16 research outputs found
Single-cell fluidic force microscopy reveals stress- dependent molecular interactions in yeast mating
Sexual agglutinins of the budding yeast Saccharomyces cerevisiae are proteins mediating cell aggregation during mating. Complementary agglutinins expressed by cells of opposite mating types “a” and “α” bind together to promote agglutination and facilitate fusion of haploid cells. By means of an innovative single-cell manipulation assay combining fluidic force microscopy with force spectroscopy, we unravel the strength of single specific bonds between a- and α-agglutinins (~100 pN) which require pheromone induction. Prolonged cell–cell contact strongly increases adhesion between mating cells, likely resulting from an increased expression of agglutinins. In addition, we highlight the critical role of disulfide bonds of the a- agglutinin and of histidine residue H273 of α-agglutinin. Most interestingly, we find that mechanical tension enhances the interaction strength, pointing to a model where physical stress induces conformational changes in the agglutinins, from a weak-binding folded state, to a strong-binding extended state. Our single-cell technology shows promises for under- standing and controlling the complex mechanism of yeast sexuality
Unzipping a Functional Microbial Amyloid
Bacterial and fungal species produce some of the best-characterized functional amyloids, that is, extracellular fibres that play key roles in mediating adhesion and biofilm formation. Yet, the molecular details underlying their mechanical strength remain poorly understood. Here, we use single-molecule atomic force microscopy to measure the mechanical properties of amyloids formed by Als cell adhesion proteins from the pathogen <i>Candida albicans</i>. We show that stretching Als proteins through their amyloid sequence yields characteristic force signatures corresponding to the mechanical unzipping of β-sheet interactions formed between surface-arrayed Als proteins. The unzipping probability increases with contact time, reflecting the time necessary for optimal inter β-strand associations. These results demonstrate that amyloid interactions provide cohesive strength to a major adhesion protein from a microbial pathogen, thereby strengthening cell adhesion. We suggest that such functional amyloids may represent a generic mechanism for providing mechanical strength to cell adhesion proteins. In nanotechnology, these single-molecule manipulation experiments provide new opportunities to understand the molecular mechanisms driving the cohesion of functional amyloid-based nanostructures
New routes to soluble magnesium amidoborane complexes
Invasive bacterial pathogens can capture host plasminogen (Plg) and allow it to form plasmin. This process is of medical importance as surface-bound plasmin promotes bacterial spread by cleaving tissue components and favors immune evasion by degrading opsonins. In Staphylococcus aureus, Plg binding is in part mediated by cell surface fibronectin-binding proteins (FnBPs), but the underlying molecular mechanism is not known. Here, we use single-cell and single-molecule techniques to demonstrate that FnBPs capture Plg by a sophisticated activation mechanism involving fibrinogen (Fg), another ligand found in the blood. We show that while FnBPs bind to Plg through weak (∼200-pN) molecular bonds, direct interaction of the adhesins with Fg through the high-affinity dock, lock, and latch mechanism dramatically increases the strength of the FnBP-Plg bond (up to ∼2,000 pN). Our results point to a new model in which the binding of Fg triggers major conformational changes in the FnBP protein, resulting in the buried Plg-binding domains being projected and exposed away from the cell surface, thereby promoting strong interactions with Plg. This study demonstrated a previously unidentified role for a ligand-binding interaction by a staphylococcal cell surface protein, i.e., changing the protein orientation to activate a cryptic biological function.IMPORTANCEStaphylococcus aureus captures human plasminogen (Plg) via cell wall fibronectin-binding proteins (FnBPs), but the underlying molecular mechanism is not known. Here we show that the forces involved in the interaction between Plg and FnBPs on the S. aureus surface are weak. However, we discovered that binding of fibrinogen to FnBPs dramatically strengthens the FnBP-Plg bond, therefore revealing an unanticipated role for Fg in the capture of Plg by S. aureus These experiments favor a model where Fg-induced conformational changes in FnBPs promote their interaction with Plg. This work uncovers a previously undescribed activation mechanism for a staphylococcal surface protein, whereby ligand-binding elicits a cryptic biological function
Single-Cell and Single-Molecule Analysis Deciphers the Localization, Adhesion, and Mechanics of the Biofilm Adhesin LapA
The large adhesin protein LapA mediates
adhesion and biofilm formation
by <i>Pseudomonas fluorescens</i>. Although adhesion is
thought to involve the long multiple repeats of LapA, very little
is known about the molecular mechanism by which this protein mediates
attachment. Here we use atomic force microscopy to unravel the biophysical
properties driving LapA-mediated adhesion. Single-cell force spectroscopy
shows that expression of LapA on the cell surface <i>via</i> biofilm-inducing conditions (<i>i.e.</i>, phosphate-rich
medium) or deletion of the gene encoding the LapG protease (LapA+
mutant) increases the adhesion strength of <i>P. fluorescens</i> toward hydrophobic and hydrophilic substrates, consistent with the
adherent phenotypes observed in these conditions. Substrate chemistry
plays an unexpected role in modulating the mechanical response of
LapA, with sequential unfolding of the multiple repeats occurring
only on hydrophilic substrates. Biofilm induction also leads to shortening
of the protein extensions, reflecting stiffening of their conformational
properties. Using single-molecule force spectroscopy, we next demonstrate
that the adhesin is randomly distributed on the surface of wild-type
cells and can be released into the solution. For LapA+ mutant cells,
we found that the adhesin massively accumulates on the cell surface
without being released and that individual LapA repeats unfold when
subjected to force. The remarkable adhesive and mechanical properties
of LapA provide a molecular basis for the “multi-purpose”
adhesion function of LapA, thereby making <i>P. fluorescens</i> capable of colonizing diverse environments
High-Resolution Imaging of Chemical and Biological Sites on Living Cells Using Peak Force Tapping Atomic Force Microscopy
Currently, there is a growing need for methods that can
quantify
and map the molecular interactions of biological samples, both with
high-force sensitivity and high spatial resolution. Force–volume
imaging is a valuable atomic force microscopy (AFM) modality for probing
specific sites on biosurfaces. However, the low speed and poor spatial
resolution of this method have severely hampered its widespread use
in life science research. We use a novel AFM mode (i.e., peak force
tapping with chemically functionalized tips) to probe the localization
and interactions of chemical and biological sites on living cells
at high speed and high resolution (8 min for 1 μm × 1 μm
images at 512 pixels × 512 pixels). First, we demonstrate the
ability of the method to quantify and image hydrophobic forces on
organic surfaces and on microbial pathogens. Next, we detect single
sensor proteins on yeast cells, and we unravel their mechanical properties
in relation to cellular function. Owing to its key capabilities (quantitative
mapping, resolution of a few nanometers, and true correlation with
topography), this novel biochemically sensitive imaging technique
is a powerful complement to other advanced AFM modes for quantitative,
high-resolution bioimaging
Mechanical Forces Guiding Staphylococcus aureus Cellular Invasion
Staphylococcus aureus can invade
various types of mammalian cells, thereby enabling it to evade host
immune defenses and antibiotics. The current model for cellular invasion
involves the interaction between the bacterial cell surface located
fibronectin (Fn)-binding proteins (FnBPA and FnBPB) and the α5β1
integrin in the host cell membrane. While it is believed that the
extracellular matrix protein Fn serves as a bridging molecule between
FnBPs and integrins, the fundamental forces involved are not known.
Using single-cell and single-molecule experiments, we unravel the
molecular forces guiding S. aureus cellular
invasion, focusing on the prototypical three-component FnBPA–Fn–integrin
interaction. We show that FnBPA mediates bacterial adhesion to soluble
Fn <i>via</i> strong forces (∼1500 pN), consistent
with a high-affinity tandem β-zipper, and that the FnBPA–Fn
complex further binds to immobilized α5β1 integrins with
a strength much higher than that of the classical Fn–integrin
bond (∼100 pN). The high mechanical stability of the Fn bridge
favors an invasion model in which Fn binding by FnBPA leads to the
exposure of cryptic integrin-binding sites <i>via</i> allosteric
activation, which in turn engage in a strong interaction with integrins.
This activation mechanism emphasizes the importance of protein mechanobiology
in regulating bacterial–host adhesion. We also find that Fn-dependent
adhesion between S. aureus and endothelial
cells strengthens with time, suggesting that internalization occurs
within a few minutes. Collectively, our results provide a molecular
foundation for the ability of FnBPA to trigger host cell invasion
by S. aureus and offer promising prospects
for the development of therapeutic approaches against intracellular
pathogens