Use of multiple singular value decompositions to analyze complex intracellular calcium ion signals

We compare calcium ion signaling ($\mathrm {Ca}^{2+}$) between two exposures; the data are present as movies, or, more prosaically, time series of images. This paper describes novel uses of singular value decompositions (SVD) and weighted versions of them (WSVD) to extract the signals from such movies, in a way that is semi-automatic and tuned closely to the actual data and their many complexities. These complexities include the following. First, the images themselves are of no interest: all interest focuses on the behavior of individual cells across time, and thus, the cells need to be segmented in an automated manner. Second, the cells themselves have 100$+$ pixels, so that they form 100$+$ curves measured over time, so that data compression is required to extract the features of these curves. Third, some of the pixels in some of the cells are subject to image saturation due to bit depth limits, and this saturation needs to be accounted for if one is to normalize the images in a reasonably unbiased manner. Finally, the $\mathrm {Ca}^{2+}$ signals have oscillations or waves that vary with time and these signals need to be extracted. Thus, our aim is to show how to use multiple weighted and standard singular value decompositions to detect, extract and clarify the $\mathrm {Ca}^{2+}$ signals. Our signal extraction methods then lead to simple although finely focused statistical methods to compare $\mathrm {Ca}^{2+}$ signals across experimental conditions.Comment: Published in at http://dx.doi.org/10.1214/09-AOAS253 the Annals of Applied Statistics (http://www.imstat.org/aoas/) by the Institute of Mathematical Statistics (http://www.imstat.org

n-3 polyunsaturated fatty acids suppress CD4+ T cell proliferation by altering phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] organization

AbstractThe mechanisms by which n-3 polyunsaturated fatty acids (n-3 PUFA), abundant in fish oil, exert their anti-inflammatory effects have not been rigorously defined. We have previously demonstrated that n-3 PUFA decrease the amount of phosphatidylinositol-(4,5)-bisphosphate, [PI(4,5)P2], in CD4+ T cells, leading to suppressed actin remodeling upon activation. Since discrete pools of PI(4,5)P2 exist in the plasma membrane, we determined whether n-3 PUFA modulate spatial organization of PI(4,5)P2 relative to raft and non-raft domains. We used Förster resonance energy transfer (FRET) to demonstrate that lipid raft mesodomains in the plasma membrane of CD4+ T cells enriched in n-3 PUFA display increased co-clustering of Lck(N10) and LAT(ΔCP), markers of lipid rafts. CD4+ T cells enriched in n-3 PUFA also exhibited a depleted plasma membrane non-raft PI(4,5)P2 pool as detected by decreased co-clustering of Src(N15), a non-raft marker, and PH(PLC-δ), a PI(4,5)P2 reporter. Incubation with exogenous PI(4,5)P2 rescued the effects on the non-raft PI(4,5)P2 pool, and reversed the suppression of T cell proliferation in CD4+ T cells enriched with n-3 PUFA. Furthermore, CD4+ T cells isolated from mice fed a 4% docosahexaenoic acid (DHA)-enriched diet exhibited a decrease in the non-raft pool of PI(4,5)P2, and exogenous PI(4,5)P2 reversed the suppression of T cell proliferation. Finally, these effects were not due to changes to post-translational lipidation, since n-3 PUFA did not alter the palmitoylation status of signaling proteins. These data demonstrate that n-3 PUFA suppress T cell proliferation by altering plasma membrane topography and the spatial organization of PI(4,5)P2

Long-Chain n-3 Fatty Acids Attenuate Oncogenic KRas-Driven Proliferation by Altering Plasma Membrane Nanoscale Proteolipid Composition

Ras signaling originates from transient nanoscale compartmentalized regions of the plasma membrane composed of specific proteins and lipids. The highly specific lipid composition of these nanodomains, termed nanoclusters, facilitates effector recruitment and therefore influences signal transduction. This suggests that Ras nanocluster proteolipid composition could represent a novel target for future chemoprevention interventions. There is evidence that consumption of fish oil containing long-chain n-3 polyunsaturated fatty acids (n-3 PUFA) such as eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17) and docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19) may reduce colon cancer risk in humans, yet the mechanism underlying this effect is unknown. Here, we demonstrate that dietary n-3 PUFA reduce the lateral segregation of cholesterol-dependent and -independent nanoclusters, suppressing phosphatidic acid-dependent oncogenic KRas effector interactions, via their physical incorporation into plasma membrane phospholipids. This results in attenuation of oncogenic Ras-driven colonic hyperproliferation in both Drosophila and murine models. These findings demonstrate the unique properties of dietary n-3 PUFA in the shaping of Ras nanoscale proteolipid complexes and support the emerging role of plasma membrane-targeted therapies. Significance: The influence of dietary long chain n-3 polyunsaturated fatty acids on plasma membrane protein nanoscale organization and KRas signaling supports development of plasma membrane-targeted therapies in colon cancer

Activation of GPER Induces Differentiation and Inhibition of Coronary Artery Smooth Muscle Cell Proliferation

BACKGROUND: Vascular pathology and dysfunction are direct life-threatening outcomes resulting from atherosclerosis or vascular injury, which are primarily attributed to contractile smooth muscle cells (SMCs) dedifferentiation and proliferation by re-entering cell cycle. Increasing evidence suggests potent protective effects of G-protein coupled estrogen receptor 1 (GPER) activation against cardiovascular diseases. However, the mechanism underlying GPER function remains poorly understood, especially if it plays a potential role in modulating coronary artery smooth muscle cells (CASMCs). METHODOLOGY/PRINCIPAL FINDINGS: The objective of our study was to understand the functional role of GPER in CASMC proliferation and differentiation in coronary arteries using from humans and swine models. We found that the GPER agonist, G-1, inhibited both human and porcine CASMC proliferation in a concentration- (10(−8) to 10(−5) M) and time-dependent manner. Flow cytometry revealed that treatment with G-1 significantly decreased the proportion of S-phase and G2/M cells in the growing cell population, suggesting that G-1 inhibits cell proliferation by slowing progression of the cell cycle. Further, G-1-induced cell cycle retardation was associated with decreased expression of cyclin B, up-regulation of cyclin D1, and concomitant induction of p21, and partially mediated by suppressed ERK1/2 and Akt pathways. In addition, G-1 induces SMC differentiation evidenced by increased α-smooth muscle actin (α-actin) and smooth muscle protein 22α (SM22α) protein expressions and inhibits CASMC migration induced by growth medium. CONCLUSION: GPER activation inhibits CASMC proliferation by suppressing cell cycle progression via inhibition of ERK1/2 and Akt phosphorylation. GPER may constitute a novel mechanism to suppress intimal migration and/or synthetic phenotype of VSMC

Alteration of EGFR Spatiotemporal Dynamics Suppresses Signal Transduction

The epidermal growth factor receptor (EGFR), which regulates cell growth and survival, is integral to colon tumorigenesis. Lipid rafts play a role in regulating EGFR signaling, and docosahexaenoic acid (DHA) is known to perturb membrane domain organization through changes in lipid rafts. Therefore, we investigated the mechanistic link between EGFR function and DHA. Membrane incorporation of DHA into immortalized colonocytes altered the lateral organization of EGFR. DHA additionally increased EGFR phosphorylation but paradoxically suppressed downstream signaling. Assessment of the EGFR-Ras-ERK1/2 signaling cascade identified Ras GTP binding as the locus of the DHA-induced disruption of signal transduction. DHA also antagonized EGFR signaling capacity by increasing receptor internalization and degradation. DHA suppressed cell proliferation in an EGFR-dependent manner, but cell proliferation could be partially rescued by expression of constitutively active Ras. Feeding chronically-inflamed, carcinogen-injected C57BL/6 mice a fish oil containing diet enriched in DHA recapitulated the effects on the EGFR signaling axis observed in cell culture and additionally suppressed tumor formation. We conclude that DHA-induced alteration in both the lateral and subcellular localization of EGFR culminates in the suppression of EGFR downstream signal transduction, which has implications for the molecular basis of colon cancer prevention by DHA

DHA reduces localization of EGFR to lipid rafts.

<p>A) YAMC cells were untreated (control) or treated with 50 µM BSA-complexed fatty acids (LA or DHA) for 72 h (12 flasks per treatment). For the final 16–18 h, cells were incubated with low serum media (0.5% FBS) with the same concentration of fatty acids. Cells were harvested from each flask, pooled (n = 12), and the plasma membrane (PM) was isolated. Following isolation, the plasma membrane was fractionated into 3 distinct fractions, high density membrane (HDM), intermediate density membrane (IDM), and lipid raft enriched membrane (LR) by gradient ultracentrifugation. Fractions were collected and an equal amount of protein from each fraction was analyzed by Western blotting using antibodies against EGFR, caveolin-1, and clathrin or using peroxidase conjugated cholera toxin B subunit (for GM-1). Quantification of band intensity was performed, and data are presented as the relative amount of EGFR in each fraction, with the sum of each fraction equaling 100. Western blots are representative of 2 independent experiments. C, control; LA, linoleic acid; DHA, docosahexaenoic acid; PM, plasma membrane; HDM, high density membrane; IDM, intermediate density membrane; LR, lipid raft enriched membrane. B) YAMC cells were treated with 50 µM BSA-complexed fatty acids for 72 h. Twenty-four h after initiating fatty acid treatment, cells were co-transfected with RFP-tH and EGFR-mGFP. Approximately 32 h after transfection, cells were incubated in low serum media (0.5% FBS) overnight prior to imaging. Images are representative of 4 independent experiments. Whole cell images of each individual channel and the merged images are shown on the left. High magnification images of the plasma membrane are shown on the right. Mander’s colocalization coefficient was calculated at the plasma membrane for the amount of EGFR-mGFP (green) colocalizing with RFP-tH (red) using Nikon Elements AR 3.2. The coefficient is the mean of n = 30–40 cells per treatment. Statistical significance between treatments (*<i>P</i><0.05) was determined using ANOVA and Tukey’s test of contrast. Bars,10 µm.</p

DHA mediates increased EGFR internalization and degradation.

<p>YAMC cells were incubated with untreated media or media supplemented with 50 µM BSA-complexed DHA for a total of 72 h. For the final 16–18 h, cells were incubated with low serum media (0.5% FBS). A) Cell surface proteins were labeled with EZ-Link Sulfo-NHS-SS-Biotin followed by stimulation with 25 ng/mL EGF for 0–30 min. After stimulation, cells were washed and biotin remaining on the cell surface was cleaved. Cell lysates were harvested, and biotinylated EGFR was quantified by ELISA using streptavidin coated plates and anti-EGFR antibody. B) Cell surface EGFR was assessed by treating cells the same as in A, and harvesting without stimulating with EGF or cleaving cell surface biotin. C) Cells were stimulated with 25 ng/mL EGF for 0–30 min and harvested. EGFR was immunoprecipitated from the total cell lysate, assessed by Western blotting for ubiquitin, and quantification of band intensity was performed. All results are representative of at least 3 independent experiments. Data represent mean±SEM. In (A) and (C), data are normalized to time 0. In B), data are normalized to control (no fatty acid). Statistical significance between treatments (*<i>P</i><0.05. **<i>P</i><0.01) was determined using Student’s <i>t</i>-test. C, control; DHA, docosahexaenoic acid.</p