Dr DAVID ALSTEENS : NANOMECHANICS OF THE YEAST CELL WALL
Dr. David Alsteens
Postdoctoral researcher of the FNRS
Tel. +32 10 47 36 61
Fax. +32 10 47 20 05
• High-resolution chemical force microscopy of living cells using peak-force tapping
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.
Peak force tapping (PFT) allows researchers to simultaneously image the structure and the physical properties (e.g. elasticity, adhesion) of the sample at high speed and high resolution, in an unprecedented manner. In PFT, the tip is oscillated in the vertical direction with an amplitude of 100-300 nm and at a frequency of 0.25-2 kHz. The z-piezo is driven with a sinusoidal waveform rather than a triangular in conventional force-distance (F-D) curves. This oscillating system allows direct force control of damaging lateral forces, which is very useful for structural imaging of soft samples. In addition, individual force curves can be analyzed to generate adhesion and mechanical maps, to a much better resolution (u to 512 × 512 pixels) than in conventional FV imaging (at best 64 × 64 pixels).
• Unraveling the structure-function relationships of the Candida albicans cell wall
The most prevalent fungal pathogens in humans Candida albicans expresses on its surface Als (Agglutinin-like sequence) proteins which mediate both yeast-to-host tissue adherence and yeast aggregation. Although adhesins often shows weak binding to specific ligands, Als mediate remarkably strong adherence. In collaboration with the Lipke group (Brooklyn College, Brooklyn, New York) and by combining single-molecule atomic force microscopy (AFM) with the tools of genetics and cell biology, we unraveled the various Als protein domains to understand how they synergize to strengthen the cell adhesion and aggregation. The complementary role of the ligand-binding domains, amyloid-forming regions and the less specific hydrophobic-effect tandem repeat domains give raise to a fascinating new mechanism of cell-activation resulting in force-activated clustering of hundreds of adhesion molecules. The amyloid heptapeptide sequence plays a crucial role in this mechanism providing a cohesive strength to the Als protein by forming molecular zipper that mediates protein interactions between cells.
Key related papers:
• "Unzipping a Functional Microbial Amyloid", ACS Nano, 2012
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. Together with the Lipke group (USA), we used single-molecule AFM to unzip amyloids formed by Als cell adhesion proteins from the pathogen Candida albicans. These experiments provide new opportunities to understand the molecular mechanisms driving the cohesion of functional amyloid-based nanostructures.
• "A Role for Amyloid in Cell Aggregation and Biofilm Formation", PLoS One, 2011
Cell adhesion molecules in Saccharomyces cerevisiae and Candida albicans contain amyloid-forming sequences that are highly conserved. We have now used site-specific mutagenesis and specific peptide perturbants to explore amyloid-dependent activity in the Candida albicans adhesin Als5p. A V326N substitution in the amyloid-forming region conserved secondary structure and ligand binding, but abrogated formation of amyloid fibrils in soluble Als5p and reduced cell surface thioflavin T fluorescence. When displayed on the cell surface, Als5p with this substitution prevented formation of adhesion nanodomains and formation of large cellular aggregates and model biofilms. In addition, amyloid nanodomains were regulated by exogenous peptides. An amyloid-forming homologous peptide rescued aggregation and biofilm activity of Als5pV326N cells, and V326N substitution peptide inhibited aggregation and biofilm activity in Als5pWT cells. Therefore, specific site mutation, inhibition by anti-amyloid peturbants, and sequence-specificity of pro-amyloid and anti-amyloid peptides showed that amyloid formation is essential for nanodomain formation and activation.
• "Force-induced formation and propagation of adhesion nanodomains in living fungal cells", Proc. Natl. Acad. Sci. USA, 2010
Understanding how cell adhesion proteins form adhesion domains is a key challenge in cell biology. Here, we use single-molecule atomic force microscopy (AFM) to demonstrate the force-induced formation and propagation of adhesion nanodomains in living fungal cells, focusing on the covalently anchored cell-wall protein Als5p from Candida albicans. We show that pulling on single adhesins with AFM tips terminated with specific antibodies triggers the formation of adhesion domains of 100–500 nm and that the force-induced nanodomains propagate over the entire cell surface. Control experiments (with cells lacking Als5p, single-site mutation in the protein, bare tips, and tips modified with irrelevant antibodies) demonstrate that Als5p nanodomains result from protein redistribution triggered by force-induced conformational changes in the initially probed proteins, rather than from nonspecific cell-wall perturbations. Als5p remodeling is independent of cellular metabolic activity because heat-killed cells show the same behavior as live cells. Using AFM and fluorescence microscopy, we also find that nanodomains are formed within ∼30 min and migrate at a speed of ∼20 nm•min−1, indicating that domain formation and propagation are slow, time-dependent processes. These results demonstrate that mechanical stimuli can trigger adhesion nanodomains in fungal cells and suggest that the force-induced clustering of adhesins may be a mechanism for activating cell adhesion.
• "Unfolding Individual Als5p Adhesion Proteins on Live Cells", ACS Nano, 2009
Elucidating the molecular mechanisms behind the strength and mechanics of cell adhesion proteins is of central importance in cell biology and offers exciting avenues for the identification of potential drug targets. Here we use single-molecule force spectroscopy to investigate the adhesive and mechanical properties of the widely expressed Als5p cell adhesion protein from the opportunistic pathogen Candida albicans. We show that the forces required to unfold individual tandem repeats of the protein are in the 150−250 pN range, both on isolated molecules and on live cells. We also find that the unfolding probability increases with the number of tandem repeats and correlates with the level of cell adherence. We suggest that the modular and flexible nature of Als5p conveys both strength and toughness to the protein, making it ideally suited for cell adhesion. The single-molecule measurements presented here open new avenues for understanding the mechanical properties of adhesion molecules from mammalian and microbial cells and may help us to elucidate their potential implications in diseases such as inflammation, cancer, and infection.
1. Articles published in peer-reviewed journals
1. D. Alsteens, V. Dupres, S. Yunus, J.P. Latgé, J.J. Heinisch, Y.F. Dufrêne, High-Resolution Imaging of Chemical and Biological Sites on Living Cells Using Peak Force Tapping Atomic Force Microscopy. Langmuir, 28 (2012), 16738-16744.
2. A. Beaussart, D. Alsteens, S. El-Kirat-Chatel, P.N. Lipke, S. Kucharíková, P. Van Dijck, Y.F. Dufrêne, Single-Molecule Imaging and Functional Analysis of Als Adhesins and Mannans during Candida albicans Morphogenesis. ACS Nano, (2012), in press.
3. C. Jimeńez-Ortigosa, V. Aimanianda, L. Muszkieta, I. Mouyna, D. Alsteens, S. Pire, R. Beau, S. Krappmann, A. Beauvais, Y.F. Dufrêne, C. Roncero, J.P. Latgé, Chitin synthases with a myosin motor-like domain control the resistance of Aspergillus fumigatus to echinocandins. Antmicrob. Agents Ch., 56 (2012), 6121-6131.
4. J.J Heinisch, P.N. Lipke, A. Beaussart, S. El Kirat Chatel, V. Dupres, D. Alsteens, and Y.F. Dufrene, Atomic force microscopy–looking at mechanosensors on the cell surface. J. Cell Sci. (2012) 125 (2012), 4189-4195.
5. D. Alsteens, C.B. Ramsook, P.N. Lipke, Y.F. Dufrêne, Unzipping a functional microbial amyloid, ACS Nano, 6 (2012), 7703-7711.
6. D. Alsteens, Microbial cells analysis by atomic force microscopy, Methods in Enzymol, 506 (2012), 3-17.
7. P.N. Lipke, M.C. Garcia, D. Alsteens, C.B. Ramsook, S.A. Klotz, Y.F; Dufrêne, Strenghtening relationships: Amyloids create adhesion nanodomains in yeasts, Trends in Microbiol., 20 (2012), 59-65.
8. M.C. Garcia, J.T. Lee, C.B. Ramsook, D. Alsteens, Y.F. Dufrêne, P.N. Lipke, A role for amyloid in cell aggregation and biofilm formation, PLOS One, 6 (2011) e17632.
9. M. Ouberai, F. El Garch, A. Bussiere, M. Riou, D. Alsteens, L. Lins, I. Baussanne, Y. F. Dufrêne, R. Brasseur, J.L. Decout, M.P. Mingeot-Leclercq, The Pseudomonas aeruginosa membranes : a target for a new amphiphilic aminoglycoside derivative?, Biochim. Biophys. Acta-Biomembr., 6 (2011), 1716-1727.
10. D. Alsteens, V. Dupres, G. Andre, Y.F. Dufrêne, Frontiers in microbial nanoscopy, Nanomedicine, 6 (2011), 395-403.
11. D. Alsteens, M.C. Garcia, P.N. Lipke, Y.F. Dufrêne, Force-induced formation and propagation of adhesion nanodomains in living fungal cells. Proc. Natl. Acad. Sci. USA, 107 (2010), 20744-20749. (paper highlighted in PNAS).
12. V. Dupres*, D. Alsteens*, G. Andre, Y.F. Dufrêne, Microbial nanoscopy: a closer look at microbial cell surfaces. Trends Microbiol., 18 (2010), 397-405. * contributed equally
13. J.J. Heinisch, V. Dupres, D. Alsteens, Y.F. Dufrêne, Measurement of the mechanical behavior of yeast membrane sensors using single-molecule atomic force microscopy, Nat. Protocols, 2010, 5, 670-673.
14. D. Alsteens, E. Dague, C. Verbelen, G. Andre, V. Dupres, Y.F. Dufrêne, Nanoscale imaging of microbial pathogens using atomic force microscopy. WIREs Nanomed. Nanobiotechnol., 1 (2009), 168-180.
15. D.J. Müller, M. Krieg, D. Alsteens, Y.F. Dufrêne, New frontiers in atomic force microscopy: analyzing interactions from single-molecules to cells. Curr. Opin. Biotechnol., 20 (2009), 4-13.
16. D.J. Müller, J. Helenius, D. Alsteens, Y.F. Dufrêne, Force probing surfaces of living cells to molecular resolution. Nat. Chem. Biol., 5 (2009), 383-390
17. V. Dupres, D. Alsteens, K. Pauwels, Y.F. Dufrêne, In Vivo imaging of S-layer nanoarrays on Corynebacterium glutamicum, Langmuir, 2009, 25, 9653-9655.
18. V. Dupres, D. Alsteens, G. Andre, C. Verbelen, Y.F. Dufrêne, Fishing single molecules on live cells, Nanotoday, 2009, 4, 262-268.
19. V. Dupres*, D. Alsteens*, S. Wilk, B. Hansen, J.J. Heinisch, Y.F. Dufrêne, The yeast Wsc1 cell surface sensor behaves like a nanospring in vivo, Nat. Chem. Biol., 2009, 5, 857-862. * contributed equally (Science editors' choice + highlight in the Editorial of the issue and in Nat. Rev. Microbiol.)
20. D. Alsteens, E. Pesavento, G. Cheuvart, V. Dupres, H. Trabelsi, P. Soumillion, Y.F. Dufrêne, Controlled manipulation of bacteriophages using single-virus force spectroscopy, ACS Nano, 2009, 3, 3063-3068.
21. D. Alsteens, V. Dupres, S.A. Klotz, N.K. Gaur, P.N. Lipke, Y.F. Dufrêne, Unfolding individual Als5p adhesion proteins on live cells, ACS Nano, 2009, 3, 1677-1682.
22. G. Francius*, D. Alsteens*, V. Dupres, S. Lebeer, S. De Keersmaecker, J. Vanderleyden, H.J. Gruber, Y.F. Dufrêne, Stretching polysaccharides on live cells using single molecule force spectroscopy, Nat. Protocols, 2009, 4, 939-946. *contributed equally
23. C. Verbelen, N. Christiaens, D. Alsteens, V. Dupres, A.R. Baulard, Y.F. Dufrêne, Molecular mapping of Lipoarabinomannans on Mycobacteria, Langmuir, 2009, 25, 4324-4327.
24. E. Dague, D. Alsteens, J.-P. Latgé, Y.F. Dufrêne, High-resolution cell surface dynamics of germinating Aspergillus fumigatus conidia, Biophys. J., 94 (2008), 656-660 (paper highlighted in Biophotonics International, Dec 07 issue).
25. G. Francius, S. Lebeer, D. Alsteens, L. Wilding, H. J. Gruber, P. Hols, S. De Keersmaecker, J. Vanderleyden, Y. Dufrêne, Detection, localization, and conformational analysis of single polysaccharide molecules on live bacteria, ACS Nano, 2 (2008), 1921-1929 (paper highlighted in the Editorial of the issue).
26. D. Alsteens, C. Verbelen, E. Dague, D. Raze, A.R. Baulard, Y.F. Dufrêne, Organization of the mycobacterial cell wall: a nanoscale view, Pflüg. Arch. Eur. J. Phy., 456 (2008), 117-125.
27. D. Alsteens, V. Dupres, K. Mc Evoy, L. Wildling, H.J. Gruber, Y.F. Dufrêne, Structure, cell wall elasticity and polysaccharide properties of living yeast cells, as probed by AFM, Nanotechnology, 2008, 19, 9.
28. E. Dague, Y. Gilbert, C. Verbelen, G. Andre, D. Alsteens, Y.F. Dufrêne, Towards a nanoscale view of fungal surfaces, Yeast, 24 (2007), 229-237.
29. D. Alsteens, E. Dague, C. Verbelen, G. Andre, G. Francius, Y.F. Dufrêne, Nanomicrobiology, Nanoscale Res. Lett., 2 (2007), 365-372.
30. E. Dague*, D. Alsteens*, J.P. Latgé, C. Verbelen, D. Raze, A.R. Baulard, Y.F. Dufrêne, Chemical force microscopy of single live cells, Nano Lett., 7 (2007), 3026-3030. *contributed equally (paper highlighted in Nature Methods).
31. D. Alsteens, E., Dague, P.G., Rouxhet, A.R., Baulard, Y.F. Dufrêne, Direct measurement of hydrophobic forces on cell surfaces using AFM, Langmuir, 23 (2007), 11977-11979.
2. Book Chapters
1. D. Alsteens, Y.F. Dufrêne, Stretching and imaging individual proteins on live cells using atomic force microscopy. In: A.F. Oberhauser (Editor), Single-molecule Studies of Proteins, Biophysics for the Life Sciences 2, Springer, New York (2013), ch. 8, 211-233.
2. E. Dague, A. Beaussart, D. Alsteens, Nanomechanics of yeast surfaces revealed by AFM. In: B. Bushan (Editor), Scanning probe microscopy in nanoscience and nanotechnology, , Springer-Verlag, Berlin Heidelberg (2013), ch. 7, 171-193.
3. C. Verbelen, V. Dupres, D. Alsteens, G. Andre, Y.F. Dufrêne, Single-molecule force spectroscopyof microbial cell envelope proteins. In: Y.F. Dufrêne (Editor), Life at the nanoscale, Pan Stanford Publishing, (2011), ch.15, 317-334.
4. D. Alsteens, V. Dupres, E. Dague, C. Verbelen, G. Andre, G. Francius, Y.F. Dufrêne, Imaging chemical groups and molecular recognition sites on live cells using AFM. In: B. Bhushan, H. Fuchs (Editors), Applied scanning probe methods XII, Springer-Verlag, Berlin Heidelberg (2009), ch. 10, 33-48.
5. A. Ebner, L. Chtcheglova, J. Tang, D. Alsteens, V. Dupres, Y.F. Dufrêne, P. Hinterdorfer, Recognition imaging using atomic force microscopy. In: P. Hinterdorfer, A. van Oijen (Editors), Handbook of Single-Molecule Biophysics, Springer-Verlag, New York Inc. (2009), ch. 18, 525-554.
6. C. Verbelen, G. Andre, X. Haulot, Y. Gilbert, D. Alsteens, E. Dague, Y.F. Dufrêne, Towards a Nanoscale View of Microbial Surfaces, Using the Atomic Force Microscope. In: B. Bhushan, H. Fuchs, M. Tomitori (Editors), Applied scanning probe methods IX, Springer-Verlag, Berlin Heidelberg (2008), ch. 17, 111-126.
LBRNA 2102 Caractérisation de surface des matériaux