Flotillins Interact with PSGL-1 in Neutrophils and, upon Stimulation, Rapidly Organize into Membrane Domains Subsequently Accumulating in the Uropod

BACKGROUND: Neutrophils polarize and migrate in response to chemokines. Different types of membrane microdomains (rafts) have been postulated to be present in rear and front of polarized leukocytes and disruption of rafts by cholesterol sequestration prevents leukocyte polarization. Reggie/flotillin-1 and -2 are two highly homologous proteins that are ubiquitously enriched in detergent resistant membranes and are thought to shape membrane microdomains by forming homo- and hetero-oligomers. It was the goal of this study to investigate dynamic membrane microdomain reorganization during neutrophil activation. METHODOLOGY/PRINCIPAL FINDINGS: We show now, using immunofluorescence staining and co-immunoprecipitation, that endogenous flotillin-1 and -2 colocalize and associate in resting spherical and polarized primary neutrophils. Flotillins redistribute very early after chemoattractant stimulation, and form distinct caps in more than 90% of the neutrophils. At later time points flotillins accumulate in the uropod of polarized cells. Chemotactic peptide-induced redistribution and capping of flotillins requires integrity and dynamics of the actin cytoskeleton, but does not involve Rho-kinase dependent signaling related to formation of the uropod. Both flotillin isoforms are involved in the formation of this membrane domain, as uropod location of exogenously expressed flotillins is dramatically enhanced by co-overexpression of tagged flotillin-1 and -2 in differentiated HL-60 cells as compared to cells expressing only one tagged isoform. Flotillin-1 and -2 associate with P-selectin glycoprotein ligand 1 (PSGL-1) in resting and in stimulated neutrophils as shown by colocalization and co-immunoprecipitation. Neutrophils isolated from PSGL-1-deficient mice exhibit flotillin caps to the same extent as cells isolated from wild type animals, implying that PSGL-1 is not required for the formation of the flotillin caps. Finally we show that stimulus-dependent redistribution of other uropod-located proteins, CD43 and ezrin/radixin/moesin, occurs much slower than that of flotillins and PSGL-1. CONCLUSIONS/SIGNIFICANCE: These results suggest that flotillin-rich actin-dependent membrane microdomains are importantly involved in neutrophil uropod formation and/or stabilization and organize uropod localization of PSGL-1

Reggie-1/flotillin-2 promotes secretion of the long-range signalling forms of Wingless and Hedgehog in Drosophila

The lipid-modified morphogens Wnt and Hedgehog diffuse poorly in isolation yet can spread over long distances in vivo, predicting existence of two distinct forms of these mophogens. The first is poorly mobile and activates short-range target genes. The second is specifically packed for efficient spreading to induce long-range targets. Subcellular mechanisms involved in the discriminative secretion of these two forms remain elusive. Wnt and Hedgehog can associate with membrane microdomains, but the function of this association was unknown. Here we show that a major protein component of membrane microdomains, reggie-1/flotillin-2, plays important roles in secretion and spreading of Wnt and Hedgehog in Drosophila. Reggie-1 loss-of-function results in reduced spreading of the morphogens, while its overexpression stimulates secretion of Wnt and Hedgehog and expands their diffusion. The resulting changes in the morphogen gradients differently affect the short- and long-range targets. In its action reggie-1 appears specific for Wnt and Hedgehog. These data suggest that reggie-1 is an important component of the Wnt and Hedgehog secretion pathway dedicated to formation of the mobile pool of these morphogens

Cyclodextrin Induces Calcium-Dependent Lysosomal Exocytosis

Cyclodextrins (CDs) have long been used to manipulate cellular cholesterol levels both in vitro and in vivo, but their direct effects at a cellular level are not well characterized. Recently, CDs have garnered much interest because of their ability to clear stored cholesterol from Niemann Pick Type C (NPC) cells and markedly prolong the life of NPC1 disease mice. Here, we investigate the hypothesis that treatment with 2-hydroxypropyl- β-cyclodextrin (HPB-CD) stimulates lysosomal exocytosis in a calcium-enhanced manner. We propose that this exocytosis is the mechanism by which HPB-CD ameliorates the endolysosomal cholesterol storage phenotype in NPC cells. These findings have significant implications for the use of HPB-CD in biochemical assays and data interpretation as well as for their use for the treatment for NPC and other disorders

Moderate performance of serum S100A12, in distinguishing inflammatory bowel disease from irritable bowel syndrome

<p>Abstract</p> <p>Background</p> <p>S100A12, a calcium-binding proinflammatory protein secreted by granulocytes, has been associated with different diseases of inflammatory origin, including inflammatory bowel disease (IBD). In this study, the utility of serum S100A12, in discriminating IBD from irritable bowel syndrome (IBS), was tested.</p> <p>Methods</p> <p>S100A12 serum levels were determined in 64 patients with ulcerative colitis (UC), 64 with Crohn's disease (CD) and 73 with IBS, by means of an enzyme-linked immunosorbent assay. S100A12 serum levels were evaluated with respect to the levels of known inflammatory markers and patients' characteristics.</p> <p>Results</p> <p>The median values of serum S100A12 levels were 68.2 ng/mL (range: 43.4-147.4) in UC, 70 ng/mL (41.4-169.8) in CD and 43.4 ng/mL (34.4-74.4) in IBS patients. UC and CD patients had significantly higher serum S100A12 levels compared to IBS patients (<it>P </it>= 0.001 for both comparisons). Moreover, a cut-off for serum S100A12 levels of 54.4 ng/mL could predict both UC and CD with a 66.7% sensitivity and a 64.4% specificity. The area under curve was estimated at 0.67 with a 95% confidence interval of 0.60-0.75 (<it>P </it>< 0.001). Considering standard activity indices, higher serum S100A12 levels in active compared to inactive IBD were observed, although the recorded difference did not reach statistical significance. C-reactive protein (CRP) and serum amyloid A (SAA) levels, showed a statistically significant positive correlation with S100A12 (r = 0.39, <it>P </it>= 0.001 and r = 0.23, <it>P </it>= 0.02 respectively).</p> <p>Conclusions</p> <p>Increased levels of circulating S100A12 are found in IBD, compared to IBS. When used to distinguish IBD from IBS adult patients, serum S100A12 levels exhibit moderate performance. On the other hand, serum S100A12 may serve as an inflammatory marker in IBD, since it is well correlated with CRP and SAA.</p

Mediterranean diet or extended fasting's influence on changing the intestinal microflora, immunoglobulin A secretion and clinical outcome in patients with rheumatoid arthritis and fibromyalgia: an observational study

BACKGROUND: Alterations in the intestinal bacterial flora are believed to be contributing factors to many chronic inflammatory and degenerative diseases including rheumatic diseases. While microbiological fecal culture analysis is now increasingly used, little is known about the relationship of changes in intestinal flora, dietary patterns and clinical outcome in specific diseases. To clarify the role of microbiological culture analysis we aimed to evaluate whether in patients with rheumatoid arthritis (RA) or fibromyalgia (FM) a Mediterranean diet or an 8-day fasting period are associated with changes in fecal flora and whether changes in fecal flora are associated with clinical outcome. METHODS: During a two-months-period 51 consecutive patients from an Integrative Medicine hospital department with an established diagnosis of RA (n = 16) or FM (n = 35) were included in the study. According to predefined clinical criteria and the subjects' choice the patients received a mostly vegetarian Mediterranean diet (n = 21; mean age 50.9 +/-13.3 y) or participated in an intermittent modified 8-day fasting therapy (n = 30; mean age 53.7 +/- 9.4 y). Quantitative aerob and anaerob bacterial flora, stool pH and concentrations of secretory immunoglobulin A (sIgA) were analysed from stool samples at the beginning, at the end of the 2-week hospital stay and at a 3-months follow-up. Clinical outcome was assessed with the DAS 28 for RA patients and with a disease severity rating scale in FM patients. RESULTS: We found no significant changes in the fecal bacterial counts following the two dietary interventions within and between groups, nor were significant differences found in the analysis of sIgA and stool ph. Clinical improvement at the end of the hospital stay tended to be greater in fasting vs. non-fasting patients with RA (p = 0.09). Clinical outcome was not related to alterations in the intestinal flora. CONCLUSION: Neither Mediterranean diet nor fasting treatments affect the microbiologically assessed intestinal flora and sIgA levels in patients with RA and FM. The impact of dietary interventions on the human intestinal flora and the role of the fecal flora in rheumatic diseases have to be clarified with newer molecular analysis techniques. The potential benefit of fasting treatment in RA and FM should be further tested in randomised trials

Meditation Awareness Training for individuals with fibromyalgia syndrome: an interpretative phenomenological analysis of participants' experiences

Fibromyalgia syndrome (FMS) is a complex and poorly understood psychosomatic pain disorder. The illness has been the subject of controversy, both in terms of the alleged lack of interest and capability of the medical community to understand and support patients with FMS, and the burden that such individuals place upon economic and healthcare re- sources. Due to the lack of convincing data for the effectiveness of extant pharmacological and non-pharmacological FMS treatments, a recent direction in FMS research has been the empirical investigation of mindfulness and other meditation-based approaches. The present qualitative study explored whether following participation in a mindfulness-based intervention, patients with FMS report experiencing changes in (i) how they experience and relate to their illness and (ii) their attitudes towards societal participation, work and unemployment. Ten individuals with FMS were randomly selected from the intervention arm of a randomised controlled trial (RCT) evaluating the effectiveness of a mindfulness-based intervention known as Meditation Awareness Training (MAT) for the treatment of FMS. Transcripts of semi-structured interviews were analysed using Interpretative Phenomenological Analysis, a robust and rigorous qualitative methodology for analysing sub jective experiences. Five super-ordinate themes emerged from the dataset: (i) reservations about participating,(ii) improvements in psychosomatic well-being,(iii) spiritual growth,(iv) awareness of impermanence and (v) increased sense of citizenship. MAT was experienced as both an acceptable and accessible intervention by individuals with FMS, and participants reported experiencing improvements in psychosocial functioning as well as an increased sense of societal responsibility. MAT appears to have utility for treating FMS and for changing the attitudes of some individuals with FMS towards community engagement and societal contribution

Mechanisms Underlying the Confined Diffusion of Cholera Toxin B-Subunit in Intact Cell Membranes

Multivalent glycolipid binding toxins such as cholera toxin have the capacity to cluster glycolipids, a process thought to be important for their functional uptake into cells. In contrast to the highly dynamic properties of lipid probes and many lipid-anchored proteins, the B-subunit of cholera toxin (CTxB) diffuses extremely slowly when bound to its glycolipid receptor GM1 in the plasma membrane of living cells. In the current study, we used confocal FRAP to examine the origins of this slow diffusion of the CTxB/GM1 complex at the cell surface, relative to the behavior of a representative GPI-anchored protein, transmembrane protein, and fluorescent lipid analog. We show that the diffusion of CTxB is impeded by actin- and ATP-dependent processes, but is unaffected by caveolae. At physiological temperature, the diffusion of several cell surface markers is unchanged in the presence of CTxB, suggesting that binding of CTxB to membranes does not alter the organization of the plasma membrane in a way that influences the diffusion of other molecules. Furthermore, diffusion of the B-subunit of another glycolipid-binding toxin, Shiga toxin, is significantly faster than that of CTxB, indicating that the confined diffusion of CTxB is not a simple function of its ability to cluster glycolipids. By identifying underlying mechanisms that control CTxB dynamics at the cell surface, these findings help to delineate the fundamental properties of toxin-receptor complexes in intact cell membranes

Statistical Analysis of Molecular Signal Recording

A molecular device that records time-varying signals would enable new approaches in neuroscience. We have recently proposed such a device, termed a “molecular ticker tape”, in which an engineered DNA polymerase (DNAP) writes time-varying signals into DNA in the form of nucleotide misincorporation patterns. Here, we define a theoretical framework quantifying the expected capabilities of molecular ticker tapes as a function of experimental parameters. We present a decoding algorithm for estimating time-dependent input signals, and DNAP kinetic parameters, directly from misincorporation rates as determined by sequencing. We explore the requirements for accurate signal decoding, particularly the constraints on (1) the polymerase biochemical parameters, and (2) the amplitude, temporal resolution, and duration of the time-varying input signals. Our results suggest that molecular recording devices with kinetic properties similar to natural polymerases could be used to perform experiments in which neural activity is compared across several experimental conditions, and that devices engineered by combining favorable biochemical properties from multiple known polymerases could potentially measure faster phenomena such as slow synchronization of neuronal oscillations. Sophisticated engineering of DNAPs is likely required to achieve molecular recording of neuronal activity with single-spike temporal resolution over experimentally relevant timescales.United States. Defense Advanced Research Projects Agency. Living Foundries ProgramGoogle (Firm)New York Stem Cell Foundation. Robertson Neuroscience Investigator AwardNational Institutes of Health (U.S.) (EUREKA Award 1R01NS075421)National Institutes of Health (U.S.) (Transformative R01 1R01GM104948)National Institutes of Health (U.S.) (Single Cell Grant 1 R01 EY023173)National Institutes of Health (U.S.) (Grant 1R01DA029639)National Institutes of Health (U.S.) (Grant 1R01NS067199)National Science Foundation (U.S.) (CAREER Award CBET 1053233)National Science Foundation (U.S.) (Grant EFRI0835878)National Science Foundation (U.S.) (Grant DMS1042134)Paul G. Allen Family Foundation (Distinguished Investigator in Neuroscience Award

Novel, Objective, Multivariate Biomarkers Composed of Plasma Amino Acid Profiles for the Diagnosis and Assessment of Inflammatory Bowel Disease

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic intestinal disorder that is associated with a limited number of clinical biomarkers. In order to facilitate the diagnosis of IBD and assess its disease activity, we investigated the potential of novel multivariate indexes using statistical modeling of plasma amino acid concentrations (aminogram). METHODOLOGY AND PRINCIPAL FINDINGS: We measured fasting plasma aminograms in 387 IBD patients (Crohn's disease (CD), n = 165; ulcerative colitis (UC), n = 222) and 210 healthy controls. Based on Fisher linear classifiers, multivariate indexes were developed from the aminogram in discovery samples (CD, n = 102; UC, n = 102; age and sex-matched healthy controls, n = 102) and internally validated. The indexes were used to discriminate between CD or UC patients and healthy controls, as well as between patients with active disease and those in remission. We assessed index performances using the area under the curve of the receiver operating characteristic (ROC AUC). We observed significant alterations to the plasma aminogram, including histidine and tryptophan. The multivariate indexes established from plasma aminograms were able to distinguish CD or UC patients from healthy controls with ROC AUCs of 0.940 (95% confidence interval (CI): 0.898-0.983) and 0.894 (95%CI: 0.853-0.935), respectively in validation samples (CD, n = 63; UC, n = 120; healthy controls, n = 108). In addition, other indexes appeared to be a measure of disease activity. These indexes distinguished active CD or UC patients from each remission patients with ROC AUCs of 0.894 (95%CI: 0.853-0.935) and 0.849 (95%CI: 0.770-0.928), and correlated with clinical disease activity indexes for CD (r(s) = 0.592, 95%CI: 0.385-0.742, p<0.001) or UC (r(s) = 0.598, 95%CI: 0.452-0.713, p<0.001), respectively. CONCLUSIONS AND SIGNIFICANCE: In this study, we demonstrated that established multivariate indexes composed of plasma amino acid profiles can serve as novel, non-invasive, objective biomarkers for the diagnosis and monitoring of IBD, providing us with new insights into the pathophysiology of the disease

Molecular Networks in FGF Signaling: Flotillin-1 and Cbl-Associated Protein Compete for the Binding to Fibroblast Growth Factor Receptor Substrate 2

Fibroblast growth factor receptor substrate 2 (FRS2α) is a signaling adaptor protein that regulates downstream signaling of many receptor tyrosine kinases. During signal transduction, FRS2 can be both tyrosine and threonine phosphorylated and forms signaling complexes with other adaptor proteins and tyrosine phosphatases. We have here identified flotillin-1 and the cbl-associated protein/ponsin (CAP) as novel interaction partners of FRS2. Flotillin-1 binds to the phosphotyrosine binding domain (PTB) of FRS2 and competes for the binding with the fibroblast growth factor receptor. Flotillin-1 knockdown results in increased Tyr phosphorylation of FRS2, in line with the inhibition of ERK activity in the absence of flotillin-1. CAP directly interacts with FRS2 by means of its sorbin homology (SoHo) domain, which has previously been shown to interact with flotillin-1. In addition, the third SH3 domain in CAP binds to FRS2. Due to the overlapping binding domains, CAP and flotillin-1 appear to compete for the binding to FRS2. Thus, our results reveal a novel signaling network containing FRS2, CAP and flotillin-1, whose successive interactions are most likely required to regulate receptor tyrosine kinase signaling, especially the mitogen activated protein kinase pathway