A modular spring-loaded actuator for mechanical activation of membrane proteins.

How cells respond to mechanical forces by converting them into biological signals underlie crucial cellular processes. Our understanding of mechanotransduction has been hindered by technical barriers, including limitations in our ability to effectively apply low range piconewton forces to specific mechanoreceptors on cell membranes without laborious and repetitive trials. To overcome these challenges we introduce the Nano-winch, a robust, easily assembled, programmable DNA origami-based molecular actuator. The Nano-winch is designed to manipulate multiple mechanoreceptors in parallel by exerting fine-tuned, low- piconewton forces in autonomous and remotely activated modes via adjustable single- and double-stranded DNA linkages, respectively. Nano-winches in autonomous mode can land and operate on the cell surface. Targeting the device to integrin stimulated detectable downstream phosphorylation of focal adhesion kinase, an indication that Nano-winches can be applied to study cellular mechanical processes. Remote activation mode allowed finer extension control and greater force exertion. We united remotely activated Nano-winches with single-channel bilayer experiments to directly observe the opening of a channel by mechanical force in the force responsive gated channel protein, BtuB. This customizable origami provides an instrument-free approach that can be applied to control and explore a diversity of mechanotransduction circuits on living cells.

Src family tyrosine kinases-driven colon cancer cell invasion is induced by Csk membrane delocalization.

The nonreceptor tyrosine kinases of the Src family (SFK) are frequently deregulated in human colorectal cancer (CRC), and they have been implicated in tumour growth and metastasis. How SFK are activated in this cancer has not been clearly established. Here, we show that the SFK-dependent invasion is induced by inactivation of the negative regulator C-terminal Src kinase, Csk. While the level of Csk was inconsistent with SFK activity in colon cancer cells, its membrane translocation, needed for efficient regulation of membrane-localized SFK activity, was impaired. Accordingly, Csk downregulation did not affect SFK oncogenic activity in these cells, whereas expression of a membrane-localized form of this kinase affected their invasive activity. Downregulation of the transmembrane and rafts-localized Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched microdomain (PAG), was instrumental for the cytoplasmic accumulation of Csk. Re-expression of PAG in cells from late-stage CRC inhibited SFK invasive activity in a Csk-dependent manner. Conversely, inactivation of its residual expression in early-stage CRC cells promoted SFK invasive activity. Finally, this mechanism was specific to CRC as Csk coupling to SFK was readily detected in breast cancer cells. Therefore, Csk mis-localization defines a novel mechanism for SFK oncogenic activation in CRC cells.

Two functionally distinct pools of Src kinases for PDGF receptor signalling.

The cytoplasmic tyrosine kinases of the Src family (SFK) play important roles in cell responses induced by growth factors, including cell growth, survival and migration. Here, we review how SFK participate in PDGF (platelet-derived growth factor) receptor signalling leading to DNA synthesis and actin assembly. Furthermore, evidence for a spatial compartmentalization of SFK signalling is also discussed.

The COOH-terminal Src kinase Csk is a tumor antigen in human carcinoma.

The cytoplasmic tyrosine kinase cSrc is involved in the regulation of many important cellular functions including cell growth and transformation, and its activity is down-regulated by phosphorylation of the Tyr530 residue by the COOH-terminal Src tyrosine kinase, Csk. Because cSrc was previously found overexpressed, activated, and in some cases mutated in carcinoma, we investigated whether it could act as a tumor antigen. We show that whereas no autoantibodies were found against cSrc or its relative Fyn, up to 20% of patients with carcinoma had high-affinity autoantibodies against Csk. Immunity mainly resulted from a secondary response, as indicated by the presence of IgG1 in the sera. Antibodies were linked to the cancer because they were not detected in healthy subjects nor in patients with unrelated diseases, and their levels decreased in the sera of patients after surgical resection. Furthermore, they behaved as early markers of epithelial transformation because they were present in sera of patients with early-stage tumors and precancerous lesions such as colorectal polyps and in sera of patients that were scored negative for other cancer serological markers (CEA, CA15-3, CA19-9, p53 antibodies). Finally the presence of these antibodies was attributed, at least in part, to a substantial elevation of Csk protein levels in the corresponding tumors. However a strong increase in Src activity was also observed in these tissues, which suggested that Csk cannot regulate Src-like activity in carcinoma. Taken together, these data demonstrate that Csk acts as an autoantigen, and the detection of anti-Csk antibodies may have potential diagnostic usefulness in the early detection and postoperative follow-up of patients with carcinoma.

A specific function for phosphatidylinositol 3-kinase alpha (p85alpha-p110alpha) in cell survival and for phosphatidylinositol 3-kinase beta (p85alpha-p110beta) in de novo DNA synthesis of human colon carcinoma cells.

We have previously shown an important function of phosphatidylinositol 3-kinase (PI3K)alpha(p85alpha-p110alpha) and PI3Kbeta (p85-alpha-p110beta) for DNA synthesis induced by various mitogens in non transformed fibroblasts and we now report a specific role of these enzymes in human colon cancer cell growth. Using antibodies specific to p110alpha and to p110beta catalytic subunits, increase in PI3Kalpha and PI3Kbeta activities was detected in 15/19 human tumour biopsies relative to adjacent normal mucosa of human colon and bladder. Increase in such activities was also observed in adenocarcinoma cell lines CaCo2, CO115, HCT 116, LS 174T and WiDr relative to non-transformed fibroblasts. Maximal PI3Kalpha activity was observed for LS 174T and PI3Kbeta activity for WiDr cells. This was partly correlated with an increase in p110alpha and p110beta protein levels both in some primary tumours and established cell lines, suggesting that PI3K overexpression is involved in enzymatic deregulation. Functional consequence of such activation was assessed by a microinjection approach. An injection of neutralizing antibody specific to p110beta in WiDr, HCT116 and CO 115 cells inhibited de novo DNA synthesis, whereas antibodies specific to p110gamma had no effect. Neutralizing antibodies specific to p110alpha induced apoptosis, a response that was reverted by treating cells with the caspase inhibitor z-VAD-fmk. However anti-p110beta and anti-p110gamma antibodies did not affect cell survival. We concluded that PI3Kalpha and PI3Kbeta play important roles in human colon cancer cell growth with a specific function for PI3Kbeta in de novo DNA synthesis and an involvement of PI3Kalpha in cell survival.

Deregulation of the cytoplasmic tyrosine kinase cSrc in the absence of a truncating mutation at codon 531 in human bladder carcinoma.

The involvement of the cytoplasmic tyrosine kinase cSrc was investigated in human bladder carcinogenesis. Kinase activity was determined in tissue lysates from bladder transitional cell carcinoma (TCC) relative to normal epithelia. Strong kinase activation was observed at all stages of carcinogenesis with a peak at the stage pT1, where tumor cells disrupt the basement membrane and invade the submucosa. In agreement with a role for cSrc in cell invasion, immunocytochemistry analysis showed a strong staining of invading cells. An increase in cSrc protein level were also found in most tumor samples, however, it did not correlate with an increase in activity (r = 0.44) suggesting that cSrc is deregulated in these tumors. Indeed, high Src activity was affinity-purified from a column (IRSVSSDGHE(p)YIYVDP-Affigel 10) that specifically retains active Src. Enzymatic regulation involves the C-terminus, recently found mutated at codon 531 in a subset of advanced human colon cancers. However, no such mutations were detected in TCC, suggesting the existence of other mechanisms for kinase activation.

Astrocytes are mainly responsible for the polyunsaturated fatty acid enrichment in blood-brain barrier endothelial cells in vitro.

To determine the respective roles of endothelial cells from brain capillaries and astrocytes in the conversion of circulating 18:2n-6 and 18:3n-3 into 20:4n-6 and 22:6n-3, respectively, a coculture of the two cell types mimicking the in vivo blood-brain barrier was used. During the culture period, endothelial cells cultured on an insert were set above the medium of a Petri dish containing or not a stabilized culture of astrocytes. Five days after confluence, labeled 18:2n-6 and 18:3n-3 (10 microM each) were added to the endothelial cells and incubated for 48 h. Analogous experiments were also performed by using each cell type cultured alone in the culture device. The distribution of radioactivity in lipids and fatty acids was studied in all the compartments of the culture device. Endothelial cells cultured alone weakly converted the precursor fatty acids into 20:4n-6 and 22:6n-3. When endothelial cells were cocultured with astrocytes, their content of polyunsaturated fatty acids increased dramatically. This effect was associated with the uptake of polyunsaturated fatty acids from the lower medium (astrocyte medium). These fatty acids were released by astrocytes after they were synthesized from the precursor fatty acids that passed through the endothelial cell monolayer into the lower medium. Polyunsaturated fatty acids were released by astrocytes as unesterified fatty acids and as phospholipids (mainly phosphatidylcholine and lysophosphatidylcholine) even when the medium was devoid of serum. These results suggest that astrocytes could play a major role in the delivery of essential polyunsaturated fatty acids to the barrier itself and to the brain.

Increased in vitro fatty acid supply and cellular transport capacities in cold-acclimated ducklings (Cairina moschata).

In cold-acclimated (CA) birds, lipids play a crucial role in regulatory thermogenesis by acting both as substrates for and activators of thermogenic processes. The capacity to supply lipids to thermogenic tissues, which could limit cold thermogenesis, was assessed in CA ducklings (5 wk old, 4 degrees C) and compared with thermoneutral controls (TN, 25 degrees C). In CA ducklings, basal lipolytic activity of adipose tissue fragments was higher (202 +/- 9 vs. 130 +/- 14 nmol glycerol released . 100 mg tissue-1 . h-1, +55%) than in TN controls, while glucagon had a much higher stimulatory effect (+140 to +500% depending on dose). This was consistent with increased plasma levels of nonesterified fatty acids (FA, +57%) and glycerol (+31%) in vivo. In vitro endothelial lipase activity per organ was higher in CA than in TN ducklings in red gastrocnemius muscle (6.3 +/- 0.6 vs. 3.5 +/- 0.3 microeq nonesterified FA released per hour, +80%) and liver (+55%). The intracellular FA-binding capacity of (12-18 kDa) proteins was higher in gastrocnemius muscle (+43%) and liver (+74%) from CA ducklings. In gastrocnemius, it was linked to a higher content (21 +/- 2 vs. 15 +/- 2 microg/mg protein, +37%) of an intracellular 15.4-kDa FA-binding protein. These in vitro results indicate that coordinated increases in FA supply from adipose tissue, cellular uptake of lipoprotein-derived FA, and intracellular FA transport capacity occur in CA ducklings endowed with higher thermogenic capacity and cold endurance.

Characterization of two different cytoplasmic protein tyrosine kinases from human breast cancer.

Two different protein tyrosine kinases were detected in the cytosolic fraction of different human tumor tissues. After partial purification, the two enzymes, which were highly active in breast tumor tissues, were characterized. One of them, soluble tyrosine kinase-1 (STK-1), represents a soluble form of the c-Src protein, which is apparently underphosphorylated on its C-terminal tyrosine residue whereas the other (STK-2) is a 48-kDa protein tyrosine kinase (PTK), which is molecularly and functionally related to the C-terminal Src kinase (Csk). These two protein tyrosine kinases clearly exhibit a different substrate specificity, and are responsible for the high tyrosine kinase activity present in the cytosolic fraction of human breast cancer. In addition, it was observed that STK-1 and STK-2 are also expressed in the breast cancer cell line, CAL-51.

Eicosapentaenoic and docosahexaenoic acids reduce PGH synthase 1 expression in bovine aortic endothelial cells.

To enlighten the mechanism of inhibition of prostacyclin (PGI2) production by n-3 fatty acids, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, cultured endothelial cells were incubated with albumin bound-EPA or -DHA for 22 h. Under these conditions, PGI2 formation in response to bradykinin, calcium ionophore or exogenous arachidonic acid was equally inhibited by 50%, suggesting that the inhibition might occur downstream the phospholipase step, likely at the level of PGH synthase and/or PGI2 synthase activities. Western blot analysis indicated that the mass of the constitutive isoform of PGH synthase (PGH synthase 1), but not PGI2 synthase, was significantly reduced in n-3 fatty acid-enriched cells. In subsequent experiments, PGH synthase 1 mRNA level, measured by northern blotting, was also decreased in n-3 supplemented cells. This reduction was not due to mRNA destabilization. None of these parameters were altered by similar enrichment with oleic acid (OA). These results suggest that EPA and DHA may affect PGH synthase 1 expression, presumably at the transcriptional level.

Docosapentaenoic acid (22:5,n-3): metabolism and effect on prostacyclin production in endothelial cells.

Eicosapentaenoic acid (EPA, 20:5,n-3) and docosahexaenoic acid (DHA, 22:6, n-3), the two main fatty acids of fish oil, have been shown to inhibit prostacyclin production and to be actively interconverted, leading to the accumulation of docosapentaenoic acid (DPA, 22:5,n-3) in endothelial cell phospholipids. We have investigated the effect of supplementing endothelial cells with DPA on their capacity to produce prostacyclin. We found that endothelial cells incubated for 22 h with 25 microM DPA bound to albumin (fatty acid/albumin ratio of 1.3) produced two-fold less prostacyclin compared to control cells when stimulated with endogenous arachidonic acid-mobilizing agents such as bradykinin and calcium ionophore A23187. Since the formation of prostacyclin from 0.1-15 microM exogenous arachidonic acid was also reduced, it is suggested that prostacyclin inhibition observed in DPA-treated cells might not proceed from a reduction of arachidonic acid availability only. Such an inhibition was already observed after 1 h incubation of the cells with DPA, and with 2-20 times lower DPA concentrations. The inhibition might depend on EPA which was formed by retroconversion of DPA.

Fatty acid composition of brain capillary endothelial cells: effect of the coculture with astrocytes.

We have investigated the fatty acid composition of brain capillary endothelial cells cultured alone or in coculture with astrocytes, using an in vitro model in which endothelial cells and astrocytes were grown from one part of a filter to another. We found that the fatty acid composition of the cocultured cerebral endothelial cells was markedly different from that of non-cocultivated endothelial cells. The most striking difference was the increase of arachidonic acid (20:4n-6) at the expense of its precursor, linoleic acid (18:2n-6). Similar modifications were found for the n-3 family of fatty acids with an increase of docosahexaenoic acid (22:6n-3) at the expense of its precursors, but the differences were less than within the n-6 fatty acids. These changes induced by the coculture were observed only in endothelial cell phospholipids, especially the phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine classes, but were not detected in phosphatidylinositols and in other lipid classes. Only the composition of the n-3 series fatty acids was altered in another capillary endothelial cell type (from adrenal cortex) cocultured with astrocytes under the same conditions. The fatty acid changes observed might be biologically relevant as they tended to make the fatty acid composition of the brain capillary endothelial cells more closely resemble that of brain microvessels.

Cross-reactivity of delta 17-6-keto-PGF1 alpha with 6-keto-PGF1 alpha antibodies.

The cross-reactivity of the PGI3 metabolite, delta 17-6-keto-PGF1 alpha, with antibodies against 6-keto-PGF1 alpha for radioimmunoassays (RIA) has been investigated. Delta 17-6-keto-PGF1 alpha was obtained either from commercial sources or after its purification from endothelial cells. In the latter case, primary cultured bovine aortic endothelial cells were incubated for 20 min at 37 degrees C with 10 microM eicosapentaenoic acid (EPA) in the presence of 2 microM 13-hydroperoxy-octadecadienoic acid, and activator of the EPA cyclooxygenation, and the 6-keto-PGF1 alpha and beta 17-6-keto-PGF1 alpha produced were separated by RP-HPLC. Then, cross-reactivities of the commercial and purified beta 17-6-keto-PGF1 alpha with 6-keto-PGF1 alpha antibodies were determined and found not to exceed 10%. In addition, the amounts of prostacyclin-related compounds detected by direct measurements in media of cells loaded with EPA were compared with those obtained after purification of 6-keto-PGF1 alpha. In accordance with the cross-reactivity data, we found that RIA in media mainly measured 6-keto-PGF1 alpha, the beta 17-6-keto-PGF1 alpha formed being undetected at 90%. It is concluded that 6-keto-PGF1 alpha antibodies generally used for RIA of 6-keto-PGF1 alpha are highly specific since they can discriminate a metabolite bearing an additional double band such as the PGI3 metabolite beta 17-6-keto-PGF1 alpha.

Interconversions and distinct metabolic fate of eicosapentaenoic, docosapentaenoic and docosahexaenoic acids in bovine aortic endothelial cells.

The anti-aggregatory activity of endothelial cells being affected by eicosapentaenoic (EPA, 20:5(n-3)) and docosahexaenoic (DHA, 22:6(n-3)) acids, the two main polyunsaturated fatty acids of fish oil, these fatty acids, as well as their intermediary, docosapentaenoic acid (DPA, 22:5(n-3)), were investigated with respect to their metabolism. Primary cultured bovine aortic endothelial cells were supplemented for 22 h at 37 degrees C with either n-3 fatty acid, and the fatty acids of cell media, of cell lipid classes, and of choline and ethanolamine glycerophospholipids (PC and PE) were quantified. Endothelial cells converted each of the three fatty acids into the two others. They were found esterified in cell lipids and partly released in cell media, the respective parts varying according to the fatty acid. For instance, half of the DPA formed from EPA and two third of the EPA formed from DPA were released in the media. Moreover, the DHA formed from EPA and DPA was not esterified but released in media. In addition, the esterified counterparts were found in either PC or PE, depending on whether they were added or formed by conversions. It is concluded that EPA, DPA and DHA are actively interconverted each others, and differ substantially in terms of distribution between media and cells, and within phospholipid classes.

Leupeptin inhibits phospholipases D and C activation in rat hepatocytes.

The relationship between phospholipase D and C activation was studied in intact rat hepatocytes and rat liver plasma membranes. In intact hepatocytes, in the presence of ethanol, vasopressin, phorbol ester, and calcium independently stimulated phosphatidylethanol (PETH) formation, a specific marker of phospholipase D activity. Leupeptin (10-1500 microM) inhibited PETH formation induced by vasopressin, but was ineffective in response to phorbol ester or calcium. Leupeptin also inhibited the formation of inositol phosphates in intact cells in response to vasopressin. In liver plasma membranes, GTP[S] induced the production of phosphatidic acid and, in the presence of ethanol, PETH. Plasma membrane-associated phospholipase D did not require calcium and was insensitive to protein kinase C inhibitors. Leupeptin inhibited PETH formation in response to GTP[S]. The inhibition by leupeptin could be overcome by increasing the concentration of GTP[S]. In plasma membranes, the inhibitory effects of leupeptin on phospholipase D occurred at doses that far exceed those required to maximally inhibit proteolysis. These data highlight a central role for phospholipase C in the activation of phospholipase D, and a minor role for a direct G-protein activation. The findings also demonstrate a novel use of leupeptin as an inhibitor of phospholipases D and C, perhaps at the level of a G protein.

The role of cytosolic Ca2+, protein kinase C, and protein kinase A in hormonal stimulation of phospholipase D in rat hepatocytes.

Ca(2+)-dependent and protein kinase C-dependent mechanisms of phospholipase D (PLD) activation were studied in rat hepatocytes by measuring phosphatidylethanol (Peth) formation in the presence of ethanol. Stimulation of Peth formation by 12-O-tetradecanoyl-phorbol 13-acetate (TPA), vasopressin, or A23187 was inhibited by multiple protein kinase C inhibitors or by protein kinase C down-regulation, indicating that this enzyme is involved in the action of all these agents. A controlled elevation of the cytosolic Ca2+ concentration ([Ca2+]cyt) over the range of 0.1-2.0 microM activated Peth formation in the absence of other agonists. Staurosporin potentiated Ca(2+)-induced Peth formation by shifting the [Ca2+]cyt dose-response curve to the left. Other protein kinase C inhibitors (calphostin C, bisindolylmaleimide) inhibited Ca(2+)-mediated Peth formation, but this inhibition was reduced in staurosporin-treated cells. Okadaic acid potentiated PLD activation by TPA, but suppressed PLD activation by elevated [Ca2+]cyt. Desensitization of TPA-induced PLD activity did not affect PLD activation by Ca2+. These data indicate that [Ca2+]cyt and protein kinase C control distinct pathways of PLD activation, but the Ca(2+)-mediated pathway is suppressed by a staurosporin-sensitive protein kinase. Both mechanisms contribute to vasopressin-induced Peth formation in intact hepatocytes. Activation of protein kinase A enhanced vasopressin-induced Peth formation, but not TPA-stimulated or Ca(2+)-stimulated stimulated Peth formation. Protein kinase A acted by enhancing hormonal Ca2+ mobilization, rather than by directly activating PLD, and thereby shifted the balance of Ca(2+)-dependent and protein kinase C-dependent activation mechanisms of PLD in intact cells.

Platelet inhibitory functions of aortic endothelial cells. Effects of eicosapentaenoic and docosahexaenoic acids.

The endothelial cell platelet inhibitory potential was assessed directly by measuring the platelet inhibition induced by platelet interaction with the cultured aortic endothelial cell. The prostacyclin content of the platelet suspensions after interaction was also quantified. We found that prostacyclin production accounted for the overall platelet inhibitory potential of the aortic cells since: (a) endothelial cells incubated with aspirin, which did not produce prostacyclin, did not inhibit platelets; (b) the prostacyclin content of platelet suspensions after interaction with endothelial cells correlated with the extent of the platelet inhibition; (c) such a platelet inhibition was reproduced by adding synthetic prostacyclin in amount equivalent to that produced by endothelial cells during the interaction. Eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids incorporated into endothelial phospholipids, decreased the ability of the cells to produce prostacyclin and to inhibit platelets, DHA being less effective than EPA.

S-adenosyl-l-methionine inhibits phosphoinositide metabolism in the rat brain synaptosomal suspensions.

S-adenosyl-l-methionine (AdoMet) has been reported to affect events linked to noradrenergic neurotransmission. In the present work, we studied the effect of AdoMet on norepinephrine (NE)-stimulated inositol phosphate production in 3H-inositol-labelled crude synaptosomal suspensions of rat brain. AdoMet (50-1000 microM) decreased both the synthesis of labelled polyphosphoinositide (30-50%) and the release of inositol mono- and bisphosphate (40-50%). The AdoMet effect was not dependent on NE concentration (10-1000 microM), suggesting that the inhibition of inositol phosphate release was not the result of a modification of the norepinephrine binding to its receptor sites. S-adenosyl-L-homocysteine (AdoHcy) (1 mM) an inhibitor of methyltransferase activities, partially inhibited (70%) the AdoMet (0.1 mM) effect, indicating that the methylation processes cannot explain all the effects observed. We conclude that, in addition to previously reported effects of AdoMet on NE transport, AdoMet may reduce NE-linked intracellular signalling.

Effect of guanine nucleotides on polyphosphoinositide synthesis in rat liver plasma membranes.

The effect of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) on PtdIns and PtdIns(4)P kinase activities was measured in rat liver plasma membranes. The addition of [32P]ATP resulted in the rapid incorporation of 32P into PtdIns(4)P and PtdIns(4,5)P2, with maximal levels reached within 30 s. GTP[S] (25-500 microM) increased the rate and magnitude of [32P]PtdIns(4)P and [32P]PtdIns(4,5)P2 formation by 50 and 120% respectively. Similar stimulatory effects were induced by guanosine 5'-[beta gamma-imido]triphosphate, GTP, GDP and guanosine 5'-[beta-thio]diphosphate. The stimulation of PtdIns phosphorylation by GTP[S] occurred in the presence of 2 mM-EGTA, a condition which fully inhibited phosphoinositide-specific phospholipase C. GTP[S] did not stimulate phosphomonoesterase activity, and its action was not due to the binding of magnesium. However, the overall ATP-hydrolysing activity of the membrane preparation was inhibited by GTP[S] and the other guanine nucleotides. There was a direct correlation between the extent of this inhibition and the stimulation of polyphosphoinositide formation. The results indicate that stimulation of polyphosphoinositide formation by guanine nucleotides in rat liver plasma membranes can be accounted for by an inhibition of ATP hydrolysis. These data are inconsistent with a specific GTP-binding protein (G-protein)-mediated stimulation of PtdIns or PtdIns(4)P kinase.