Alsteens David

 Research project of Dr David Alsteens

Nanomechanics of the yeast cell wall

Unraveling the structure-function relationships of the yeast cell wall is a major challenge in current microbiology and offers exciting prospects in biomedicine. A key question is to understand how cell wall proteins respond to mechanical force and how this response could be related to function. I am interested in gaining insight into the nanoscale surface properties of the yeast cell wall, with an emphasis on the cell adhesion protein Als5p from the pathogen Candida albicans. The methodology involves developing atomic force microscopy (AFM) techniques for live cell analysis [1, 2], in combination with genetics and cell biology tools.

In collaboration with the Lipke group (Brooklyn College, Brooklyn, New York), we recently used AFM to unfold single Als5p cell adhesion proteins, both in vitro and in vivo [3]. We found that force extension curves for isolated Als proteins showed sawtooth patterns with well-defined force peaks, each peak corresponding to the force-induced unfolding of an individual tandem repeat engaged in cell-cell aggregation. The modular and flexible nature of Als may convey both strength and toughness to the protein, making it ideally-suited for cell adhesion. Next we used single-molecule AFM to investigate the clustering of Als5p proteins in live cells [4]. Remarkably, we found that mechanical stimuli can trigger the formation and propagation of adhesion nanodomains in live cells (Figure). Pulling on single Als adhesins with AFM tips terminated with specific antibodies was shown to induce the formation of nanoadhesomes, i.e. adhesion domains of 100-500 nm size. In addition, the force-induced nanodomains were found to propagate over the entire cell surface. This study suggests that clustering of cell adhesion proteins in response to mechanical stimuli may be a general mechanism for activating cell adhesion in pathogens and offer exciting prospects in therapeutics for developing new antimicrobial strategies. 

Imaging the dynamic clustering of cell adhesion proteins on the surface of a living cell. A) Single C. albicans adhesins were localized and stretched using an AFM tip bearing specific antibodies, on different locations of a single yeast cell. B) Molecular recognition map recorded on a cell that was never probed, thus not subjected to force. Coloured pixels document the detection of single proteins (the blue and red pixels correspond to forces in the 0-150 pN and 150-300 pN range, respectively). Most proteins were isolated and evenly distributed, without any clear evidence for clustering. C) Subsequent recognition map recorded on a remote area of the same but force-stimulated cell. Proteins in the second map were no longer isolated but clustered into nanodomains referred to as “nanoadhesomes”.

[1] D.J. Muller et al., Atomic force microscopy as a multifunctional molecular toolbox in nanobiotechnology, Nature Nanotechnology, 3, 261-269 (2008).
[2] D.J. Muller et al., Force probing surfaces of living cells to molecular resolution, Nature Chemical Biology, 5, 383-390 (2009).
[3] D. Alsteens et al., Unfolding individual Als5p adhesion proteins on live cells, ACS Nano, 3, 1677-1682 (2009).
[4] D. Alsteens et al., Force-induced formation and propagation of adhesion nanodomains in living fungal cells, Proceedings of the National Academy of Sciences U.S.A., 107, 20744-20749 (2010).