Single-cell analysis of phosphoinositide 3-kinase and phosphatase and tensin homolog activation

Jiang, Dechen; Sims, Christopher E.; Allbritton, Nancy L.

doi:10.1039/c005362g

Cited by 19 publications

(32 citation statements)

References 75 publications

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“…TSP buffer was tested since SDC is often used to disperse lipids [24,27]. ECB and PBS buffers were chosen as these buffers are cell compatible and are often used as the sample matrix when cells or lysates are separated by CE [29,34]. BFL (1 nM), PIP2FL (10 nM) and PIP3FL (10 nM) were dissolved in each of the sample matrices and then separated in the buffer system optimized for the phosphoinositide analytes (NaH 2 PO 4 32 mM, pH 7.3 and 20% 1-propanol).…”

Section: Resultsmentioning

confidence: 99%

“…Metabolites and enzyme activities have been monitored by a variety of investigators using CE-LIF applied to cell lysates and single cells [30–36]. The use of CE in analyzing PI3K and sphingosine kinase activity for in vitro and cell-based assays has also been demonstrated [24,34,37]. Electrophoresis in a microfluidic device has likewise been applied to phospholipid separations.…”

Section: Introductionmentioning

confidence: 99%

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Separation of fluorescently labeled phosphoinositides and sphingolipids by capillary electrophoresis

Wang

Jiang

Sims

et al. 2012

Journal of Chromatography B

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Phosphoinositides (PIs) and sphingolipids regulate many aspects of cell behavior and are often involved in disease processes such as oncogenesis. Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) is emerging as an important tool for enzymatic assays of the metabolism of these lipids, particularly in cell-based formats. Previous separations of phosphoinositide lipids by CE required a complex buffer with polymer additives which had the disadvantages of high cost and/or short shelf life. Further a simultaneous separation of these classes of lipids has not been demonstrated in a robust buffer system. In the current work, a simple separation buffer based on NaH2PO4 and 1-propanol was optimized to separate two sphingolipids and multiple phosphoinositides by CE. The NaH2PO4 concentration, pH, 1-propanol fraction, and a surfactant additive to the buffer were individually optimized to achieve simultaneous separation of the sphingolipids and phosphoinositides. Fluorescein-labeled sphingosine (SFL) and sphingosine 1-phosphate (S1PFL), fluorescein-labeled phosphatidyl-inositol 4,5-bisphosphate (PIP2) and phosphatidyl-inositol 3,4,5-trisphosphate (PIP3), and bodipy-fluorescein (BFL)-labeled PIP2 and PIP3 were separated pairwise and in combination to demonstrate the generalizability of the method. Theoretical plate numbers achieved were as high as 2×105 in separating fluorophore-labeled PIP2 and PIP3. Detection limits for the 6 analytes were in the range of 10−18 to 10−20 mol. The method also showed high reproducibility, as the relative standard deviation of the normalized migration time for each analyte in the simultaneous separation of all 6 compounds was less than 1%. The separation of a mixture composed of diacylglycerol (DAG) and multiple phosphoinositides was also demonstrated. As a final test, fluorescent lipid metabolites formed within cells loaded with BFLPIP2 were separated from a cell lysate as well as a single cell. This simple and robust separation method for SFL and S1PFL and various metabolites of phosphoinositide-related signal transduction is expected to enable improved enzymatic assays for biological and clinical applications.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Separation of fluorescently labeled phosphoinositides and sphingolipids by capillary electrophoresis

Wang

Jiang

Sims

et al. 2012

Journal of Chromatography B

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“…A variety of cellular metabolites have been assayed using single-cell CZE, including thiol-containing compounds [16], small molecules such as glucose [17], dopamine [18], and amino acids [19], as well as measures of enzyme and peptidase activity [2023]. Single-cell CZE has also been used to study different classes of lipid metabolism in cytosolic lysates and single cells [24], such as sphingolipids [25,26], glycosphingolipids [27], and phosphatidylinositides [28]. …”

Section: Introductionmentioning

confidence: 99%

Chemical fixation to arrest phospholipid signaling for chemical cytometry

Proctor

Sims

Allbritton

2017

Journal of Chromatography A

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Chemical cytometry is a powerful tool for measuring biological processes such as enzymatic signaling at the single cell level. Among these technologies, single-cell capillary zone electrophoresis (CZE) has emerged as a powerful tool to assay a wide range of cellular metabolites. However, analysis of dynamic processes within cells remains challenging as signaling pathways are rapidly altered in response to changes in the cellular environment, including cell manipulation and storage. To address these limitations, we describe a method for chemical fixation of cells to stop the cellular reactions to preserve the integrity of key signaling molecules or reporters within the cell and to enable the cell to act as a storage reservoir for the reporter and its metabolites prior to assay by single-cell CZE. Fluorescent phosphatidylinositol 4,5-bisphosphate reporters were loaded into cells and the cells were chemically fixed and stored prior to analysis. The reporter and its metabolites were electrophoretically separated by singlecell CZE. Chemical fixation parameters such as fixative, fixation time, storage solution, storage duration, and extraction solution were optimized. When cells were loaded with a fluorescent C6- or C16-PIP2 followed by glutaraldehyde fixation and immediate analysis, 24 ± 2% and 139 ± 12% of the lipid was recoverable, respectively, when compared to an unfixed control. Storage of the cells for 24 h yielded recoverable lipid of 61 ± 3% (C6-PIP2) and 55 ± 5% (C16-PIP2) when compared to cells analyzed immediately after fixation. The metabolites observed with and without fixation were identical. Measurement of phospholipase C activity in single leukemic cells in response to an agonist demonstrated the capability of chemical fixation coupled to singlecell CZE to yield an accurate snapshot of cellular reactions with the probe. This methodology enables cell assay with the reporter to be separated in space and time from reporter metabolite quantification while preserving assay integrity.

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“…The relative changes in cellular localizations and intensities of these biosensors reflect the dynamic metabolism of the corresponding PIs, and have been used to monitor activity of PI metabolic enzymes. Recently, fluorescent PI derivatives have been coupled with capillary electrophoresis separation technique to develop a unique assay for PI signaling[32-34]. In this assay, the substrate and product forms of the fluorescent PI derivatives are separated and quantified to measure cellular enzyme activity.…”

Section: Introductionmentioning

confidence: 99%

Required hydrophobicity of fluorescent reporters for phosphatidylinositol family of lipid enzymes

et al. 2017

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The phosphatidylinositol (PtdIns) family of lipids plays important roles in cell differentiation, proliferation and migration. Abnormal expression, mutation or regulation of their metabolic enzymes has been associated with various human diseases such as cancer, diabetes and bipolar disorder. Recently, fluorescent derivatives have increasingly been used as chemical probes to monitor either lipid localization or enzymatic activity. However, the requirements of a good probe have not been well defined, particularly modifications on the diacylglycerol side chain partly due to challenges in generating PtdIns lipids. We have synthesized a series of fluorescent PtdIns(4,5)P2 (PIP2) and PtdIns (PI) derivatives with various length of side chains and tested their capacity as substrates for PI3KIα and PI4KIIα, respectively. Both capillary electrophoresis and thin layer chromatography were used to analyze enzymatic reactions. For both enzymes, the fluorescent probe with a longer side chain functions as a better substrate than that with a shorter chain, and works well in the presence of the endogenous lipid, highlighting the importance of hydrophobicity of side chains in fluorescent phosphoinositide reporters. This comparison is consistent with their interactions with lipid vesicles, suggesting that the binding of a fluorescent lipid with liposome serves as a standard for assessing its utility as a chemical probe for the corresponding endogenous lipid. These findings are likely applicable to other lipid enzymes where the catalysis takes place at the lipid-water interface.

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Single-cell analysis of phosphoinositide 3-kinase and phosphatase and tensin homolog activation

Cited by 19 publications

References 75 publications

Separation of fluorescently labeled phosphoinositides and sphingolipids by capillary electrophoresis

Separation of fluorescently labeled phosphoinositides and sphingolipids by capillary electrophoresis

Chemical fixation to arrest phospholipid signaling for chemical cytometry

Required hydrophobicity of fluorescent reporters for phosphatidylinositol family of lipid enzymes

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