Spatiotemporal analysis of cell response to a rigidity gradient: a quantitative study using multiple optical tweezers

We investigate the dynamic response of single cells to weak and local rigidities, applied at controlled adhesion sites. Using multiple latex beads functionalized with fibronectin, and each trapped in its own optical trap, we study the reaction in real time of single 3T3 fibroblast cells to asymmetrical tensions in the tens of pN · μm⁻¹ range. We show that the cell feels a rigidity gradient even at this low range of tension, and over time develops an adapted change in the force exerted on each adhesion site. The rate at which force increases is proportional to trap stiffness. Actomyosin recruitment is regulated in space and time along the rigidity gradient, resulting in a linear relationship between the amount of recruited actin and the force developed independently in trap stiffness. This time-regulated actomyosin behavior sustains a constant and rigidity-independent velocity of beads inside the traps. Our results show that the strengthening of extracellular matrix-cytoskeleton linkages along a rigidity gradient is regulated by controlling adhesion area and actomyosin recruitment, to maintain a constant deformation of the extracellular matrix.     

Is conspecific substrate marking a long‐term external memory of previously colonized overwintering sites in Harmonia axyridis?

The multicoloured Asian ladybeetle, Harmonia axyridis (Pallas), aggregates inside dwellings during winters to survive the cold. This beetle uses chemical cues coming from congeners to select an overwintering site. Recent research has shown that they preferentially gather at places where conspecifics previously laid a substrate marking made up of saturated and unsaturated hydrocarbons. Some authors have reported that H. axyridis colonizes the same overwintering sites from 1 year to another. Herein, the hypothesis that this substrate marking is used by H. axyridis to settle in the same aggregation sites from one winter to another was tested. To this aim, the temporal modification in the chemical profile of the hydrocarbon marking was studied by performing chromatographic analyses. After 1 year, the overall profile was modified qualitatively and quantitatively: the unsaturated hydrocarbons were no longer detected while some saturated hydrocarbons were still present in large quantities. In a behavioural assay conducted in the laboratory, the 12‐month‐old marking did not induce the aggregation of H. axyridis. This result indicates that the chemical markings left by conspecifics during a previous aggregation period in an overwintering site are not sufficient to induce the gathering of the newly arriving individuals.     

[3H] BOC [NLE28,31]CCK27−33, a new highly labelle ligand for CCK receptors: Binding on brain and on pancreas

Preliminary results on the binding of [³H]Boc[Nle^28,31]CCK_27^?33, designated [³H]Boc[diNle]CCK₇, on mouse brain and rat pancreas membranes are presented. This new ligand for CCK receptors possesses a high specific activity (144 Ci/mmole), and binds in a saturable manner to mouse brain (Kd = 0.49 nM, Bmax = 49 fmoles/mg protein) and rat pancreas (Kd = 4.4 nM, Bmax = 696 fmoles/mg protein). Unlabelled Boc[diNle]CCK₇ displaces [¹²⁵I]CCK₈ from its binding sites on mouse brain membranes with a high affinity, slightly superior to that of CCK₈. The order of potencies to displace [³H]Boc[diNle]CCK₇ from its binding sites was the same on mouse brain and rat pancreas: $\text{[}{}^3\text{HBoc[diNle]CCK}{}_7\text{>}\text{CCK}{}_8\text{,}\text{Boc-CCK}{}_7\text{>}\text{non-sulfated CCK}{}_8$, the pancreas binding sites being more discriminative than the brain binding sites. Thus, [³H]Boc[diNle]CCK₇ is a very promising new probe for the characterization of CCK receptors and their interaction with different CCK fragments.     

Normal human epidermal keratinocytes express in vitro specific molecular forms of (pro)filaggrin recognized by rheumatoid arthritis-associated antifilaggrin autoantibodies.

BACKGROUND: The so-called antikeratin antibodies and the antiperinuclear factor are the most specific serological markers of rheumatoid arthritis (RA). They were recently shown to be largely the same autoantibodies and to recognize human epidermal filaggrins and profilaggrin-related proteins of buccal epithelial cells (collectively referred to as (pro)filaggrin). MATERIALS AND METHODS: To further characterize the target antigens, we investigated their expression by normal human epidermal keratinocytes cultured in differentiating conditions, using immunofluorescence and immunoblotting with RA sera and three different monoclonal antibodies to (pro)filaggrin. RESULTS: On the cornified, stratified epithelial sheets obtained in vitro, RA sera with anti(pro)filaggrin autoantibodies (AFA) produced granular staining of the stratum granulosum and diffuse staining of the stratum corneum. The antigens recognized by RA sera strictly colocalized with (pro)filaggrin in keratohyalin granules. Following sequential extraction of the proteins from the epithelial sheets, the RA sera and the three monoclonal antibodies to (pro)filaggrin, recognized a series of low-salt-soluble molecules, including a neutral/acidic isoform of filaggrin and several proteins with sizes and pI intermediates between this isoform and profilaggrin. They also recognized urea-soluble high-molecular-weight profilaggrin-related molecules. CONCLUSIONS: These results show that in vitro epidermal keratinocytes express various molecular forms of (pro) filaggrin that bear epitopes targeted by AFA of RA sera, and that some of these are absent from epidermis. Moreover, these epitopes, which are present on the keratohyalin granules of buccal epithelial cells but not on those of epidermal cells, are present on the granules of the cultured keratinocytes. This work completes the molecular characterization of the proteins targeted by AFA.     

Occurrence of two cholecystokinin binding sites in guinea-pig brain cortex

Saturation experiments of the highly potent cholecystokinin analogue [3H]Boc(diNle28,31)CCK27-33 ([3H]BNDL-CCK7, 100 Ci/mmol) with guinea pig brain cortex in a large concentration range (0.05 nM to 30 nM) show the presence of two different binding sites (A site: KD = 0.13 nM, Bmax = 35 fmol/mg; B site: KD = 6.4 nM, Bmax = 92 fmol/mg). Both sites exhibit different sensitivity to sodium ions and therefore can be selectively investigated at [3H]BDNL-CCK7 concentration lower than 1 nM for the A site in Tris buffer and in Krebs buffer for the B site. The selectivity factors KIB/KIA of various CCK related peptides vary from 58 for CCK4 to 26 for CCK8 and 4 for the antagonist (Nle28,31) CCK27-32-NH2. The occurrence of two different CCK binding sites in the brain could explain biphasic pharmacological effects of CCK8.     

Conformational analysis of cholecystokinin CCK26-33 and related fragments by 1H NMR spectroscopy, fluorescence-transfer measurements, and calculations

The conformational behavior of CCK7, Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2, and CCK8, Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2, in their sulfated and unsulfated forms, was studied both by 1H NMR spectroscopy in dimethyl-d6 sulfoxide and water and by fluorescence-transfer measurements at pH 7. In neutral conditions, both experimental methods show that these peptides exist preferentially in folded forms with beta and gamma turns around the sequence Gly-Trp-Met-Asp and Met-Asp-Phe-NH2, respectively. The presence of stable folded conformations is supported by through-space effects during the titration of the ionizable groups and by the weak temperature dependency of some amide protons not only in dimethyl sulfoxide but also in water. The folding of the C-terminal part, already shown in CCK5, seems to be a common conformational characteristic in CCK peptides. The N-terminal part of CCK8 presents an equilibrium between beta and gamma turns, whereas this part of the peptide is more flexible in CCK7. The low quantum yield of Tyr and the large mean distance (R = 15 A) between Tyr and Trp, determined by fluorescence-transfer measurements, support the occurrence of folded conformations pushing the aromatic rings far from each other. Interestingly, the introduction of the sulfate group enhances the folding tendency even in aqueous medium. The larger amide temperature dependency and the decrease in the R distance at acidic pH suggest that an intramolecular ionic interaction involving the N-terminal amino group and the beta-carboxyl groups of Asp32 stabilize the folded forms. Metropolis calculations performed on CCK8 support the existence of stable folded conformations closely related to those deduced from experimental data.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophysiological studies with new CCK analogs: correlation with binding affinity on B-type receptors

The electrophysiological effects of Boc-D-Asp-Tyr(SO3H)-Nle-D-Lys-Trp-Nle-Asp-Phe-NH2 (compound I) and Boc-gamma-D-Glu-Tyr(SO3H)-Nle-D-Lys-Trp-Nle-Asp-Phe-NH2 (compound II), two cyclic cholecystokinin analogs with high selectivity for CCK-B receptors, as well as the effects of the linear enzyme-resistant analog Boc-[Nle28,Nle31]-CCK7 (BDNL), were compared with those of CCK8 using extracellular recordings in rat hippocampal slices in vitro. Bath applications of the three synthetic compounds resulted in concentration-dependent and reversible increases in single-unit activity. Comparison of equieffective concentrations yielded the following potency rank order: BDNL greater than CCK8 greater than compound II greater than compound I. There was a close correlation (r = .96, slope = 0.98) between the excitatory activities of the analogs and their potencies in displacing radiolabelled CCK8 from CCK-B receptors on rat brain membranes.     

Mitochondria and aging: dilution is the solution

Metabolic component depletion in model systems results in life-span extension, which has been difficult to reconcile with human metabolic pathologies. Recently, Rea et al. (2007) have shown that mitochondrial electron transport chain RNAi phenotypes in the worm C. elegans are dose dependent, providing an alternative view of mitochondrial function in longevity and metabolic diseases.