Cleavage of focal adhesion kinase by different proteases during SRC-regulated transformation and apoptosis. Distinct roles for calpain and caspases

Integrin-associated focal adhesion complexes provide the main adhesive links between the cellular actin cytoskeleton and the surrounding extracellular matrix. In vitro, cells utilize a complex temporal and spatially regulated mechanism of focal adhesion assembly and disassembly required for cell migration. Recent studies indicate that members of both calpain and caspase protease families can promote limited proteolytic cleavage of several components of focal adhesions leading to disassembly of these complexes. Such mechanisms that influence cell adhesion may be deregulated under pathological conditions characterized by increased cell motility, such as tumor invasion. v-Src-induced oncogenic transformation is associated with loss of focal adhesion structures and transition to a less adherent, more motile phenotype, while inactivating temperature-sensitive v-Src in serum-deprived transformed cells leads to detachment and apoptosis. In this report, we demonstrate that v-Src-induced disassembly of focal adhesions is accompanied by calpain-dependent proteolysis of focal adhesion kinase. Furthermore, inhibitors of calpain repress v-Src-induced focal adhesion disruption, loss of substrate adhesion, and cell migration. In contrast, focal adhesion loss during detachment and apoptosis induced after switching off temperature-sensitive v-Src in serum-deprived transformed cells is accompanied by caspase-mediated proteolysis of focal adhesion kinase. Thus, calpain and caspase differentially regulate focal adhesion turnover during Src-regulated cell transformation, motility, and apoptosis.     

Expression, purification, and in vitro characterization of recombinant salmon insulin-like growth factor-II

The insulin-like growth factors, IGF-I and IGF-II, are single chain polypeptides, which are structurally related to proinsulin and promote proliferation and differentiation of cells in many vertebrate species. Previous attempts to produce recombinant salmon IGF-II (rsIGF-II) were compromised by low expression levels and co-purification of incorrectly cleaved protein with the authentic recombinant product. In this study, a gene containing the coding region for Atlantic salmon (Salmo salar) IGF-II was cloned into a modified pET32a expression vector and transformed into Escherichia coli BL21 trxB (DE3) cells. Upon growth and induction (with IPTG) of the transformant, recombinant salmon IGF-II (rsIGF-II) was expressed as an insoluble, 28kDa thioredoxin.sIGF-II fusion protein linked by a protease cleavage motif (trx.FAHY.sIGF-II) in inclusion bodies. The inclusion bodies were subsequently solubilized and the fusion protein was purified by Ni-affinity chromatography. Recombinant IGF-II (7.8kDa) was then released from the fusion partner using H64A subtilisin BPN' protease and purified by reversed-phase HPLC. Homogeneity of the final recombinant product was confirmed by N-terminal amino acid sequencing, ion-spray mass spectrometry, SDS-polyacrylamide gel electrophoresis, and analytical reversed-phase HPLC. The biological activity of rsIGF-II was demonstrated in cultured rat L6 myoblasts and was found to be approximately 9- and 5-fold less potent than recombinant human IGF-I and recombinant salmon IGF-I, respectively, a result similar to that demonstrated previously with other recombinant fish IGF-II's in non-homologous cell lines.     

Automated three-dimensional reconstruction of keyhole limpet hemocyanin type 1

We have reconstructed a three-dimensional map of keyhole limpet hemocyanin isoform 1 (KLH1), using our automated data collection software, Leginon, integrated with particle selection algorithms, and the SPIDER reconstruction package. KLH1, a 7.9 MDa macromolecule, is an extracellular respiratory pigment composed of two asymmetric decamers, and presents an overall D(5) point-group symmetry. The reconstruction is in agreement with previous data published on molluscan hemocyanins. The reconstructed map (11.3A resolution, 3sigma criterion) was used to fit an available X-ray crystallography structure of Octopus dofleini Odg, solved at 2.3A [J. Mol. Biol. 278 (4) (1998) 855], with satisfactory results. The results validate the approach of automating the cryoEM process and demonstrate that the quality of the images acquired and the particles selected is comparable to those obtained using manual methods. Several problems remain to be solved however before these results can be generalized.     

Automated molecular microscopy: the new Leginon system

We report here on the current state of our efforts in automated molecular microscopy. Our primary automated data acquisition software system, Leginon, has been completely redesigned over the past two years. The new distributed system has been developed using the Python programming language and is compatible with both Linux and Windows operating systems. The new flexible architecture was designed to allow for the development of customized data collection protocols, several of which are described here. The system has been used to acquire data for approximately 150 experiments and we have demonstrated the capacity for high throughput data acquisition by acquiring images of more than 100,000 particles in a single session at the microscope.     

v-Src's hold over actin and cell adhesions

The oncoprotein v-Src and its cellular homologue (c-Src) are tyrosine kinases that modulate the actin cytoskeleton and cell adhesions. Through the concerted action of their protein-interaction and kinase domains, they are targeted to cell matrix integrin adhesions or cadherin-dependent junctions between epithelial cells, where they phosphorylate substrates that induce adhesion turnover and actin re-modelling. Recent experiments have defined some of the key targets and effector pathways that mediate the pleiotropic oncogenic effects of v-Src.     

A new species of Dyrosaurus (Crocodylomorpha, Dyrosauridae) from the early Eocene of Morocco: phylogenetic implications

A new dyrosaurid is described from the Ypresian of the phosphatic deposits of the Oulad Abdoun Basin of Morocco. It is based on numerous cranial and postcranial remains, allowing an almost complete reconstruction. This new Dyrosaurus species, Dyrosaurus maghribensis sp. nov. , is currently only known from Morocco. It differs from D. phosphaticus , present in contemporaneous levels of Algeria and Tunisia, by several autapomorpies, including a smooth dorsal margin of the parietal and widely opened choanae. A phylogenetic analysis, using 47 taxa and 234 morphological characters, shows the dyrosaurids as the sister taxon of pholidosaurids, which include Elosuchus , Sarcosuchus , Terminonaris and Pholidosaurus , and the thalattosuchians. Goniopholididae is a non-monophyletic group; however, if dyrosaurids are not included in the analysis, the result differs and the goniopholidids form a distinct clade. If Thalattosuchia is excluded, both Goniopholididae and Pholidosauridae become paraphyletic assemblages. Thus, phylogenetic problems remain with respect to longirostrine clade, and more attention should be paid to resolving their evolutionary relationships amongst the crocodyliforms.  © 2006 The Linnean Society of London, Zoological Journal of the Linnean Society , 2006, 148 , 603–656.     

Differences in the sensitivity of brown trout, Salmo trutta L., and rainbow trout, Salmo gairdneri Richardson, to physiological doses of cortisol

Interspecific differences in the stress response of fish may be due, in part, to differences in the sensitivity of target tissues to cortisol. The relative response of brown and rainbow trout to a standardized dose of cortisol was assessed by monitoring condition (K factor), the number of circulating lymphocytes and mortality due to disease, following cortisol treatment. Cortisol implantation resulted in a significant decline in K factor and number of circulating lymphocytes in immature brown trout, but not in immature rainbow trout, despite plasma cortisol levels being similar in both cases. Cortisol implantation in mature brown and rainbow trout significantly increased the mortality rate due to bacterial and fungal infection compared with control fish. Furthermore, the mortality rate due to disease was significantly greater in brown trout than rainbow trout, despite both groups receiving similar doses of steroid.     

Factors Mediating the Vitamin B12 Requirement of Euglena

SYNOPSIS. Deprived of vitamin B₁₂, Euglena gracilis strain Z ceases to divide which we believe to be a function of the light regime: division inhibition occurs more quickly in continuous light than in alternating (6L : 6D) light and not at all in total darkness. This phenomenon is dependent on the carbon source; cells grown in glutamate-malate medium do not divide regardless of the culture conditions while dl -lactate as carbon source permits growth in darkness in the absence of B₁₂. Conditions which lead to an increased O₂ or decreased CO₂ tension in the medium, such as agitation in darkness or incubation in red or white light, result in inhibition of division. This inhibition can be reversed by re-transferring the cells to still culture in the dark or, in the case of light-induced blockage, by the addition of DCMU.     

Deconvolution of pigment and physiologically related photochemical reflectance index variability at the canopy scale over an entire growing season

The sensitivity of the photochemical reflectance index (PRI) to leaf pigmentation and its impacts on its potential as a proxy for light‐use efficiency (LUE) have recently been shown to be problematic at the leaf scale. Most leaf‐to‐leaf and seasonal variability can be explained by such a confounding effect. This study relies on the analysis of PRI light curves that were generated at the canopy scale under natural conditions to derive a precise deconvolution of pigment‐related and physiologically related variability in the PRI. These sources of variability were explained by measured or estimated physiologically relevant variables, such as soil water content, that can be used as indicators of water availability and canopy chlorophyll content. The PRI mainly reflected the variability in the pigment content of the canopy. However, the corrected PRI, which was obtained by subtracting the pigment‐related seasonal variability from the PRI measurement, was highly correlated with the upscaled LUE measurements. Moreover, the sensitivity of the PRI to the leaf pigment content may mask the PRI versus LUE relationship or result in an artificial relationship that reflects the relationship of chlorophyll versus LUE, depending on the species phenology.     

Response of Tetraselmis suecica to nutrient and grazer manipulation

Three methods of algal quantification (direct cell counts, chlorophyll a extraction, in vivo fluorescence) were used to evaluate the response of the unicellular green flagellate Tetraselmis suecica to nutrients and grazers. Nutrient enrichment enhanced total cell counts, chlorophyll a concentration and in vivo and DCMU-fluorescence. Photosynthetic efficiency was reduced in the complete F2 medium as indicated by the high level of in vivo fluorescence, whereas photosynthetic efficiency was increased by the introduction of mussels to the F2 medium. The addition of mussels significantly increased the proportion of non-motile cells, but did not reduce the total cell count. The effect of mussel grazing on algae could be underestimated if only total cells were counted or only the chlorophyll a concentration was measured. The results indicate that these three methods measure different properties of an algal culture and are complementary to each other in assessing the quality and quantity of an algal population. Direct algal counting offers a reliable numerical assessment for cell population abundance. Chlorophyll a concentration was closely correlated to the total cell count. In the presence of mussels, in vivo fluorescence did not correlate with either algal cell counts or chlorophyll a concentration, indicating that the measurement of in vivo fluorescence may be misleading for estimating algal abundance under different culture conditions. This revised version was published online in August 2006 with corrections to the Cover Date.