Talin is a structural component of focal adhesion sites and is thought to be engaged in multiple protein interactions at the cytoplasmic face of cell/matrix contacts. Talin is a major link between integrin and the actin cytoskeleton and was shown to play an important role in focal adhesion assembly. Consistent with the view that talin must be activated at these sites, we found that phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate (PI4,5P 2 ) bound to talin in cells in suspension or at early stages of adhesion, respectively. When phosphoinositides were associated with phospholipid bilayer, talin/phosphoinositide association was restricted to PI4,5P 2 . This association led to a conformational change of the protein. Moreover, the interaction between integrin and talin was greatly enhanced by PI4,5P 2 -induced talin activation. Finally, sequestration of PI4,5P 2 by a specific pleckstrin homology domain confirms that PI4,5P 2 is necessary for proper membrane localization of talin and that this localization is essential for the maintenance of focal adhesions. Our results support a model in which PI4,5P 2 exposes the integrin-binding site on talin. We propose that PI4,5P 2 -dependent signaling modulates assembly of focal adhesions by regulating integrin-talin complexes. These results demonstrate that activation of the integrinbinding activity of talin requires not only integrin engagement to the extracellular matrix but also the binding of PI4,5P 2 to talin, suggesting a possible role of lipid metabolism in organizing the sequential assembly of focal adhesion components.
Protein kinase CK2 is a multifunctional enzyme which has long been described as a stable heterotetrameric complex resulting from the association of two catalytic (␣ or ␣) and two regulatory () subunits. To track the spatiotemporal dynamics of CK2 in living cells, we fused its catalytic ␣ and regulatory  subunits with green fluorescent protein (GFP). Both CK2 subunits contain nuclear localization domains that target them independently to the nucleus. Imaging of stable cell lines expressing low levels of GFP-CK2␣ or GFP-CK2 revealed the existence of CK2 subunit subpopulations exhibiting differential dynamics. Once in the nucleus, they diffuse randomly at different rates. Unlike CK2, CK2␣ can shuttle, showing the dynamic nature of the nucleocytoplasmic trafficking of the kinase. When microinjected in the cytoplasm, the isolated CK2 subunits are rapidly translocated into the nucleus, whereas the holoenzyme complex remains in this cell compartment, suggesting an intramolecular masking of the nuclear localization sequences that suppresses nuclear accumulation. However, binding of FGF-2 to the holoenzyme triggers its nuclear translocation. Since the substrate specificity of CK2␣ is dramatically changed by its association with CK2, the control of the nucleocytoplasmic distribution of each subunit may represent a unique potential regulatory mechanism for CK2 activity.Protein kinase CK2 is a ubiquitous serine/threonine protein kinase, generally described as a stable ␣ 2  2 tetramer, where ␣ and  are the catalytic and regulatory subunits, respectively (3). Although its signaling function has long remained obscure, the importance of CK2 is suggested by the evolutionary conservation of the enzyme and by the fact that the disruption of both Saccharomyces cerevisiae genes encoding CK2 catalytic subunits is a lethal event (29). In addition to its role in embryonic development and terminal differentiation, the enzyme is required for normal cell cycle progression (20,30). At last, a function of CK2 in cell survival has recently emerged (1).Many of the identified CK2 substrates that are critical for cell proliferation and viability are localized in different cellular compartments. However, there is controversy as to the localization of CK2 and where its substrates are phosphorylated. Although the current prevailing view of CK2 is a tetrameric enzyme, accumulating evidence also indicates that free populations of both CK2 subunits can exist and exert specific functions in the cell (18, 37). At least in vitro, CK2 exerts a central role in modulating the catalytic activity of CK2 (26). Consequently, it is suspected that in vivo, the substrate specificity of the enzyme is likely to be determined both by subcellular localization and by affinity for its regulatory subunit that brings the kinase in proximity to the substrate.In a previous study, the behavior of CK2 subunits fused to GFP was characterized in living cells (25). The expressed fusion proteins were functional and interacted with endogenous CK2. Both subunits were mostl...
We have shown previously that ADP released upon platelet adhesion mediated by ␣ IIb  3 integrin triggers accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns-3,4-P 2 ) (Gironcel, D., Racaud-Sultan, C., Payrastre, B., Haricot, M., Borchert, G., Kieffer, N., Breton, M., and Chap, H. (1996) FEBS Lett. 389, 253-256). ADP has also been involved in platelet spreading. Therefore, in order to study a possible role of phosphoinositide 3-kinase in platelet morphological changes following adhesion, human platelets were pretreated with specific phosphoinositide 3-kinase inhibitors LY294002 and wortmannin. Under conditions where PtdIns-3,4-P 2 synthesis was totally inhibited (25 M LY294002 or 100 nM wortmannin), platelets adhered to the fibrinogen matrix, extended pseudopodia, but did not spread. Moreover, addition of ADP to the medium did not reverse the inhibitory effects of phosphoinositide 3-kinase inhibitors on platelet spreading. Although synthetic dipalmitoyl PtdIns-3,4-P 2 and dipalmitoyl phosphatidylinositol 3,4,5-trisphosphate restored only partially platelet spreading, phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P 2 ) was able to trigger full spreading of wortmannin-treated adherent platelets. Following 32 P labeling of intact platelets, the recovery of [ 32 P]PtdIns-4,5-P 2 in anti-talin immunoprecipitates from adherent platelets was found to be decreased upon treatment by wortmannin. These results suggest that the lipid products of phosphoinositide 3-kinase are required but not sufficient for ADP-induced spreading of adherent platelets and that PtdIns-4,5-P 2 could be a downstream messenger of this signaling pathway.Platelets play a key role in hemostasis by their capacities to adhere and to aggregate in response to vascular injury. The most abundant platelet integrin, the ␣ IIb  3 complex, is largely responsible for platelet aggregation after binding of soluble fibrinogen. Moreover, ␣ IIb  3 integrin is required for a complete and irreversible platelet adhesion to the subendothelial matrix (1). In this case, its preferential ligand is the von Willebrand factor, but under certain conditions fibrinogen or fibrin could also act as adhesion substrates. In its resting state, ␣ IIb  3 integrin is able to recognize immobilized fibrinogen. However, the interaction of soluble fibrinogen with ␣ IIb  3 complex requires a previous conformational change of the integrin due to an inside-out signaling pathway. When platelets adhere in vitro to a fibrinogen matrix, they undergo several irreversible morphological changes such as rounding and spreading. These responses are sustained by a cytoskeletal reorganization including extension of filopodia, lamellipodia, and controlled orientation of stress fibers. It has been shown that a concomitant granular secretion of ADP from adherent platelets was necessary for spreading (2), and that it controlled specific signals, i.e. p125 FAK and PtdIns 3-kinase 1 activations (2, 3). Data from Haimovich et al. (4) show that tyrosine phosphorylation of p125 FAK tyrosine kin...
The spruce budworm, Choristoneura fumiferana, Clem., is the most significant defoliating pest of boreal balsam fir (Abies balsamea (L.) Mill.) and spruce (Picea sp.) in North America. Historically, spruce budworm outbreaks have been managed via a reactive, foliage protection approach focused on keeping trees alive rather than stopping the outbreak. However, recent theoretical and technical advances have renewed interest in proactive population control to reduce outbreak spread and magnitude, i.e., the Early Intervention Strategy (EIS). In essence, EIS is an area-wide management program premised on detecting and controlling rising spruce budworm populations (hotspots) along the leading edge of an outbreak. In this article, we lay out the conceptual framework for EIS, including all of the core components needed for such a program to be viable. We outline the competing hypotheses of spruce budworm population dynamics and discuss their implications for how we manage outbreaks. We also discuss the practical needs for such a program to be successful (e.g., hotspot monitoring, population control, and cost-benefit analyses), as well as the importance of proactive communications with stakeholders.
The spotted wing drosophila Drosophila suzukii Matsumura (Diptera: Drosophilidae), a pest of berries stone fruits, invaded North America and Europe in 2008. Current control methods rely mainly on insecticides. The sterile insect technique (SIT) has potential as an additional control tactic for the integrated management of D. suzukii. As a step towards the development of the SIT, this study aimed at finding the optimum irradiation dose to sterilize D. suzukii under controlled laboratory conditions. Four-day-old D. suzukii pupae were irradiated 12 to 24 hours prior to adult emergence in a 60Co Gamma Cell 220 and in a 137Cs Gamma Cell 3000 with doses of 30, 50, 70, 80, 90, 100 or 120 Gy. Emergence rate (88.1%), percent of deformed flies (4.0%) and survival curves were not affected by the tested irradiation doses. However, some reproductive parameters of the flies were affected by irradiation. Females irradiated with a dose of 50 Gy or more had almost no fecundity. When non-irradiated females were mated with irradiated males, egg hatch decreased exponentially with irradiation dose from 82.6% for the untreated control males to 4.0% for males irradiated with 120 Gy. Mortality of F1 individuals from the irradiated treatment also occurred during larval and pupal stages, with an egg to adult survival of 0.2%. However, descendants produced by the irradiated generation were fertile. These results are an encouraging first experimental step towards the development of the SIT for the management of D. suzukii populations.
Accumulating evidence indicates that in addition to the classical complex, the catalytic and regulatory subunits of CK2 can also exist as free populations in living cells. The association of recombinant CK2 subunits in vitro has been characterized, providing evidence for the first time for their targeted interactions in living cells. The data also suggest that the regulation by phosphorylation of many CK2 substrates may strongly depend on the dynamic localization/association of its subunits.
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