We investigated the focal adhesion proteins paxillin and Fak, and the cell-cell adhesion protein cadherin in developing zebrafish (Danio rerio) embryos. Cadherins are expressed in presomitic mesoderm where they delineate cells. The initiation of somite formation coincides with an increase in the phosphorylation of Fak, and the accumulation of Fak, phosphorylated Fak, paxillin, and fibronectin at nascent somite boundaries. In the notochord, cadherins are expressed on cells during intercalation, and phosphorylated Fak accumulates in circumferential rings where the notochord cells contact laminin in the perichordal sheath. Subsequently, changes in the orientations of collagen fibers in the sheath suggest that Fak-mediated adhesion allows longitudinal expansion of the notochord, but not lateral expansion, resulting in notochord elongation. Novel observations showed that focal adhesion kinase and paxillin concentrate at sites of cell-cell adhesion in the epithelial enveloping layer and may associate with actin cytoskeleton at epithelial junctions containing cadherins. Fak is phosphorylated at these epithelial junctions but is not phosphorylated on Tyr397, implicating a noncanonical mechanism of regulation. These data suggest that Fak and paxillin may function in the integration of cadherin-based and integrin-based cell adhesion during the morphogenesis of the early zebrafish embryo.

The pituitary adenylate cyclase-activating polypeptide (PACAP)-selective PAC1 receptor (Adcyap1r1) is a G protein-coupled receptor (GPCR) that activates adenylyl cyclase and PLC. Similar to many other GPCRs, our previous studies showed that the PAC1 receptor is internalized after ligand binding to form signaling endosomes, which recruit additional second messenger pathways. Using a human embryonic kidney (HEK 293) PAC1Hop1-EGFP receptor cell line, we have examined how different PAC1 receptor signaling mechanisms contribute to MEK/ERK activation. Unlike PAC1 receptor-stimulated adenylyl cyclase/cAMP production in the plasma membrane, PACAP-mediated ERK phosphorylation was partly dependent on receptor internalization, as determined by treatment with pharmacological inhibitors of endocytosis or temperature reduction, which also suppressed receptor internalization. Stimulation of cAMP generation by forskolin or exposure to the cell-permeable cAMP analogs 8-bromo-cAMP and dibutyryl cAMP had minimal effects on ERK phosphorylation in this system. The ability of reduced temperature (24°C) to consistently suppress ERK activation to a greater extent than the endocytosis inhibitors Pitstop 2 and dynasore indicated that other mechanisms, in addition to PAC1 internalization/endosome activation, were involved. Inhibition of PAC1 receptor-stimulated PLC/diacylglycerol/PKC signaling by bisindoylmaleimide I also attenuated ERK phosphorylation, and direct PKC activation with phorbol ester increased ERK phosphorylation in a temperature-dependent manner. Inhibition of PAC1 receptor endocytosis and PKC activation completely blocked PACAP-stimulated ERK activation. PACAP augmented phosphorylated ERK staining uniformly over the cytoplasm and nucleus, and PKC signaling facilitated nuclear phosphorylated ERK translocation. In sum, our results show that PACAP/PAC1 receptor endocytosis and PLC/diacylglycerol/PKC activation represent two complementary mechanisms contributing to PACAP-induced ERK activation.

The structure of complex I from Yarrowia lipolytica was determined by three-dimensional electron microscopy. A random conical data set was collected from deep stain embedded particles. More than 14000 image pairs were analyzed. Through extensive classification combined with three-dimensional reconstruction, it was possible for the first time to show a much more detailed substructure of the complex. The peripheral arm is subdivided in at least six domains. The membrane arm shows two major protrusions on its matrix facing side and exhibits a channel like feature on the side facing the cytoplasm. Structures resembling a tether connecting the subunits near the catalytic center with the protrusions of the membrane arm provide a second connection between matrix and membrane domain.

Pharmacological manipulations in the Drosophila embryo have been hindered by the impermeability of the eggshell. The ultimate barrier to delivery of small molecule solutes to the embryo is the waxy layer that lies beneath the external chorion layers and encases the underlying vitelline membrane of the eggshell. Conventional protocols call for heptane or octane to permeablize the dechorionated eggshell however, these solvents are toxic and can result in low viability. Furthermore, heptane and octane require transition of the embryo between aqueous and organic phase solvents making it challenging to avoid desiccation. Here we describe an embryo permeabilization solvent (EPS) composed of D-limonene and plant-derived surfactants that is water miscible and highly effective in rendering the dechorionated eggshell permeable. EPS permeabilization enables embryo uptake of several different dyes of various molecular mass up to 995 Daltons. We find that the embryo undergoes an age dependent decrease in the ability to be permeabilized in the first six to eight hours after egg laying. This apparent developmental change in the vitelline membrane contributes to the heterogeneity in permeabilization seen even among closely staged embryos. However, using fluorescent properties of Rhodamine B dye and various conditions of EPS treatment we demonstrate the ability to obtain optimally permeabilized viable embryos. We also demonstrate the ability to assess teratogenic activity of several compounds applied to embryos in vitro, using both early and late developmental endpoints. Application of the method to transgenic strains carrying GFP reporter genes results in a robust readout of pharmacological alteration of embryogenesis. The straightforward and rapid nature of the manipulations needed to prepare batches of permeabilized embryos has the potential of establishing the Drosophila embryo as an alternative model in toxicology and for small molecule screening in a high throughput format.

The structures of the NADH dehydrogenases from Bos taurus and Aquifex aeolicus have been determined by 3D electron microscopy, and have been analyzed in comparison with the previously determined structure of Complex I from Yarrowia lipolytica. The results show a clearly preserved domain structure in the peripheral arm of complex I, which is similar in the bacterial and eukaryotic complex. The membrane arms of both eukaryotic complexes show a similar shape but also significant differences in distinctive domains. One of the major protuberances observed in Y. lipolytica complex I appears missing in the bovine complex, while a protuberance not found in Y. lipolytica connects in bovine complex I a domain of the peripheral arm to the membrane arm. The structural similarities of the peripheral arm agree with the common functional principle of all complex Is. The differences seen in the membrane arm may indicate differences in the regulatory mechanism of the enzyme in different species.