Live cell imaging with protein domains capable of recognizing phosphatidylinositol 4,5-bisphosphate; a comparative study

<p>Abstract</p> <p>Background</p> <p>Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)<it>P</it>₂] is a critically important regulatory phospholipid found in the plasma membrane of all eukaryotic cells. In addition to being a precursor of important second messengers, PtdIns(4,5)<it>P</it>₂also regulates ion channels and transporters and serves the endocytic machinery by recruiting clathrin adaptor proteins. Visualization of the localization and dynamic changes in PtdIns(4,5)<it>P</it>₂levels in living cells is critical to understanding the biology of PtdIns(4,5)<it>P</it>₂. This has been mostly achieved with the use of the pleckstrin homology (PH) domain of PLCδ1 fused to GFP. Here we report on a comparative analysis of several recently-described yeast PH domains as well as the mammalian Tubby domain to evaluate their usefulness as PtdIns(4,5)<it>P</it>₂imaging tools.</p> <p>Results</p> <p>All of the yeast PH domains that have been previously shown to bind PtdIns(4,5)<it>P</it>₂showed plasma membrane localization but only a subset responded to manipulations of plasma membrane PtdIns(4,5)<it>P</it>₂. None of these domains showed any advantage over the PLCδ1PH-GFP reporter and were compromised either in their expression levels, nuclear localization or by causing peculiar membrane structures. In contrast, the Tubby domain showed high membrane localization consistent with PtdIns(4,5)<it>P</it>₂binding and displayed no affinity for the soluble headgroup, Ins(1,4,5)P₃. Detailed comparison of the Tubby and PLCδ1PH domains showed that the Tubby domain has a higher affinity for membrane PtdIns(4,5)<it>P</it>₂and therefore displays a lower sensitivity to report on changes of this lipid during phospholipase C activation.</p> <p>Conclusion</p> <p>These results showed that both the PLCδ1PH-GFP and the GFP-Tubby domain are useful reporters of PtdIns(4,5)<it>P</it>₂changes in the plasma membrane, with distinct advantages and disadvantages. While the PLCδ1PH-GFP is a more sensitive reporter, its Ins(1,4,5)P₃binding may compromise its accuracy to measure PtdIns(4,5)<it>P</it>₂changes. The Tubby domain is more accurate to report on PtdIns(4,5)<it>P</it>₂but its higher affinity and lower sensitivity may limit its utility when phospholipase C activation is only moderate. These studies also demonstrated that similar changes in PtdIns(4,5)<it>P</it>₂levels in the plasma membrane can differentially regulate multiple effectors if they display different affinities to PtdIns(4,5)<it>P</it>₂.</p

Multiplicity fluctuations in nuclear collisions at 158 A GeV

System size dependence of multiplicity fluctuations of charged particles produced in nuclear collisions at 158 A GeV was studied in the NA49 CERN experiment. Results indicate a non-monotonic dependence of the scaled variance of the multiplicity distribution with a maximum for semi-peripheral Pb+Pb interactions with number of projectile participants of about 35. This effect is not observed in a string-hadronic model of nuclear collision HIJING.Comment: Presented at "Focus on Multiplicity", 17-19 of June, Bari, Ital

Bose-Einstein correlations of pion pairs in central Pb+Pb collisions at CERN SPS energies

Measurements of Bose-Einstein correlations of pion pairs in central Pb+Pb collisions were performed with the NA49 detector at the CERN SPS for beam energies of 20A, 30A, 40A, 80A, and 158A GeV. Correlation functions were measured in the longitudinally co-moving ``out-side-long'' reference frame as a function of rapidity and transverse momentum in the forward hemisphere of the reaction. Radius and correlation strength parameters were obtained from fits of a Gaussian parametrization. The results show a decrease of the radius parameters with increasing transverse momentum characteristic of strong radial flow in the pion source. No striking dependence on pion-pair rapidity or beam energy is observed. Static and dynamic properties of the pion source are obtained from simultaneous fits with a blast-wave model to radius parameters and midrapidity transverse momentum spectra. Predictions of hydrodynamic and microscopic models of Pb+Pb collisions are discussed.Comment: 22 pages, 23 figure

System size dependence of strange particle yields and spectra at sqrt(s)=17.3 GeV

Yields and spectra of strange hadrons (K+, K-, phi, Lambda and Antilambda) as well as of charged pions were measured in near central C+C and Si+Si collisions at 158 AGeV beam energy with the NA49 detector. Together with earlier data for p+p, S+S and Pb+Pb reactions the system size dependence can be studied. Relative strangeness production rises fast and saturates at about 60 participating nucleons; the net hyperon spectra show an increasing shift towards midrapidity for larger colliding nuclei. An interpretation based on the formation of coherent systems of increasing volume is proposed. The transverse mass spectra can be described by a blast wave ansatz. Increasing flow velocity is accompanied by decreasing temperatures for both kinetic and chemical freeze out. The increasing gap between inelastic and elastic decoupling leaves space for rescattering.Comment: 8 pages, 6 figures, Proceedings of the Hot Quarks 2004 worksho

High p_T Spectra of Identified Particles Produced in Pb+Pb Collisions at 158GeV/nucleon Beam Energy

Transverse momentum spectra of pi^{+/-}, p, pbar, K^{+/-}, K^0_s and Lambda at midrapidity were measured at high p_T in Pb+Pb collisions at 158GeV/nucleon beam energy by the NA49 experiment. Particle yield ratios (p/pi, K/pi and Lambda/K^0_s) show an enhancement of the baryon/meson ratio for p_T>2GeV/c. The nuclear modification factor R_{CP} is extracted and compared to RHIC measurements and pQCD calculations.Comment: Quark Matter 2005 parallel section proceeding

Event-by-Event Fluctuations of Particle Ratios in Central Pb+Pb Collisions at 20 to 158 AGeV

In the vicinity of the QCD phase transition, critical fluctuations have been predicted to lead to non-statistical fluctuations of particle ratios, depending on the nature of the phase transition. Recent results of the NA49 energy scan program show a sharp maximum of the ratio of K+ to Pi+ yields in central Pb+Pb collisions at beam energies of 20-30 AGeV. This observation has been interpreted as an indication of a phase transition at low SPS energies. We present first results on event-by-event fluctuations of the kaon to pion and proton to pion ratios at beam energies close to this maximum.Comment: 4 pages, 4 figures, Quark Matter 2004 proceeding

Increased Production of the Soluble Tumor-Associated Antigens CA19-9, CA125, and CA15-3 in Rheumatoid Arthritis

Some tumor-associated antigens (TAAs) are expressed on inflammatory cells. We previously detected carcinoembryonic antigen (CEA; CD66) in the rheumatoid (RA) synovium. The production of CEA, CA19-9, CA125, and CA15.3, may be increased in patients with RA, scleroderma, lupus, and SjÖgren's syndrome (SS). Some of these TAAs contain sialylated carbohydrate motifs and they are involved in tumor-associated cell adhesion and metastasis. We assessed levels of TAAs in the sera of RA patients and healthy subjects. Serum TAA levels were correlated with disease markers including serum rheumatoid factor (RF), C-reactive protein (CRP), and anti-CCP antibody levels, DAS28, age disease duration. TAAs including CEA, CA15-3, CA72-4, CA125, and CA19-9, and neuron-specific enolase (NSE) were assessed by immunoassay in the sera of 75 patients with RA and 50 age- and sex-matched healthy controls. Normal upper limits for these TAAs were 3.4 Μg/L, 25 kU/L, 6.9 kU/L, 35 kU/L, 34 kU/L, and 16.3 Μg/L, respectively. There were significantly more RA patients showing abnormally high levels of CA125 (10.8% versus 7.1%), CA19-9 (8.1% versus 0%), and CA15-3 (17.6% versus 14.3%) in comparison to controls ( P < 0.05). The mean absolute serum levels of CA125 (23.9 ± 1.8 versus 16.8 ± 2.2 kU/L) and CA19-9 (14.2 ± 1.2 versus 10.5 ± 1.6 kU/L) were also significantly higher in RA compared to controls ( P < 0.05). Among RA patients, serum CEA showed significant correlation with RF ( r = 0.270; P < 0.05). None of the assessed TAAs showed any correlation with CRP, anti-CCP, DAS28, age or disease duration. The concentration of some TAAs may be elevated in the sera of patients with established RA in comparison to healthy subjects. CEA, CA19-9, CA125, and CA15-3 contain carbohydrate motifs and thus they may be involved in synovitis-associated adhesive events. Furthermore, some TAAs, such as CEA, may also correlate with prognostic factors, such as serum RF levels.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73224/1/annals.1422.037.pd

Strangeness production at SPS energies

We present a summary of measurements of strange particles performed by the experiment NA49 in central and minimum bias Pb+Pb collisions in the beam energy range 20A - 158A GeV. New results on Xi production in central Pb+Pb collisions and on Lambda, Xi production in minimum bias collisions are shown. Transverse mass spectra and rapidity distributions of strange particles at different energies are compared. The energy dependence of the particle yields and ratios is discussed. NA49 measurements of the Lambda and Xi enhancement factors are shown for the first time.Comment: Submitted to J. Phys. G (Proceedings of the 9th International Conference on Strangeness in Quark Matter, Los Angeles, USA, March 26-31, 2006). 9 pages, 9 figure

High p_T Spectra of Identified Particles Produced in Pb+Pb Collisions at 158A GeV Beam Energy

Results of the NA49 collaboration on the production of hadrons with large transverse momentum in Pb+Pb collisions at 158A GeV beam energy are presented. A range up to p_T = 4 GeV/c is covered. The nuclear modification factor R_CP is extracted for pions, kaons and protons, and the baryon to meson ratios p/pi+, pbar/pi- and Lambda/K^0_s are studied. All results are compared to other measurements at SPS and RHIC and to theoretical calculations.Comment: Submitted to J. Phys. G (Proceedings of the 9th International Conference on Strangeness in Quark Matter, Los Angeles, USA, March 26-31, 2006). 5 pages, 3 figure

Baryon Stopping and Charged Particle Distributions in Central Pb+Pb Collisions at 158 GeV per Nucleon

Net proton and negative hadron spectra for central \PbPb collisions at 158 GeV per nucleon at the CERN SPS were measured and compared to spectra from lighter systems. Net baryon distributions were derived from those of net protons, utilizing model calculations of isospin contributions as well as data and model calculations of strange baryon distributions. Stopping (rapidity shift with respect to the beam) and mean transverse momentum \meanpt of net baryons increase with system size. The rapidity density of negative hadrons scales with the number of participant nucleons for nuclear collisions, whereas their \meanpt is independent of system size. The \meanpt dependence upon particle mass and system size is consistent with larger transverse flow velocity at midrapidity for \PbPb compared to \SS central collisions.Comment: This version accepted for publication in PRL. 4 pages, 3 figures. Typos corrected, some paragraphs expanded in response to referee comments, to better explain details of analysi