Differential contribution of troponin I phosphorylation sites to the endothelin-modulated contractile response

Cardiac troponin I is a phosphorylation target for endothelin-activated protein kinase C. Earlier work in cardiac myocytes expressing nonphosphorylatable slow skeletal troponin I provided evidence that protein kinase C-mediated cardiac troponin I phosphorylation accelerates relaxation. However, replacement with the slow skeletal isoform also alters the myofilament pH response and the Ca2+ transient, which could influence endothelin-mediated relaxation. Here, differences in the Ca2+ transient could not explain the divergent relaxation response to endothelin in myocytes expressing cardiac versus slow skeletal troponin I nor could activation of Na+/H+ exchange. Three separate clusters within cardiac troponin I are phosphorylated by protein kinase C, and we set out to determine the contribution of the Thr144 and Ser23/Ser24 clusters to the endothelin-mediated contractile response. Myocyte replacement with a cardiac troponin I containing a Thr144 substituted with the Pro residue found in slow skeletal troponin I resulted in prolonged relaxation in response to acute endothelin compared with control myocytes. Ser23/Ser24 also is a target for protein kinase C phosphorylation of purified cardiac troponin I, and although this cluster was not acutely phosphorylated in intact myocytes, significant phosphorylation developed within 1 h after adding endothelin. Replacement of Ser23/Ser24 with Ala indicated that this cluster contributes significantly to relaxation during more prolonged endothelin stimulation. Overall, results with these mutants provide evidence that Thr144 plays an important role in the acute acceleration of relaxation, whereas Ser23/Ser24 contributes to relaxation during more prolonged activation of protein kinase C by endothelin.     

Do low oxygen environments facilitate marine invasions? Relative tolerance of native and invasive species to low oxygen conditions

Biological invasions are one of the biggest threats to global biodiversity. Marine artificial structures are proliferating worldwide and provide a haven for marine invasive species. Such structures disrupt local hydrodynamics, which can lead to the formation of oxygen‐depleted microsites. The extent to which native fauna can cope with such low oxygen conditions, and whether invasive species, long associated with artificial structures in flow‐restricted habitats, have adapted to these conditions remains unclear. We measured water flow and oxygen availability in marinas and piers at the scales relevant to sessile marine invertebrates (mm). We then measured the capacity of invasive and native marine invertebrates to maintain metabolic rates under decreasing levels of oxygen using standard laboratory assays. We found that marinas reduce water flow relative to piers, and that local oxygen levels can be zero in low flow conditions. We also found that for species with erect growth forms, invasive species can tolerate much lower levels of oxygen relative to native species. Integrating the field and laboratory data showed that up to 30% of available microhabitats within low flow environments are physiologically stressful for native species, while only 18% of the same habitat is physiologically stressful for invasive species. These results suggest that invasive species have adapted to low oxygen habitats associated with manmade habitats, and artificial structures may be creating niche opportunities for invasive species.     

Continued feeding on <Emphasis Type="Italic">Diporeia</Emphasis> by deepwater sculpin in Lake Huron

Monitoring changes in diets of fish is essential to understanding how food web dynamics respond to changes in native prey abundances. In the Great Lakes, Diporeia, a benthic macroinvertebrate and primary food of native benthivores, declined following the introduction of invasive Dreissena mussels and these changes were reflected in fish diets. We examined the diets of deepwater sculpin Myoxocephalus thompsonii collected in bottom trawls during 2010–2014 in the main basin of Lake Huron, and compared these results to an earlier diet study (2003–2005) to assess if their diets have continued to change after a prolonged period of Dreissena mussel invasion and declined Diporeia densities. Diporeia, Mysis, Bythotrephes, and Chironomidae were consumed regularly and other diet items included ostracods, copepods, sphaerid clams, and fish eggs. The prey-specific index of relative importance calculated for each prey group indicated that Mysis importance increased at shallow (≤55 m) and mid (64–73 m) depths, while Diporeia importance increased offshore (≥82 m). The average number of Diporeia consumed per fish increased by 10.0% and Mysis decreased by 7.5%, while the frequency of occurrence of Diporeia and Mysis remained comparable between time periods. The weight of adult deepwater sculpin (80 mm and 100 mm TL bins) increased between time periods; however, the change in weight was only significant for the 80 mm TL group (p?<?0.01). Given the historical importance of Diporeia in the Great Lakes, the examination of deepwater sculpin diets provides unique insight into the trophic dynamics of the benthic community in Lake Huron.     

Large, rapidly evolving intergenic spacers in the mitochondrial DNA of the salamander family Ambystomatidae (Amphibia: Caudata)

We report the presence, in the mitochondrial DNA (mtDNA) of all of the sexual species of the salamander family Ambystomatidae, of a shared 240- bp intergenic spacer between tRNAThr and tRNAPro. We place the intergenic spacer in context by presenting the sequence of 1,746 bp of mtDNA from Ambystoma tigrinum tigrinum, describe the nucleotide composition of the intergenic spacer in all of the species of Ambystomatidae, and compare it to other coding and noncoding regions of Ambystoma and several other vertebrate mtDNAs. The nucleotide substitution rate of the intergenic spacer is approximately three times faster than the substitution rate of the control region, as shown by comparisons among six Ambystoma macrodactylum sequences and eight members of the Ambystoma tigrinum complex. We also found additional inserts within the intergenic spacers of five species that varied from 87-444 bp in length. The presence of the intergenic spacer in all sexual species of Ambystomatidae suggests that it arose at least 20 MYA and has been a stable component of the ambystomatid mtDNA ever since. As such, it represents one of the few examples of a large and persistent intergenic spacer in the mtDNA of any vertebrate clade.      

Fitness consequences of larval traits persist across the metamorphic boundary

Metamorphosis is thought to provide an adaptive decoupling between traits specialized for each life-history stage in species with complex life cycles. However, an increasing number of studies are finding that larval traits can carry-over to influence postmetamorphic performance, suggesting that these life-history stages may not be free to evolve independently of each other. We used a phenotypic selection framework to compare the relative and interactive effects of larval size, time to hatching, and time to settlement on postmetamorphic survival and growth in a marine invertebrate, Styela plicata. Time to hatching was the only larval trait found to be under directional selection, individuals that took more time to hatch into larvae survived better after metamorphosis but grew more slowly. Nonlinear selection was found to act on multivariate trait combinations, once again acting in opposite directions for selection acting via survival and growth. Individuals with above average values of larval traits were most likely to survive, but surviving individuals with intermediate larval traits grew to the largest size. These results demonstrate that larval traits can have multiple, complex fitness consequences that persist across the metamorphic boundary; and thus postmetamorphic selection pressures may constrain the evolution of larval traits.     

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission^2,10,11. This cell to cell transfer between CD4⁺ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell ⁸. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions^1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4⁺ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3ζ, ZAP70, LAT, SLP-76, Itk, and PLCγ¹⁵.In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.