No abstract

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 regulation of matrix metalloproteinases (MMP) has been studied extensively due to the fundamental roles these zinc-endopeptidases play in diverse physiological and pathological processes. However, phosphorylation has not previously been considered as a potential modulator of MMP activity. The ubiquitously expressed MMP-2 contains 29 potential phosphorylation sites. Mass spectrometry reveals that at least five of these sites are phosphorylated in hrMMP-2 expressed in mammalian cells. Treatment of HT1080 cells with an activator of protein kinase C results in a change in MMP-2 immunoreactivity on 2D immunoblots consistent with phosphorylation, and purified MMP-2 is phosphorylated by protein kinase C in vitro. Furthermore, MMP-2 from HT1080 cell-conditioned medium is immunoreactive with antibodies directed against phosphothreonine and phosphoserine, which suggests that it is phosphorylated. Analysis of MMP-2 activity by zymography, gelatin dequenching assays, and measurement of kinetic parameters shows that the phosphorylation status of MMP-2 significantly affects its enzymatic properties. Consistent with this, dephosphorylation of MMP-2 immunoprecipitated from HT1080 conditioned medium with alkaline phosphatase significantly increases its activity. We conclude that MMP-2 is modulated by phosphorylation on multiple sites and that protein kinase C may be a regulator of this protease in vivo.

The assembly of myosin into higher order structures is dependent upon accessory factors that are often tissue-specific. UNC-45 acts as such a molecular chaperone for myosin in the nematode Caenorhabditis elegans, in both muscle and non-muscle contexts. Although vertebrates contain homologues of UNC-45, their requirement for muscle function has not been assayed. We identified a zebrafish gene, unc45b, similar to a mammalian unc-45 homologue, expressed exclusively in striated muscle tissue, including the somites, heart and craniofacial muscle. Morpholino-oligonucleotide-mediated knockdown of unc45b results in paralysis and cardiac dysfunction. This paralysis is correlated with a loss of myosin filaments in the sarcomeres of the trunk muscle. Morphants lack circulation, heart looping and display severe cardiac and yolk-sac edema and also demonstrate ventral displacement of several jaw cartilages. Overall, this confirms a role for unc45b in zebrafish motility consistent with a function in myosin thick filament assembly and stability and uncovers novel roles for this gene in the function and morphogenesis of the developing heart and jaw. These results suggest that Unc45b acts as a chaperone that aids in the folding of myosin isoforms required for skeletal, cranial and cardiac muscle contraction.

The transition from quiescence (G 0 ) into the cell division cycle is marked by accelerated phospholipid turnover. We examined the rates of phosphatidylcholine (PC) synthesis and the activity, membrane affinity, and intracellular localization of the rate-limiting enzyme in the synthesis of PC, CTP:phosphocholine cytidylyltransferase (CT) during this transition. The addition of serum to quiescent IIC9 fibroblasts resulted in a wave of PC synthesis beginning at ϳ10 min, peaking at ϳ3 h with a >10-fold increase in rate, and declining to near basal rates by 10 h. CT activity, monitored in situ, was elevated ϳ3-fold between 1 and 2 h postserum. Neither CT mass nor its phosphorylation state changed during the surge in PC synthesis and CT activity. On the other hand, the ratio of particulate/soluble CT surged and then receded in concert with the wave of PC synthesis. During quiescence, CT was confined to the nucleus, as assessed by indirect immunofluorescence. Within 10 min after serum stimulation, a portion of the CT fluorescence appeared in the cytoplasm, where it intensified until ϳ4 h postserum. Thereafter, the cytoplasmic CT signal waned, while the nuclear signal increased, and by 8 h CT was once again predominantly nuclear. The dynamics of CT's apparent translocation in and out of the nucleus paralleled the wave of PC synthesis and the solubility changes of CT. Cytoplasmic CT co-localized with BiP, a resident endoplasmic reticulum protein, in a double labeling experiment. These data suggest that the wave of PC synthesis that accompanies the G 0 3 G 1 transition is regulated by the coordinated changes in CT activity, membrane affinity, and intracellular distribution. We describe for the first time a redistribution of CT from the nucleus to the ER that correlates with an activation of the enzyme. We propose that this movement is required for the stimulation of PC synthesis during entry into the cell cycle.