Golgi antiapoptotic protein (GAAP) is a novel regulator of cell death that is highly conserved in eukaryotes and present in some poxviruses, but its molecular mechanism is unknown. Given that alterations in intracellular Ca 2؉ homeostasis play an important role in determining cell sensitivity to apoptosis, we investigated if GAAP affected Ca 2؉ signaling. Overexpression of human (h)-GAAP suppressed staurosporine-induced, capacitative Ca 2؉ influx from the extracellular space. In addition, it reduced histamine-induced Ca 2؉ release from intracellular stores through inositol trisphosphate receptors. h-GAAP not only decreased the magnitude of the histamine-induced Ca 2؉ fluxes from stores to cytosol and mitochondrial matrices, but it also reduced the induction and frequency of oscillatory changes in cytosolic Ca 2؉ . Overexpression of h-GAAP lowered the Ca 2؉ content of the intracellular stores and decreased the efficacy of IP 3 , providing possible explanations for the observed results. Opposite effects were obtained when h-GAAP was knocked down by siRNA. Thus, our data demonstrate that h-GAAP modulates intracellular Ca 2؉ fluxes induced by both physiological and apoptotic stimuli.

ATP is the "principal energy currency" in metabolism and the most versatile small molecular regulator of cellular activities. Although already much is known about the role of ATP in fundamental processes of living systems, data about its compartmentalization are rather scarce, and we still have only very limited understanding of whether patterns in the distribution of intracellular ATP concentration ("ATP inhomogeneity") do exist and have a regulatory role. Here we report on the analysis of coupling of local ATP supply to regulation of actomyosin behavior, a widespread and dynamic process with conspicuous high ATP dependence, which is central to cell shape changes and cell motility. As an experimental model, we use embryonic fibroblasts from knock-out mice without major ATP-ADP exchange enzymes, in which we (re)introduce the ATP/ADP exchange enzyme adenylate kinase-1 (AK1) and deliberately manipulate its spatial positioning by coupling to different artificial location tags. By transfection-complementation of AK1 variants and comparison with yellow fluorescent protein controls, we found that motility and spreading were enhanced in cells with AK1 with a focal contact guidance tag. Intermediary enhancement was observed in cells with membrane-targeted or cytosolic AK1. Use of a heterodimer-inducing approach for transient translocation of AK1 to focal contacts under conditions of constant global AK1 activity in the cell corroborated these results. Based on our findings with these model systems, we propose that local ATP supply in the cell periphery and "on site" fuelling of the actomyosin machinery, when maintained via enzymes involved in phosphoryl transfer, are codetermining factors in the control of cell motility.Maintenance of adequate ATP supply is of crucial importance for the mechanisms of structural remodeling in cells with high shape plasticity, especially under high energy-demanding circumstances (1). During processes like cell motility or phagocytosis, the cell movement and cellular shape changes require active restructuring of the actin cytoskeleton. By spatially controlled polymerization of ATP-bound G-actin monomers to the plus end of growing actin filaments and formation of multiple branches, a dense network is built, which is called the actin cortex, based on its specific localization within the cell (2). In this network, ATP hydrolysis and release of P i drive actin filament dynamics by modulating filament stability and determine nucleotide-dependent filament conformation and interaction(s) with regulatory actin-binding proteins (3). Furthermore, force generation needed for contraction and cell movement is controlled by myosin and nonmuscle myosin ATPases (4), and also upstream signaling, involving small GTPases, is contingent upon nucleotide exchange (5). Taken together, actomyosin dynamics is overall an energy-demanding process, directly coupled to ATP availability.Indeed, actomyosin-based processes may consume a major fraction of cellular energy (6). Moreover, the coupling between global ATP suppl...

Background: The Warburg phenotype in cancer cells has been long recognized, but there is still limited insight in the consecutive metabolic alterations that characterize its establishment. We obtained better understanding of the coupling between metabolism and malignant transformation by studying mouse embryonic fibroblast-derived cells with loss-of-senescence or H-RasV12/E1A-transformed phenotypes at different stages of oncogenic progression.

BackgroundCreatine Kinases (CK) catalyze the reversible transfer of high-energy phosphate groups between ATP and phosphocreatine, thereby playing a storage and distribution role in cellular energetics. Brain-type CK (CK-B) deficiency is coupled to loss of function in neural cell circuits, altered bone-remodeling by osteoclasts and complement-mediated phagocytotic activity of macrophages, processes sharing dependency on actomyosin dynamics.Methodology/Principal FindingsHere, we provide evidence for direct coupling between CK-B and actomyosin activities in cortical microdomains of astrocytes and fibroblasts during spreading and migration. CK-B transiently accumulates in membrane ruffles and ablation of CK-B activity affects spreading and migration performance. Complementation experiments in CK-B-deficient fibroblasts, using new strategies to force protein relocalization from cytosol to cortical sites at membranes, confirmed the contribution of compartmentalized CK-B to cell morphogenetic dynamics.Conclusion/SignificanceOur results provide evidence that local cytoskeletal dynamics during cell motility is coupled to on-site availability of ATP generated by CK-B.

ADP-ribosylation factor (Arf) proteins are small guanosine triphosphatases (GTPases) that act as major regulators of intracellular vesicular trafficking and secretory organelle pathway integrity. Like all small monomeric GTPases, Arf proteins cycle between a GDP-bound and a GTP-bound state, and this cycling is catalysed by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins. While the class I Arfs, especially Arf1, have been studied extensively, little is known as yet about the function and regulation of class II Arfs, Arf4 and Arf5. In this study, we show that Arf proteins show class-specific dynamic behaviour. Moreover, unlike class I Arfs, membrane association of class II Arfs is resistant to inhibition of large Arf GEFs by Brefeldin A. Through the construction of Arf chimeric proteins, evidence is provided that the N-terminal amphipathic helix and a class-specific residue in the conserved interswitch domain determine the membrane-binding properties of class I and class II Arf proteins. Our results show that fundamental differences exist in behaviour and regulation of these small GTPases.