Tensegrity behaviour of cortical and cytosolic cytoskeletal components in twisted living adherent cells

The present study is an attempt to relate the multicomponent response of the cytoskeleton (CSK), evaluated in twisted living adherent cells, to the heterogeneity of the cytoskeletal structure - evaluated both experimentally by means of 3D reconstructions, and theoretically considering the predictions given by two tensegrity models composed of (four and six) compressive elements and (respectively 12 and 24) tensile elements. Using magnetic twisting cytometry in which beads are attached to integrin receptors linked to the actin CSK of living adherent epithelial cells, we specifically measured the elastic CSK response at quasi equilibrium state and partitioned this response in terms of cortical and cytosolic contributions with a two-component model (i.e., a series of two Voigt bodies). These two CSK components were found to be prestressed and exhibited a stress-hardening response which both characterize tensegrity behaviour with however significant differences: compared to the cytosolic component, the cortical cytoskeleton appears to be a faster responding component, being a less prestressed and easily deformable structure. The discrepancies in elastic behaviour between the cortical and cytosolic CSK components may be understood on the basis of prestress tensegrity model predictions, given that the length and number of constitutive actin elements are taken into account.     

Recycling of RNA binding iron regulatory protein 1 into an aconitase after nitric oxide removal depends on mitochondrial ATP

Iron regulatory proteins (IRPs) control iron metabolism by specifically interacting with iron-responsive elements (IREs) on mRNAs. Nitric oxide (NO) converts IRP-1 from a [4Fe-4S] aconitase to a trans-regulatory protein through Fe-S cluster disassembly. Here, we have focused on the fate of IRE binding IRP1 from murine macrophages when NO flux stops. We show that virtually all IRP-1 molecules from NO-producing cells dissociated from IRE and recovered aconitase activity after re-assembling a [4Fe-4S] cluster in vitro. The reverse change in IRP-1 activities also occurred in intact cells no longer exposed to NO and did not require de novo protein synthesis. Likewise, inhibition of mitochondrial aconitase via NO-induced Fe-S cluster disassembly was also reversed independently of protein translation after NO removal. Our results provide the first evidence of Fe-S cluster repair of NO-modified aconitases in mammalian cells. Moreover, we show that reverse change in IRP-1 activities and repair of mitochondrial aconitase activity depended on energized mitochondria. Finally, we demonstrate that IRP-1 activation by NO was accompanied by both a drastic decrease in ferritin levels and an increase in transferrin receptor mRNA levels. However, although ferritin expression was recovered upon IRP-1-IRE dissociation, expression of transferrin receptor mRNA continued to rise for several hours after stopping NO flux.     

Low molecular weight peptides restore the procoagulant activity of factor VIII in the presence of the potent inhibitor antibody ESH8

Following repeated administration of factor VIII (FVIII), a significant number of hemophilia A patients develop antibodies (Abs), inhibiting the procoagulant activity of infused FVIII. We have designed an approach based on the blocking of the deleterious activity of these Abs by peptide decoys mimicking the anti-FVIII Ab epitopes. Here, the well characterized inhibitory monoclonal Ab ESH8 served as a model. Several phage peptide libraries were screened for specific binding to ESH8. Seven constrained dodecapeptide sequences were obtained. Six sequences carried the consensus motif, hydrophobic-(Y/F)GKTXL. This motif showed a certain similarity with the (2231)QVDFQKTMKV(2240) sequence of the C(2) domain. In the seventh sequence, YCNPSIGDKNCR, the residues GDKN are similar to the sequence (2267)DGHQ(2270). Upon inspection of the C(2) domain crystallographic structure, the two stretches QVDFQKTMKV and DGHQ appeared close together in space and might constitute a discontinuous epitope. Corresponding synthetic peptides were able to inhibit the binding of ESH8 to FVIII in a specific and dose-dependent manner. Moreover, the ability of the selected peptides to neutralize the inhibitory activity of ESH8 was demonstrated in functional tests as well as in vivo in a murine model of hemophilia A. This study demonstrates the potential of this approach to neutralize the activity of potent inhibitory Abs.     

Genetic control of extracellular protease synthesis in the yeast Yarrowia lipolytica

Depending on the pH of the growth medium, the yeast Yarrowia lipolytica secretes an acidic protease or an alkaline protease, the synthesis of which is also controlled by carbon, nitrogen, and sulfur availability, as well as by the presence of extracellular proteins. Previous results have indicated that the alkaline protease response to pH was dependent on YlRim101p, YlRim8p/YlPalF, and YlRim21p/YlPalH, three components of a conserved pH signaling pathway initially described in Aspergillus nidulans. To identify other partners of this response pathway, as well as pH-independent regulators of proteases, we searched for mutants that affect the expression of either or both acidic and alkaline proteases, using a YlmTn1-transposed genomic library. Four mutations affected only alkaline protease expression and identified the homolog of Saccharomyces cerevisiae SIN3. Eighty-nine mutations affected the expression of both proteases and identified 10 genes. Five of them define a conserved Rim pathway, which acts, as in other ascomycetes, by activating alkaline genes and repressing acidic genes at alkaline pH. Our results further suggest that in Y. lipolytica this pathway is active at acidic pH and is required for the expression of the acidic AXP1 gene. The five other genes are homologous to S. cerevisiae OPT1, SSY5, VPS28, NUP85, and MED4. YlOPT1 and YlSSY5 are not involved in pH sensing but define at least a second protease regulatory pathway.     

B cell positive selection by soluble self-antigen

It is well established that autoreactive B cells undergo negative selection. This stands in paradox with the high frequency of so-called natural autoreactive B cells producing low affinity polyreactive autoantibodies with recurrent specificities, suggesting that these B cells are selected on the basis of their autoreactivity. We previously described two transgenic mouse lines (with and without IgD) producing a human natural autoantibody (nAAb) that binds ssDNA and human Fcgamma. In the absence of human IgG, nAAb-transgenic B cells develop normally. By crossing these mice with animals expressing knockin chimeric IgG with the human Fcgamma, we now show that the constitutive expression of chimeric IgG promotes the increase of nAAb-expressing B cells. This positive selection is critically dependent on the presence of IgD, occurs in the spleen, and concerns all mature B cell subsets, with a relative preferential enrichment of marginal zone B cells. These data support the view that soluble self-Ags can result in positive clonal selection.     

Cloning and characterization of a phospholipase C from the C(4) plant Digitaria sanguinalis

As a PLC activity was implicated in the light transduction pathway that controls C(4) photosynthesis in Digitaria sanguinalis, a full length PLC cDNA (DsPLC2) was cloned. The proteins encoded by the two possible open reading frames were produced in Escherichia coli; they both harbour a PLC activity but with different response to Ca(2+) concentration, and with different sensitivity to the PLC inhibitor U-73122.