Biochemical composition of outer layers.

<p>Panel A. Oil red staining of fibres. Silk fibres were treated with water (upper filament) or ether extracted (lower filament) followed by oil red staining. Bar corresponds to 2000 nm. Panel B. Concavalin A (Con A) staining of fibres. Fibres gently washed in phosphate buffered saline or vigorously washed in 0.1% Triton X-100 were reacted with biotinylated Con A. The presence of α-methyl mannoside (α-MM), an inhibitor of Con A, is indicated by “I”. Bound Con A was visualised by gold conjugated streptavidin and SEM. The bar equals 500 nm.</p

Solubilisation of polymerised spidroins.

<p>Dragline silk from <i>N. clavipes</i> was incubated with the indicated solvents and the soluble fraction was analysed by gel-electrophoresis followed by Coomassie staining (A) and western blotting employing antibodies S1Rx and enhanced luminescence (ECL) for detection. The molecular weights of marker proteins are indicated in kilo Daltons (kDa).</p

Protein composition of silk layers.

<p>Filter strips obtained by western blotting and loaded with material extracted from the indicated fibre layers were stained with Ponceau S (Pon) or reacted with Concavalin A (ConA), pre-immune serum (PIS), S1Rx, S2Rx and S-pbs. The running position of a 200 kDa marker is indicated by the lines.</p

Compositions and functions of silk layers from the dragline of <i>N. clavipes</i>

<p>MiSp: minor ampullate spidroin; MaSp: major ampullate spidroin</p

Treatment of fibres with chaotropic agents and acids.

<p>Panel A. Light Microscopy (LM) phase images of fibres treated with mixtures of different ratios between HAc and HCl. Bar corresponds to 4000 nm. Minor (mi, reinforcement) and major (ma, dragline) ampullate fibres are indicated. Panel B. Light Microscopy (LM) phase contrast images of dragline treated with LiSCN at the stated concentrations for 1 minute. Bar corresponds to 8000 nm. Panel C. Scanning Electron Microscope (SEM) images of dragline incubated for the indicated times with 99% Hfip. Bar corresponds to 1000 nm. Panel D. Video contrast images of draglines (left unstressed, right fractured by applying stress) soaked in 8 M urea and counterstained with Coomassie Brilliant Blue. Bar equals 4000 nm.</p

Localization of components.

<p>Panel A. Fluorescence labelling of silk fibres. Reactivities of Concavalin A (ConA), pre-immune serum (PIS), S-pbs, S1Rx and S2Rx to lesions of vigorously washed silk filaments. Phase contrast (Ph), immunfluorescence (IF) and merged images are shown. The bar indicates 4000 nm. Panel B. Immunogold staining of silk fibre cross-sections. Shown are transmission electron microscopy (TEM) images of dragline cross sections reacted with Concavalin A (ConA) or the indicated antibodies. The bar equals 200 nm.</p

Model of the multilayer organisation of dragline silk.

<p>A dragline can be divided into a shell and a core with five major layers of different material composition (from exterior to interior): a lipid coat, a glyco coat, a skin, an outer core and inner core. The approximate relative extensions of each layer are indicated.</p

S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci

<p>The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is an essential process in maintenance of chromosomal stability. A key player of HR is the strand exchange factor RAD51 whose assembly at sites of DNA damage is tightly regulated. We detected an endogenous complex of RAD51 with the calcium-binding protein S100A11, which is localized at sites of DNA repair in HaCaT cells as well as in normal human epidermal keratinocytes (NHEK) synchronized in S phase. In biochemical assays, we revealed that S100A11 enhanced the RAD51 strand exchange activity. When cells expressing a S100A11 mutant lacking the ability to bind Ca²⁺, a prolonged persistence of RAD51 in repair sites and nuclear γH2AX foci was observed suggesting an incomplete DNA repair. The same phenotype became apparent when S100A11 was depleted by RNA interference. Furthermore, down-regulation of S100A11 resulted in both reduced sister chromatid exchange confirming the restriction of the recombination capacity of the cells, and in an increase of chromosomal aberrations reflecting the functional requirement of S100A11 for the maintenance of genomic stability. Our data indicate that S100A11 is involved in homologous recombination by regulating the appearance of RAD51 in DSB repair sites. This function requires the calcium-binding activity of S100A11.</p