Research project Dr Vincent Dupres
Nanomechanics and clustering of yeast sensors
In the yeast Saccharomyces cerevisiae, the plasma membrane protein Wsc1 functions as a sensor of the cell wall status. Wsc1 carries a cysteine motif near the amino terminus, followed by a serine/threonine-rich, highly mannosylated extracellular region, a single transmembrane domain (TMD) and a relatively short cytoplasmic tail. It had been proposed that Wsc1 might act as a mechanosensor, activating stress pathways in response to physical changes in the cell wall, but direct evidence for such a mechanism remained elusive.
Together with Prof. Jürgen Heinisch (Universität Osnabrück), we used atomic force microscopy [1,2] to stretch single Wsc1 sensors on living yeast cells, revealing that it behaves like a nanospring capable of resisting high mechanical force and of responding to cell surface stress [3,4] (Figure). Force-extension curves recorded for individual sensors showed a constant force region, followed by a linear region in which force is directly proportional to extension, thus characteristic of a Hookean spring. The use of mutants with reduced glycosylation resulted in severe alterations in protein spring properties, supporting the important role of glycosylation at the extracellular serine/threonine-rich region. Lowering the salt concentration or increasing temperature resulted in a substantial reduction of the sensor spring constant, indicating that Wsc1 is sensitive to cell surface stress.
We are currently using AFM to reveal the lateral clustering of Wsc1 sensors in living cells [5]. Recently, individual wild-type sensors were first localized on the cell surface, revealing that they form clusters of ~200 nm size. Analyses of three different mutants indicated that the cysteine-rich domain of Wsc1 has a crucial, not yet anticipated function in sensor clustering and signalling. Clustering of Wsc1 was strongly enhanced in deionized water or at elevated temperature, suggesting its relevance in proper stress response. This study indicates that in yeast, signalling is coupled to the localized enrichment of sensors within membrane patches, for which the term "nanosensosomes" was proposed.
AFM measures the mechanical behavior of yeast proteins. (a) Stretching single His-tagged Wsc1 sensors using an AFM tip functionalized with Ni++-NTA groups. (b) Representative force-extension revealing that Wsc1 behaves as a linear nanospring.
[1] V. Dupres et al., Fishing single molecules on live cells, Nanotoday, 4, 262-268 (2009).
[2] V. Dupres et al., Microbial nanoscopy: a closer look at microbial cell surfaces, Trends in Microbiology, 18, 397-405 (2010).
[3] V. Dupres et al., The yeast Wsc1 cell surface sensor behaves like a nanospring in vivo, Nature Chemical Biology, 5, 857-862 (2009).
[4] J.J. Heinisch et al., Measurement of the mechanical behaviour of yeast membrane sensors using single-molecule atomic force microscopy, Nature Protocols, 5, 670-677 (2010).
[5] J.J. Heinisch et al., Single-molecule atomic force microscopy reveals clustering of the yeast plasma-membrane sensor Wsc1, PlosOne, 5, e11104 (2010).
