution demonstrated suppression after RNAi knockdown of Abi1 by 95% (p = 0.001). Importantly, treatment with STI571 did abolish Abi1 Y435-phosphorylation, suppressed the matrix dissolution, decreased fibronectin attachment, and suppressed cell invasion through reconstituted extracellular matrix.ConclusionOur data indicate that phosphorylated Abi1 contributes to the invasive properties of colorectal cancer.

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High-Content Screening

Cited by 41 publications

References 0 publications

Detailed interrogation of trypanosome cell biology via differential organelle staining and automated image analysis

Detailed interrogation of trypanosome cell biology via differential organelle staining and automated image analysis

TOR Complex 2-Regulated Protein Kinase Fpk1 Stimulates Endocytosis via Inhibition of Ark1/Prk1-Related Protein Kinase Akl1 in Saccharomyces cerevisiae

Expression and Y435-phosphorylation of Abelson interactor 1 (Abi1) promotes tumour cell adhesion, extracellular matrix degradation and invasion by colorectal carcinoma cells

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