Phosphorylation of ezrin on Thr567 is required for the synergistic activation of cell spreading by EPAC1 and protein kinase A in HEK293T cells

Recent studies have demonstrated that the actin binding protein, ezrin, and the cAMP-sensor, EPAC1, cooperate to induce cell spreading in response to elevations in intracellular cAMP. To investigate the mechanisms underlying these effects we generated a model of EPAC1-dependent cell spreading based on the stable transfection of EPAC1 into HEK293T (HEK293T–EPAC1) cells. We found that direct activation of EPAC1 with the EPAC-selective analogue, 8-pCPT-2′-O-Me-cAMP (007), promoted cell spreading in these cells. In addition, co-activation of EPAC1 and PKA, with a combination of the adenylate cyclase activator, forskolin, and the cAMP phosphodiesterase inhibitor, rolipram, was found to synergistically enhance cell spreading, in association with cortical actin bundling and mobilisation of ezrin to the plasma membrane. PKA activation was also associated with phosphorylation of ezrin on Thr567, as detected by an electrophoretic band mobility shift during SDS-PAGE. Inhibition of PKA activity blocked ezrin phosphorylation and reduced the cell spreading response to cAMP elevation to levels induced by EPAC1-activation alone. Transfection of HEK293T–EPAC1 cells with inhibitory ezrin mutants lacking the key PKA phosphorylation site, ezrin-Thr567Ala, or the ability to associate with actin, ezrin-Arg579Ala, promoted cell arborisation and blocked the ability of EPAC1 and PKA to further promote cell spreading. The PKA phospho-mimetic mutants of ezrin, ezrin-Thr567Asp had no effect on EPAC1-driven cell spreading. Our results indicate that association of ezrin with the actin cytoskeleton and phosphorylation on Thr567 are required, but not sufficient, for PKA and EPAC1 to synergistically promote cell spreading following elevations in intracellular cAMP

Leading infrared logarithms for sigma-model with fields on arbitrary Riemann manifold

We derive non-linear recursion equation for the leading infrared logarithms (LL) in four dimensional sigma-model with fields on an arbitrary Riemann manifold. The derived equation allows one to compute leading infrared logarithms to essentially unlimited loop order in terms of geometric characteristics of the Riemann manifold. We reduce the solution of the SU(oo) principal chiral field in arbitrary number of dimensions in the LL approximation to the solution of very simple recursive equation. This result paves a way to the solution of the model in arbitrary number of dimensions at N-->ooComment: Talk given by MVP at the conference devoted to memory of A.N. Vasilie

Categorizing click trains to increase taxonomic precision in echolocation click loggers

L.R. and K.J.P. were supported by Marine Scotland Science and the Marine Alliance for Science and Technology for Scotland (MASTS) pooling initiative and their support is gratefully acknowledged. MASTS is funded by the Scottish Funding Council (grant reference HR09011) and contributing institutions.Passive acoustic monitoring is an efficient way to study acoustically active animals but species identification remains a major challenge. C-PODs are popular logging devices that automatically detect odontocete echolocation clicks. However, the accompanying analysis software does not distinguish between delphinid species. Click train features logged by C-PODs were compared to frequency spectra from adjacently deployed continuous recorders. A generalized additive model was then used to categorize C-POD click trains into three groups: broadband click trains, produced by bottlenose dolphin (Tursiops truncatus) or common dolphin (Delphinus delphis), frequency-banded click trains, produced by Risso's (Grampus griseus) or white beaked dolphins (Lagenorhynchus albirostris), and unknown click trains. Incorrect categorization rates for broadband and frequency banded clicks were 0.02 (SD 0.01), but only 30% of the click trains met the categorization threshold. To increase the proportion of categorized click trains, model predictions were pooled within acoustic encounters and a likelihood ratio threshold was used to categorize encounters. This increased the proportion of the click trains meeting either the broadband or frequency banded categorization threshold to 98%. Predicted species distribution at the 30 study sites matched well to visual sighting records from the region.PostprintPeer reviewe

Low-frequency components in harbor porpoise (Phocoena phocoena) clicks : communication signal, by-products, or artifacts?

Author Posting. © Acoustical Society of America, 2008. This article is posted here by permission of Acoustical Society of America for personal use, not for redistribution. The definitive version was published in Journal of the Acoustical Society of America 124 (2008): 4059-4068, doi:10.1121/1.2945154.Underwater sound signals for biosonar and communication normally have different source properties to serve the purposes of generating efficient acoustic backscatter from small objects or conveying information to conspecifics. Harbor porpoises (Phocoena phocoena) are nonwhistling toothed whales that produce directional, narrowband, high-frequency (HF) echolocation clicks. This study tests the hypothesis that their 130 kHz HF clicks also contain a low-frequency (LF) component more suited for communication. Clicks from three captive porpoises were analyzed to quantify the LF and HF source properties. The LF component is 59 (S.E.M=1.45 dB) dB lower than the HF component recorded on axis, and even at extreme off-axis angles of up to 135°, the HF component is 9 dB higher than the LF component. Consequently, the active space of the HF component will always be larger than that of the LF component. It is concluded that the LF component is a by-product of the sound generator rather than a dedicated pulse produced to serve communication purposes. It is demonstrated that distortion and clipping in analog tape recorders can explain some of the prominent LF components reported in earlier studies, emphasizing the risk of erroneous classification of sound types based on recording artifacts.This work was supported by the Carlsberg Foundation and Oticon, and via a Steno Scholarship from the Danish Natural Science Research Council to PTM

Adenylate Cyclase Toxin Promotes Internalisation of Integrins and Raft Components and Decreases Macrophage Adhesion Capacity

Bordetella pertussis, the bacterium that causes whooping cough, secretes an adenylate cyclase toxin (ACT) that must be post-translationally palmitoylated in the bacterium cytosol to be active. The toxin targets phagocytes expressing the CD11b/CD18 integrin receptor. It delivers a catalytic adenylate cyclase domain into the target cell cytosol producing a rapid increase of intracellular cAMP concentration that suppresses bactericidal functions of the phagocyte. ACT also induces calcium fluxes into target cells. Biochemical, biophysical and cell biology approaches have been applied here to show evidence that ACT and integrin molecules, along with other raft components, are rapidly internalized by the macrophages in a toxin-induced calcium rise-dependent process. The toxin-triggered internalisation events occur through two different routes of entry, chlorpromazine-sensitive receptor-mediated endocytosis and clathrin-independent internalisation, maybe acting in parallel. ACT locates into raft-like domains, and is internalised, also in cells devoid of receptor. Altogether our results suggest that adenylate cyclase toxin, and maybe other homologous pathogenic toxins from the RTX (Repeats in Toxin) family to which ACT belongs, may be endowed with an intrinsic capacity to, directly and efficiently, insert into raft-like domains, promoting there its multiple activities. One direct consequence of the integrin removal from the cell surface of the macrophages is the hampering of their adhesion ability, a fundamental property in the immune response of the leukocytes that could be instrumental in the pathogenesis of Bordetella pertussis

Mitochondrial Control Region and microsatellite analyses on harbour porpoise (Phocoena phocoena) unravel population differentiation in the Baltic Sea and adjacent waters

The population status of the harbour porpoise (Phocoena phocoena) in the Baltic area has been a continuous matter of debate. Here we present the by far most comprehensive genetic population structure assessment to date for this region, both with regard to geographic coverage and sample size: 497 porpoise samples from North Sea, Skagerrak, Kattegat, Belt Sea, and Inner Baltic Sea were sequenced at the mitochondrial Control Region and 305 of these specimens were typed at 15 polymorphic microsatellite loci. Samples were stratified according to sample type (stranding vs. by-caught), sex, and season (breeding vs. non-breeding season). Our data provide ample evidence for a population split between the Skagerrak and the Belt Sea, with a transition zone in the Kattegat area. Among other measures, this was particularly visible in significant frequency shifts of the most abundant mitochondrial haplotypes. A particular haplotype almost absent in the North Sea was the most abundant in Belt Sea and Inner Baltic Sea. Microsatellites yielded a similar pattern (i.e., turnover in occurrence of clusters identified by STRUCTURE). Moreover, a highly significant association between microsatellite assignment and unlinked mitochondrial haplotypes further indicates a split between North Sea and Baltic porpoises. For the Inner Baltic Sea, we consistently recovered a small, but significant separation from the Belt Sea population. Despite recent arguments that separation should exceed a predefined threshold before populations shall be managed separately, we argue in favour of precautionary acknowledging the Inner Baltic porpoises as a separate management unit, which should receive particular attention, as it is threatened by various factors, in particular local fishery measures. © Springer Science+Business Media B.V. 2009

An alternative pathway for alphavirus entry

The study of alphavirus entry has been complicated by an inability to clearly identify a receptor and by experiments which only tangentially and indirectly examine the process, producing results that are difficult to interpret. The mechanism of entry has been widely accepted to be by endocytosis followed by acidification of the endosome resulting in virus membrane-endosome membrane fusion. This mechanism has come under scrutiny as better purification protocols and improved methods of analysis have been brought to the study. Results have been obtained that suggest alphaviruses infect cells directly at the plasma membrane without the involvement of endocytosis, exposure to acid pH, or membrane fusion. In this review we compare the data which support the two models and make the case for an alternative pathway of entry by alphaviruses

FRET biosensor uncovers cAMP nano-domains at β-adrenergic targets that dictate precise tuning of cardiac contractility

Compartmentalized cAMP/PKA signalling is now recognized as important for physiology and pathophysiology, yet a detailed understanding of the properties, regulation and function of local cAMP/PKA signals is lacking. Here we present a fluorescence resonance energy transfer (FRET)-based sensor, CUTie, which detects compartmentalized cAMP with unprecedented accuracy. CUTie, targeted to specific multiprotein complexes at discrete plasmalemmal, sarcoplasmic reticular and myofilament sites, reveals differential kinetics and amplitudes of localized cAMP signals. This nanoscopic heterogeneity of cAMP signals is necessary to optimize cardiac contractility upon adrenergic activation. At low adrenergic levels, and those mimicking heart failure, differential local cAMP responses are exacerbated, with near abolition of cAMP signalling at certain locations. This work provides tools and fundamental mechanistic insights into subcellular adrenergic signalling in normal and pathological cardiac function