A ROS-dependent mechanism promotes CDK2 phosphorylation to drive progression through S phase.

Reactive oxygen species (ROS) at the right concentration promote cell proliferation in cell culture, stem cells, and model organisms. However, the mystery of how ROS signaling is coordinated with cell cycle progression and integrated into the cell cycle control machinery on the molecular level remains unsolved. Here, we report increasing levels of mitochondrial ROS during the cell cycle in human cell lines that target cyclin-dependent kinase 2 (CDK2). Chemical and metabolic interferences with ROS production decrease T-loop phosphorylation on CDK2 and so impede its full activation and thus its efficient DNA replication. ROS regulate CDK2 activity through the oxidation of a conserved cysteine residue near the T-loop, which prevents the binding of the T-loop phosphatase KAP. Together, our data reveal how mitochondrial metabolism is coupled with DNA replication and cell cycle progression via ROS, thereby demonstrating how KAP activity toward CDKs can be cell cycle regulated.

Metabolic interaction of hydrogen peroxide and hypoxia in zebrafish fibroblasts.

Cells require oxygen for aerobic metabolism, which may also result in the production of reactive oxygen species (ROS) as a by-product. Under low oxygen conditions, ROS formation has been reported to either increase or decrease. We addressed this physiological response for the first time in zebrafish embryonic fibroblasts (Z3) and used a hydrogen peroxide (H2O2)-specific fluorescent protein (roGFP2-Orp1) either targeted to the mitochondria or expressed in the cytosol. Microfluidic live-cell imaging measurements showed that oxygen deprivation in Z3 cells results in decreased or stable H2O2 levels within the mitochondria or the cytosol, respectively, and that the reductive shift recorded in the mitochondrial matrix is directly dependent on oxygen concentration. The response was accompanied by a transient increase in extracellular acidification rate (ECAR) and a lower cellular reducing potential as assessed by the viability stain alamarBlue. Complex I and III inhibition with Rotenone and Antimycin A led to H2O2 production under normoxia but these inhibitors were not able to avert the reductive shift under hypoxia. Only by system-wide inhibition of flavin-containing oxidases with Diphenyleneiodonium (DPI) were we able to decrease the reductive shift, while selective inhibition of NADPH oxidases with the inhibitor Apocynin had no effect on the hypoxia response. Since DPI also led to a strong increase in ECAR we found that, in order to keep the cytosolic H2O2 levels stable, glycolytic metabolism was of fundamental importance. According to our experiments with the glucose-6-phosphate dehydrogenase inhibitor 6-Aminonicotinamide, this was attributable to the pentose phosphate pathway producing reducing equivalents required for ROS degradation.

Sex-specific prey partitioning in breeding piscivorous birds examined via a novel, noninvasive approach.

Piscivorous birds frequently display sex-specific differences in their hunting and feeding behavior, which lead to diverging impacts on prey populations. Cormorants (Phalacrocoracidae), for example, were previously studied to examine dietary differences between the sexes and males were found to consume larger fish in coastal areas during autumn and winter. However, information on prey partitioning during breeding and generally on sex-specific foraging in inland waters is missing. Here, we assess sex-specific prey choice of Great Cormorants (Phalacrocorax carbo) during two subsequent breeding seasons in the Central European Alpine foreland, an area characterized by numerous stagnant and flowing waters in close proximity to each other. We developed a unique, noninvasive approach and applied it to regurgitated pellets: molecular cormorant sexing combined with molecular fish identification and fish-length regression analysis performed on prey hard parts. Altogether, 364 pellets delivered information on both, bird sex, and consumed prey. The sexes differed significantly in their overall prey composition, even though Perca fluviatilis, Rutilus rutilus, and Coregonus spp. represented the main food source for both. Albeit prey composition did not indicate the use of different water bodies by the sexes, male diet was characterized by higher prey diversity within a pellet and the consumption of larger fish. The current findings show that female and male cormorants to some extent target the available prey spectrum at different levels. Finally, the comprehensive and noninvasive approach has great potential for application in studies of other piscivorous bird species.