The generation of knock-in mice expressing fluorescently tagged galanin receptors 1 and 2

The neuropeptide galanin has diverse roles in the central and peripheral nervous systems, by activating the G protein-coupled receptors Gal(1), Gal(2) and the less studied Gal(3) (GalR1–3 gene products). There is a wealth of data on expression of Gal(1–3) at the mRNA level, but not at the protein level due to the lack of specificity of currently available antibodies. Here we report the generation of knock-in mice expressing Gal(1) or Gal(2) receptor fluorescently tagged at the C-terminus with, respectively, mCherry or hrGFP (humanized Renilla green fluorescent protein). In dorsal root ganglia (DRG) neurons expressing the highest levels of Gal(1)-mCherry, localization to the somatic cell membrane was detected by live-cell fluorescence and immunohistochemistry, and that fluorescence decreased upon addition of galanin. In spinal cord, abundant Gal(1)-mCherry immunoreactive processes were detected in the superficial layers of the dorsal horn, and highly expressing intrinsic neurons of the lamina III/IV border showed both somatic cell membrane localization and outward transport of receptor from the cell body, detected as puncta within cell processes. In brain, high levels of Gal(1)-mCherry immunofluorescence were detected within thalamus, hypothalamus and amygdala, with a high density of nerve endings in the external zone of the median eminence, and regions with lesser immunoreactivity included the dorsal raphe nucleus. Gal(2)-hrGFP mRNA was detected in DRG, but live-cell fluorescence was at the limits of detection, drawing attention to both the much lower mRNA expression than to Gal(1) in mice and the previously unrecognized potential for translational control by upstream open reading frames (uORFs)

Modest Interference with Actin Dynamics in Primary T Cell Activation by Antigen Presenting Cells Preferentially Affects Lamellal Signaling

Dynamic subcellular distributions of signaling system components are critical regulators of cellular signal transduction through their control of molecular interactions. Understanding how signaling activity depends on such distributions and the cellular structures driving them is required for comprehensive insight into signal transduction. In the activation of primary murine T cells by antigen presenting cells (APC) signaling intermediates associate with various subcellular structures, prominently a transient, wide, and actin-associated lamellum extending from an interdigitated T cell:APC interface several micrometers into the T cell. While actin dynamics are well established as general regulators of cellular organization, their role in controlling signaling organization in primary T cell:APC couples and the specific cellular structures driving it is unresolved. Using modest interference with actin dynamics with a low concentration of Jasplakinolide as corroborated by costimulation blockade we show that T cell actin preferentially controls lamellal signaling localization and activity leading downstream to calcium signaling. Lamellal localization repeatedly related to efficient T cell function. This suggests that the transient lamellal actin matrix regulates T cell signaling associations that facilitate T cell activation

Assessment of the usefulness of a diagnostic test: A survey of patient preference for diagnostic techniques in the evaluation of intestinal inflammation

BACKGROUND: In order to assess the usefulness of radiolabeled white cell scanning in the diagnosis of intestinal inflammation, subjects were asked to rank several dimensions of preference for white cell scanning in relation to other diagnostic tests. Two groups were surveyed: one known to have inflammatory bowel disease and the second not familiar in most cases with the tests. Subjects were asked to rank preference for each of seven tests: radiolabeled white cell scan, colonoscopy, barium enema, sigmoidoscopy, enteroclysis, stool analysis and laparotomy for the diagnosis of IBD and impressions of discomfort, embarrassment, inconvenience and danger related to each test. Mean rank scores were calculated, test ranks compared within groups and significance determined by the Wilcoxon rank test. RESULTS: Significant differences were seen in overall preference for white cell scan over barium enema and colonoscopy (p < 0.01) in both survey groups. Perceived discomfort and embarrassment demonstrated similar rankings. CONCLUSION: This patient preference combined with the reported accuracy of white cell scanning further establishes the usefulness of this means of IBD diagnosis

Low dose JASP treatment only modestly interferes with interface actin accumulation.

<p><b>(A)</b> Principal actin and signaling distributions are schematically represented: The APC above the T cell is not shown. Central reflects a large central signaling complex, peripheral the part of the actin network stabilizing the interface edge. Diffuse reflects cortical accumulation, invagination enrichment in a transient large T cell invagination suggested to contribute to early signal resetting [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.ref020" target="_blank">20</a>]. Asymmetric reflects individual small membrane extensions and the lamellal pattern represents accumulation in a large transient actin sheet (‘lamellum’) extending from the undulating T cell:APC interface μm deep into the T cell, as characterized in detail in an accompanying manuscript. <b>(B, D)</b> Representative interactions of 5C.C7 T cells expressing GFP-actin with peptide-loaded CH27 APCs (10μM MCC) over the indicated time relative to formation of a tight cell conjugate are given in control T cells (B) and T cells treated with 40nM Jasplakinolide (D). DIC images are shown on top and top-down maximum projections of 3D fluorescence data are shown in the bottom panels in a rainbow-like, false-color intensity scale (increasing from blue to red)(scale bar = 5μm). (<b>C, E</b>) 5C.C7 T cells expressing GFP-actin were stimulated with peptide loaded CH27 APCs (10μM MCC). The percentage of T cells showing accumulation in defined patterns [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.ref003" target="_blank">3</a>] among all cell couples analyzed across multiple experiments as schematically represented in (A) is shown under the (C) control (number of cell couples analyzed across multiple independent experiments, n = 80) or (E) 40nM Jasplakinolide (n = 53) condition. (<b>F</b>) 5C.C7 T cells expressing GFP-actin were stimulated with peptide loaded CH27 APCs (10μM MCC) in the presence of 40nM Jasplakinolide (n = 25) or DMSO (n = 25). The accumulation index, a measure for the amount of GFP-actin recruited to the cellular interface, is plotted relative to the time of tight cell conjugate formation. (<b>G</b>) GFP-actin amounts relative to maximum as a function of the distance from the interface were measured from the same live cell 5C.C7:CH27 conjugates as in C, E. The measurements were made separately for the center (delineated by the 25% and 75% marks across the interface diameter) and periphery (remainder) of the interface and are shown accordingly. Control data are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.g002" target="_blank">Fig 2C</a> of the accompanying manuscript.</p

Low dose JASP treatment preferentially interferes with lamellal signaling localization.

<p>(<b>A</b>) A representative interaction of a 5C.C7 T cell expressing PLCδPH-GFP (PIP₂ sensor) with 40nM Jasplakinolide (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.s007" target="_blank">S3 Video</a><b>)</b> is given similar to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.g001" target="_blank">Fig 1B</a>. (<b>B</b>) The corresponding pattern classification is given similar to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.g003" target="_blank">Fig 3C</a> Control data are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.g001" target="_blank">Fig 1F</a> in the accompanying manuscript. (number of cell couples analyzed across multiple independent experiments, n = 46). (<b>C</b>) A representative interaction of a 5C.C7 T cell expressing Themis-GFP with 40nM Jasplakinolide (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.s008" target="_blank">S4 Video</a>) is given similar to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.g001" target="_blank">Fig 1B</a>. (<b>D</b>) The corresponding pattern classification is given similar to B (n = 32)(control data from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.ref026" target="_blank">26</a>]). (<b>E)</b> 5C.C7 T cells expressing Themis-GFP were activated with CH27 APCs (10 μM MCC agonist peptide) and subjected to a FRAP experiment. Average fitted recovery curves and recovery half times are given for Themis with (n = 19) and without (n = 18) low dose JASP treatment. (<b>F</b>) A representative interaction of a 5C.C7 T cell expressing Vav1-GFP with 40nM Jasplakinolide (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.s009" target="_blank">S5 Video</a>) is given similar to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.g001" target="_blank">Fig 1B</a>. (<b>G</b>) The corresponding pattern classification is given similar to B upon addition of peripheral control data (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.g003" target="_blank">Fig 3C</a> in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.ref024" target="_blank">24</a>])(n = 50). Error bars are s.e.m.. Significance was determined by Student’s t-test and is indicated by asterisks (****p<0.0001).</p

Costimulation blockade diminishes recruitment of signaling intermediates to the lamellal pattern.

<p>5C.C7 T cells were activated by CH27 APCs and 10 μM MCC peptide upon costimulation blockade with 10 μg/ml anti-CD80 and anti-CD86. The percentage of T cells showing accumulation in defined patterns among all cell couples analyzed across multiple experiments are shown similar to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.g005" target="_blank">Fig 5D</a> for (<b>A</b>) SLP-76-GFP (number of cell couples analyzed across multiple independent experiments, n = 52) (<b>B</b>) PLCδPH-GFP (PIP₂ sensor) (n = 59), (<b>C</b>) NFκB p65 (n = 55), and (<b>D</b>) Vav1-GFP (n = 51). Broken lines indicate interface accumulation under control conditions as a reference (Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.g001" target="_blank">1E, 1F</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.g004" target="_blank">4D</a> of the accompanying manuscript and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.g003" target="_blank">Fig 3C</a> in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.ref024" target="_blank">24</a>]). Error bars are s.e.m..</p

Low dose JASP treatment interferes with lamellal SLP-76 localization.

<p><b>(A, B)</b> Representative interactions of 5C.C7 T cells expressing SLP-76-GFP with peptide-loaded CH27 APCs (10μM MCC) under control conditions (A) or in the presence of 40nM Jasplakinolide <b>(</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.s006" target="_blank">S2 Video</a>)(B) over the indicated time relative to formation of a tight cell conjugate is given as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.g001" target="_blank">Fig 1B</a>. The control data are a reproduction of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.g001" target="_blank">Fig 1B</a> in the accompanying manuscript. (<b>C</b>) The corresponding pattern classification is given upon treatment with 40nM Jasplakinolide similar to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.g001" target="_blank">Fig 1D</a> (number of cell couples analyzed across multiple independent experiments, n = 50). Control ‘any interface’, ‘central’ and ‘lamellal’ accumulation curves from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.g001" target="_blank">Fig 1E</a> in the accompanying manuscript are plotted as a reference in broken lines. <b>(D)</b> 5C.C7 T cells expressing SLP-76-GFP were activated with peptide-loaded CH27 APCs (10μM MCC). The fluorescence intensity relative to maximum as a function of the distance from the interface, measured from live cell conjugates upon control (n = 19) and low dose JASP treatment (n = 15) at 1min across the entire interface, is given. Control data are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.g001" target="_blank">Fig 1G</a> of the accompanying manuscript. <b>(E</b>) 5C.C7 T cell:CH27 APC conjugates under control conditions or treated with 40nM Jasplakinolide were fixed at the 2min time point and stained for F-actin (Phalloidin), pSLP-76 (Y128), and APCs with Cell Trace Violet (representative images are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.g004" target="_blank">Fig 4A and 4B</a>). pSLP-76 (Y128) cluster intensity is given for DMSO control (n = 17) and low dose JASP (n = 17) treated cell conjugates. (<b>F</b>) Analyzing the same cell couples as in E, the frequencies of APC proximal (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.g004" target="_blank">Fig 4B</a>) and lamellal (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.g004" target="_blank">Fig 4A</a>) clusters are given. (<b>G</b>) Analyzing the same cell couples as in E, the distance of lamellal pSLP-76 clusters from the interface for DMSO control (32 clusters) and residual lamellal clusters in low dose JASP treated cell conjugates (12 clusters) is plotted (whiskers = min and max). Significance was determined by Student’s t-test and is indicated by asterisks (*p<0.05, ***p<0.001).</p

Low dose JASP treatment selectively affects lamellal signaling.

<p>(<b>A-D</b>) 5C.C7 T cells were activated with CH27 APCs (10μM MCC) with vehicle (solid black) or 40nM Jasplakinolide (broken grey). Phosphorylation levels for (A) SLP-76, (B) Vav1, (C) PKCθ, and (D) LAT were determined after 1, 2, and 5min. Graphs depict phosphorylation levels normalized to the max value jointly for control and Jasplakinolide treatment (at least 3 independent experiments). Corresponding representative immunoblots are adjacent to each graph. Complete blots are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.s002" target="_blank">S2A–S2D Fig</a><b>(E-H)</b> 5C.C7 T cells were activated with CH27 APCs (10μM MCC) with vehicle (solid black) or 10nM LatA (broken grey). Phosphorylation levels for (E) SLP-76, (F) Vav1, (G) PKCθ, and (H) LAT were determined after 1, 2, and 5min and are given as in A-D. Complete blots are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133231#pone.0133231.s002" target="_blank">S2G–S2J Fig</a> Error bar are s.e.m.. Significance was determined by Student’s t-test and is indicated by asterisks (*p<0.05,**p<0.001,***p<0.0001).</p