Unfolding individual als5p adhesion proteins on live cells

Abstract

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.

Figure 1

Unfolding isolated Als5p proteins. (a) Representation of an Als molecule projecting outward from the C. albicans cell wall by means of the stalk region. The tandem repeat (TR) region is comprised of multiple glycosylated 36-amino acid repeats that are arranged in anti-parallel β-sheets. As shown in the inset, modelling with ROSETTA and LINUS consistently predicted independently folded three-stranded anti-parallel β−sheet domains for each repeat. Thus, the TR region consists of 6 such repeats, which would unfold independently. The amyloidogenic Threonine-rich region (T) is the most conserved sequence in Als proteins and connects to the ligand-binding Ig-like region which possesses three equal-sized β-sheet rich domains. (b) Principle of the SMFS experiments. Ig-T-TR₆ fragments were attached on a gold surface and stretched via their Ig domains using an Ig-T-tip. (c) Force-extension curves obtained by stretching single Ig-T-TR₆ showed periodic features reflecting the sequential unfolding of the TR domains (upper traces). Force peaks were well-described by the worm-like-chain model (inset; red line), using a persistence length of 0.4 nm: F(x) = k_bT/l_p [0.25(1-x/L_c)⁻² + x/L_c − 0.25], where L_c and l_p are the contour length and persistence length of the molecule, k_b is the Boltzmann constant and T the absolute temperature. Addition of urea dramatically altered the unfolding peaks due to hydrogen bond disruption (lower traces). All curves were recorded using a loading rate of 10,000 pN/s and an interaction time of 500 ms. Similar data were obtained using more than ten different tips and four independent samples.

Figure 2

Unfolding forces depend on pulling speed and interaction time. (a) Unfolding forces of individual TR domains as a function of the loading rate (constant interaction time of 500 ms). The unfolding force F is related to the loading rate (k_{c v}) by: $F=\frac{k_{b}T}{x_u}\text{ln}\left(\frac{k_{c}v{x}_u}{k_{b}T{K}_u^0}\right)$ where k_c is the spring constant, v is the pulling speed, x_u is the width of the potential barrier, and $k_u^0$ is the mechanical unfolding rate constant under zero force. Thus, x_u can be determined from the slope of this plot and $k_u^0$ from the intercept. Data represent the mean ± s.e.m. (n = 50). (b) Dependence of the unfolding probability on the interaction time (constant loading rate of 10,000 pN/s). The data can be fitted with an exponential function (red line).

Figure 3

Unfolding Als5p proteins on live cells. (a) Force-extension curves recorded between an Ig-T-tip and the surface of S. cerevisiae cells expressing Als5p with six, four, two and no TR repeats. All curves were recorded using a loading rate of 10,000 pN/s and an interaction time of 500 ms. Similar data were obtained using more than ten different tips and ten cells from independent cultures. (b) Higher magnification curve documenting the sequential unfolding of six TR domains. (c) Both the unfolding probability and the level of yeast adherence increased with the number of TR repeats, indicating there was a good agreement between single-molecule and microscale assays.

Figure 4

Mapping unfolding forces on cell surfaces. (a) Left column: AFM topographic images recorded in buffer with an Ig-T-tip showing single S. cerevisiae cells expressing different numbers of TR repeats. Cells were trapped into the pores of a porous polymer membrane for in vivo imaging. Columns 2–4: representative unfolding maps (256 force curves; n = 256) recorded on 500 nm × 500 nm areas on top of the cells (loading rate of 10,000 pN/s, interaction time of 500 ms). Each bright pixel reflects the detection and unfolding of single Als5p proteins. The unfolding probability is clearly proportional to the number of TR repeats. (b) The probability of unfolding increased with interaction time (inset: unfolding maps obtained at increasing times).