The Gαq/11 Proteins Contribute to T Lymphocyte Migration by Promoting Turnover of Integrin LFA-1 through Recycling

The role of Gαi proteins coupled to chemokine receptors in directed migration of immune cells is well understood. In this study we show that the separate class of Gαq/11 proteins is required for the underlying ability of T cells to migrate both randomly and in a directed chemokine-dependent manner. Interfering with Gαq or Gα11 using dominant negative cDNA constructs or siRNA for Gαq causes accumulation of LFA-1 adhesions and stalled migration. Gαq/11 has an impact on LFA-1 expression at plasma membrane level and also on its internalization. Additionally Gαq co-localizes with LFA-1- and EEA1-expressing intracellular vesicles and partially with Rap1- but not Rab11-expressing vesicles. However the influence of Gαq is not confined to the vesicles that express it, as its reduction alters intracellular trafficking of other vesicles involved in recycling. In summary vesicle-associated Gαq/11 is required for the turnover of LFA-1 adhesion that is necessary for migration. These G proteins participate directly in the initial phase of recycling and this has an impact on later stages of the endo-exocytic pathway

A novel coumarin-labelled peptide for sensitive continuous assays of the matrix metalloproteinases

Abstract(7-methoxycoumarin-4-yl)Acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-l-2,3-diaminopropionyl)-Ala-Arg-NH2 (Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2) has been synthesised as a fluorogenic substrate for the matrix metalloproteinases. The highly flourescent 7-methoxycoumarin group is efficiently quenched by energy transfer to the 2,4-dinitrophenyl group. The punctuated metalloproteinase (PUMP, EC 3,4,24,23) cleaves the substrate at the Gly-Leu bond with a 190-fold increase in fluorescence (λcm 328 nm, λcm 393 nm). In assays of the human matrix metalloproteinases, Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 is about 50 to 100 times more sensitive than dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-d-Arg-NH2 and continuous assays can be made at enzyme concentrations comparable to those used with macromolecular substrates. Specificity constants (kcal/Km) are reported for both synthetic substrates with PUMP, collagenase, stromelysin and 72 kDa gelatinase

Predicting Early Disease Recurrence of Pancreatic Cancer following Surgery: Determining the Role of NUDT15 as a Prognostic Biomarker

Surgical resection remains the only curative treatment strategy for Pancreatic Ductal Adenocarcinoma (PDAC). A proportion of patients succumb to early disease recurrence post-operatively despite receiving adjuvant chemotherapy. The ability to identify these high-risk individuals at their initial diagnosis, prior to surgery, could potentially alter their treatment algorithm. This unique patient cohort may benefit from neo-adjuvant chemotherapy, even in the context of resectable disease, as this may secure systemic control over their disease burden. It may also improve patient selection for surgery. Using the Cancer Genome Atlas dataset, we first confirmed the poor overall survival associated with early disease recurrence (p < 0.0001). The transcriptomic profiles of these tumours were analysed, and we identified key aberrant signalling pathways involved in early disease relapse; downregulation across several immune signalling pathways was noted. Differentially expressed genes that could serve as biomarkers were identified (BPI, C6orf58, CD177, MCM7 and NUDT15). Receiver operating characteristic curves were constructed in order to identify biomarkers with a high diagnostic ability to identify patients who developed early disease recurrence. NUDT15 expression had the highest discriminatory capability as a biomarker (AUC 80.8%). Its expression was confirmed and validated in an independent cohort of patients with resected PDAC (n = 13). Patients who developed an early recurrence had a statistically higher tumour expression of NUDT15 when compared to patients who did not recur early (p < 0.01). Our results suggest that NUDT15 can be used as a prognostic biomarker that can stratify patients according to their risk of developing early disease recurrence

Predicting Early Disease Recurrence of Pancreatic Cancer following Surgery: Determining the Role of NUDT15 as a Prognostic Biomarker

Surgical resection remains the only curative treatment strategy for Pancreatic Ductal Adenocarcinoma (PDAC). A proportion of patients succumb to early disease recurrence post-operatively despite receiving adjuvant chemotherapy. The ability to identify these high-risk individuals at their initial diagnosis, prior to surgery, could potentially alter their treatment algorithm. This unique patient cohort may benefit from neo-adjuvant chemotherapy, even in the context of resectable disease, as this may secure systemic control over their disease burden. It may also improve patient selection for surgery. Using the Cancer Genome Atlas dataset, we first confirmed the poor overall survival associated with early disease recurrence (p n = 13). Patients who developed an early recurrence had a statistically higher tumour expression of NUDT15 when compared to patients who did not recur early (p < 0.01). Our results suggest that NUDT15 can be used as a prognostic biomarker that can stratify patients according to their risk of developing early disease recurrence

The Gαq/11 proteins control both random and directed T cell migration.

<p>(<b>A</b>) Random migration on ICAM-1 of HSB2 T cells ± transfection with vector control or DN Gαq, DN Gα11 or both cDNAs showing a representative experiment of single cell tracking patterns (n = 15 cells per condition) and speed of migration (mean ±s.d.), n = 5 experiments; (<b>B</b>) Western blot showing expression level of Gαq ± treatment of HSB2 cells with either control siRNA or Gαq siRNA and α-tubulin to represent the sample loading control; (<b>C</b>) Primary T cells treated with either control siRNA or Gαq siRNA showing single cell tracking pattern (n = 15 cells per condition) and random migration on ICAM-1 (mean ± s.d.), n = 3 experiments; (<b>D</b>) Transwell assay showing extent of chemotactic response to CXCL12 of primary T cells treated with either control siRNA or Gαq siRNA, n = 3 experiments.</p

Association of Gαq/11 with intracellular vesicles and LFA-1 internalization.

<p>(<b>A</b>) Biotinylated HSB2 T cells migrating on ICAM-1 over 40 min with quantification of the effect on membrane LFA-1 internalization of transfection with siRNA Gαq compared with control siRNA, n = 3 experiments; (<b>B</b>) Representative confocal microscopy image at substrate level of T lymphoblast polarized on ICAM-1 and fixed before staining with rabbit anti-Gαq and Alexa488 goat anti-rabbit IgG; shown with Rainbow scale of graded fluorescence intensity; black asterisk = leading edge; scale bar = 10 µm;</p

Effect of Gαq siRNA knockdown on intracellular vesicles.

<p>(<b>A</b>) DIC and confocal microscopy images of HSB2 T cells on ICAM-1 showing distribution and intensity of staining of LFA-1, EEA1, Rab11 and Rap1 following transfection with either control or Gαq siRNAs; results are representative of n = 3 experiments, scale bar = 10 µm; (<b>B</b>) Comparison of membrane LFA-1 expression per comparable cell area in T cells treated either with control or DN Gαq/11 cDNAs.</p

TIRF images of T lymphoblasts polarized on ICAM-1.

<p>Single cell images of T lymphoblasts viewed at ICAM-1 level by TIRF showing co-localization of Gαq expression (green) with LFA-1 and endosomal vesicle markers EEA1 (red) and merge of both colors. There is an absence of detection of Rab11 and Rap1 at TIRF level; the presented images are from the same experiment as illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038517#pone-0038517-g005" target="_blank">Fig. 5</a>; images are representative of n = 3 experiments; scale bar = 10 µm.</p

Quantification of the effect of Gαq siRNA knockdown on expression of other vesicle markers.

<p>Mean fluorescence intensity (MFI) measurements were quantified from 10 T cells per sample type for integrin LFA-1, Rab5 effector EEA1 and GTPases Rab11 and Rap1. The HSB2 T cells were transfected with either control or Gαq siRNAs for 48 hr prior to attaching to and migrating on ICAM-1.</p>*<p>Mean fluorescence intensity ± SD.</p