Oligomers and fibrils formation differentiated by ThT fluorescence.

<p>ThT fluorescence intensity was monitored to follow fibrillogenesis of Aβ(1–40) and Aβ(1–40)E22G in the presence and in the absence of 2 mM Ca²⁺. Black bars, Aβ(1–40) in phosphate buffer (“–Ca²⁺ condition”); light grey bars, Aβ(1–40) in 2 mM CaCl₂; dark grey bars, Aβ(1–40)E22G in phosphate buffer; light blue bars, Aβ(1–40)E22G in CaCl₂. Shown are averages of values obtained in four independent experiments; error bars indicating the standard error of the average.</p

ATR-FTIR spectra of Aβ(1–40) and Aβ(1–40)E22G.

<p>FTIR spectra of Aβ(1–40) and Aβ(1–40)E22G were taken in the presence and in the absence of added Ca²⁺, showing the amide I region of the spectra (1600–1700 cm⁻¹). Aliquots of 2 µl were taken from each sample at <i>t</i> = 0, 2, 6, 24, 48, 72, and 96 h (shown in blue, green, red, cyan, purple, mustard, and dark blue, respectively). The data shown here were collected in one continuous experiment and are representative of three independent trials.</p

Single-molecule force spectroscopy experiments unravel the nanomechanics of microbial adhesins.

<p>Series of force-distance profiles obtained by stretching adhesins from various microbial species (see text for details): single adhesion peaks reflecting specific recognition (A), sawtooth patterns with multiple force peaks corresponding to the force-induced unfolding of protein secondary structures (B), constant force plateaus originating from the mechanical unzipping of amyloid interactions formed between multiple adhesins (C), and single large adhesion force peaks with linear shapes obtained by pulling on Gram-positive bacterial pili (D). The arrows emphasize the characteristic force peaks in each case.</p

Morphological comparison of Aβ(1–40) and Aβ(1–40)E22G.

<p>Contact mode AFM images (5 µm × 5 µm, Z scale 15 nm) of Aβ(1–40) and Aβ(1–40)E22G peptides on mica, recorded either in phosphate buffer or in MOPS buffer with Ca²⁺. Samples of Aβ(1–40) and Aβ(1–40)E22G in the presence and absence of added Ca²⁺ (marked as “+Ca²⁺” or “−Ca²⁺”, respectively) at <i>t</i> = 0, 6, or 72 h. Closer views (1 µm × 1 µm, Z scale 15 nm) of oligomers, protofibrils and fibrils are shown as insets in the panel of <i>t</i> = 72 h (C, F, I, L). Images A, D, G, J were taken at <i>t</i> = 0; images B, E, H, K were taken at <i>t</i> = 6 h. Peptide concentration was the same in all samples.</p

Subcellular localization of OatA^TM10-YFP and OatB^TM10-YFP fusions.

<p>A) Schematic representation of fusion proteins (YFP, yellow star) and corresponding micrographs in phase contrast (PC) microscopy and fluorescence microscopy (YFP). Upper panels, <i>oatA</i> mutant producing cytoplasmic YFP (OatA^−/YFP) used as control; middle panels, <i>oatA</i> mutant producing OatA^TM1–10-YFP (OatA^−/OatA^TM1–10-YFP); lower panels, <i>oatB</i> mutant producing OatB^TM1–10-YFP (OatB^−/OatB^TM1–10-YFP). Induction of expression was performed with 10 ng/ml of nisin. Bar scale, 2.0 µm. B) Fluorescence ratio (FR; AU, arbitrary unit) between the fluorescence measured at mid-cell position and pole. A^−/A™, <i>oatA</i> mutant producing OatA^TM1–10-YFP and B^−/B™, <i>oatB</i> mutant producing OatB^TM1–10-YFP. Lines represent the mean value (n = 20, 3 independent replicates).</p

Morphological aberrations induced by the overproduction of different variants of OatA in the <i>oatA</i> mutant.

<p>Induction was performed with 20 ng/ml of nisin. A) Selection of cells showing curvature (labeled C), asymmetrical septation (labeled A), dual septation (labeled D) observed in bright field (BF) or phase contrast (PC) microscopy and fluorescent microscopy (FM4–64, membrane staining). A^−/A⁺⁺⁺, <i>oatA</i> mutant overexpressing <i>oatA</i>^WT; A^−/A^{* +++}, <i>oatA</i> mutant overexpressing <i>oatA</i>^D510A/S511A; A^−/A™⁺⁺⁺, <i>oatA</i> mutant overexpressing <i>oatA</i>^TM1–10<i>::yfp</i>. Bar scale, 2.0 µm. B) Selection of two tripartite cells (I and II) showing a central mini-cell (labeled MC) resulting from a dual septation event observed in bright field (BF) microscopy and fluorescent microscopy (YFP, OatA^TM1–10-YFP fluorescence; DAPI, DNA staining).</p

Effect of expression of <i>min::yfp</i> fusions on cell morphology and subcellular localization.

<p>Effect of expression of <i>minC::yfp</i> (A, MinC-YFP) and <i>minD::yfp</i> (B, MinD-YFP) in <i>L. plantarum</i> wild-type (WT) and <i>oatA</i> mutant (OatA⁻) without nisin induction (0 ng/ml) and with 2.5 ng/ml of nisin. Micrographs were obtained in bright field (BF) microscopy and fluorescence microscopy (YFP). For MinD-YFP (nisin 2.5 ng/ml), minicells are indicated by arrows in WT and three selected branched cells (insets) are added for OatA⁻. Bar scale, 2.0 µm.</p

Study of the uncoupling between elongation and septation phases by time-lapse experiments.

<p>A) Cell length (µm) of the mother cell during the division process for the wild type (solid line) and the <i>oatA</i> mutant (dashed line). Each line corresponds to one individual cell. Time (min) was arbitrarily fixed to zero at the last view before any detectable cell invagination in bright field. Correspondences between time and division state are drawn upside of the graph for the wild type. One representative time-lapse experiment of three independent experiments for each strain (n ≥3 for each). All examined cells of the wild type and the <i>oatA</i> mutant (n = 15 for each) display their respective phenotype. B) Cell length increase (µm) measured during the division process after 5 min (T10–T5; T5, initial invagination), 10 min (T15–T5), and 15 min (T20–T5; T20, final septation). Time intervals are reported in panel A. Symbols: WT, wild-type; A⁻, <i>oatA</i> mutant; A^−/A⁺<i>oatA</i> mutant complemented with <i>oatA</i>^WT; A^−/A^*<i>oatA</i> mutant complemented with <i>oatA</i>^D510A/S511A; A^−/A™, <i>oatA</i> mutant complemented with <i>oatA</i>^TM1–10<i>::yfp</i>. All the variants of <i>oatA</i> are expressed at low basal level in absence of the nisin inducer. Mean values of 5 cells in each time-lapse experiment. Significance based on <i>t</i>-test; **, p-value <0.01.</p

Bacterial strains and plasmids.

#<p>Cm^r and Amp^r indicate resistance to chloramphenicol and ampicillin, respectively.</p

Temporal localization of OatA^TM1–10-YFP during the cell cycle.

<p>Upper panels, selection of cells at different division stages observed in bright field (BF) microscopy and fluorescent microscopy (FM4–64, membrane staining; YFP, OatA^TM1–10-YFP fluorescence). Induction of <i>oatA^TM1–10::yfp</i> expression was performed with 10 ng/ml of nisin. I, septal localization of the YFP fusion prior to membrane invagination; II, co-localization of the YFP fusion and the septal membrane; III, polar localization at the end of septation; IV, reinitiation of septal localization in daughter cells. Bar scale, 1.0 µm. Lower panel, schematic representation of the cell cycle. Colors and numbers refer to above micrographs. Yellow represent a merge between FM4–64 and YFP fluorescences.</p