Metabolic Rate and Oxygen Radical Levels Increase But Radical Generation Rate Decreases with Male Age in Drosophila melanogaster Sperm

Oxidative damage increases with age in a variety of cell types, including sperm, which are particularly susceptible to attack by reactive oxygen species (ROS). While mitochondrial respiration is the main source of cellular ROS, the relationship between the rates of aerobic metabolism and ROS production, and how this relationship may be affected by age, both in sperm and in other cell types, is unclear. Here, we investigate in Drosophila melanogaster sperm, the effects of male age on (i) the level of hydrogen peroxide in the mitochondria, using a transgenic H2O2 reporter line; (ii) the in situ rate of non-H2O2 ROS production, using a novel biophysical method; and (iii) metabolic rate, using fluorescent lifetime imaging microscopy. Sperm from older males had higher mitochondrial ROS levels and a higher metabolic rate but produced ROS at a lower rate. In comparison, a somatic tissue, the gut epithelium, also showed an age-related increase in mitochondrial ROS levels but a decrease in metabolic rate. These results support the idea of a tissue-specific optimal rate of aerobic respiration balancing the production and removal of ROS, with aging causing a shift away from this optimum and leading to increased ROS accumulation. Our findings also support the view that pathways of germline and somatic aging can be uncoupled, which may have implications for male infertility treatments.

Changing Times: Fluorescence-lifetime Analysis of Amyloidogenic SF-IAPP Fusion Protein

The fluorescence lifetime of the superfolder green fluorescent protein (SF) and the SF protein fused with islet amyloid polypeptide (SF-IAPP) were studied in polyacrylamide gel. It was shown that the SF average fluorescence lifetime under these conditions slightly differs from that of the SF-IAPP monomer. SF-IAPP does not lose the ability to form amyloid-like fibrils; meanwhile, the average fluorescence lifetime of the fusion protein in fibrils is reduced. We propose the application of Fluorescent-lifetime Imaging Microscopy (FLIM) to the measurement of average fluorescence lifetimes of fusion proteins (amyloidogenic protein–SF) in the context of studies using cellular models of conformational diseases.

Crystal structure of the complete integrin αVβ3 ectodomain plus an α/β transmembrane fragment

We determined the crystal structure of 1TM-αVβ3, which represents the complete unconstrained ectodomain plus short C-terminal transmembrane stretches of the αV and β3 subunits. 1TM-αVβ3 is more compact and less active in solution when compared with ΔTM-αVβ3, which lacks the short C-terminal stretches. The structure reveals a bent conformation and defines the α–β interface between IE2 (EGF-like 2) and the thigh domains. Modifying this interface by site-directed mutagenesis leads to robust integrin activation. Fluorescent lifetime imaging microscopy of inactive full-length αVβ3 on live cells yields a donor–membrane acceptor distance, which is consistent with the bent conformation and does not change in the activated integrin. These data are the first direct demonstration of conformational coupling of the integrin leg and head domains, identify the IE2–thigh interface as a critical steric barrier in integrin activation, and suggest that inside-out activation in intact cells may involve conformational changes other than the postulated switch to a genu-linear state.

CysLT2 receptors interact with CysLT1 receptors and down-modulate cysteinyl leukotriene–dependent mitogenic responses of mast cells

Cysteinyl leukotrienes (cys-LTs) induce inflammation through 2 G protein–coupled receptors (GPCRs), CysLT1 and CysLT2, which are coexpressed by most myeloid cells. Cys-LTs induce proliferation of mast cells (MCs), transactivate c-Kit, and phosphorylate extracellular signal-regulated kinase (ERK). Although MCs express CysLT2, their responses to cys-LTs are blocked by antagonists of CysLT1. We demonstrate that CysLT2 interacts with CysLT1, and that knockdown of CysLT2 increases CysLT1 surface expression and CysLT1-dependent proliferation of cord blood–derived human MCs (hMCs). Cys-LT–mediated responses were absent in MCs from mice lacking CysLT1 receptors, but enhanced by the absence of CysLT2 receptors. CysLT1 and CysLT2 receptors colocalized to the plasma membranes and nuclei of a human MC line, LAD2. Antibody-based fluorescent lifetime imaging microscopy confirmed complexes containing both receptors based on fluorescence energy transfer. Negative regulation of CysLT1-induced mitogenic signaling responses of MCs by CysLT2 demonstrates physiologically relevant functions for GPCR heterodimers on primary cells central to inflammation.