Single-molecule atomic force microscopy reveals clustering of the yeast plasma-membrane sensor Wsc1

Abstract

Signalling is a key feature of living cells which frequently involves the local clustering of specific proteins in the plasma membrane. How such protein clustering is achieved within membrane microdomains ("rafts") is an important, yet largely unsolved problem in cell biology. The plasma membrane of yeast cells represents a good model to address this issue, since it features protein domains that are sufficiently large and stable to be observed by fluorescence microscopy. Here, we demonstrate the ability of single-molecule atomic force microscopy to resolve lateral clustering of the cell integrity sensor Wsc1 in living Saccharomyces cerevisiae cells. We first localize individual wild-type sensors on the cell surface, revealing that they form clusters of approximately 200 nm size. Analyses of three different mutants indicate that the cysteine-rich domain of Wsc1 has a crucial, not yet anticipated function in sensor clustering and signalling. Clustering of Wsc1 is strongly enhanced in deionized water or at elevated temperature, suggesting its relevance in proper stress response. Using in vivo GFP-localization, we also find that non-clustering mutant sensors accumulate in the vacuole, indicating that clustering may prevent endocytosis and sensor turnover. This study represents the first in vivo single-molecule demonstration for clustering of a transmembrane protein in S. cerevisiae. Our findings indicate that in yeast, like in higher eukaryotes, signalling is coupled to the localized enrichment of sensors and receptors within membrane patches.

Figure 1. Single-molecule mapping reveals clustering of Wsc1 in live cells.

(a) AFM deflection image of a yeast cell trapped into a porous polymer membrane, recorded in buffer solution (sodium acetate + sucrose 100 mM; pH 4.75) at 25°C. As shown in the left cartoon, the cells express elongated, fully functional Wsc1-Mid2 hybrid sensor bearing an His-tag. (b) Representative adhesion force maps obtained by scanning 1 µm×1 µm areas on different cells with a Ni⁺⁺-NTA-tip in buffer solution. The heterogeneous distribution of the bright pixels, which represent the detection of single sensors, clearly documents the formation of nanoscale clusters (highlighted by dotted red lines). We define a cluster as a group of sensors containing at least 10 molecules (bright pixels) either in direct contact with each other or separated by no more than one dark pixel. All maps were obtained using a retraction speed of 1,500 nm s⁻¹ corresponding to a loading rate of 9,000 pN s⁻¹, and an interaction time of 500 ms. The data shown are representative of results obtained on 12 different cells using 14 different tips.

Figure 2. Clustering of Wsc1 is stimulated under stressing conditions.

(a, b) Adhesion force maps (1 µm×1 µm) recorded with a Ni⁺⁺-NTA-tip either in buffer solution at 37°C (a, heat shock) or in deionized water at 25°C (b, hypoosmotic shock). Stressing conditions strongly enhance Wsc1 clustering (clusters are highlighted by dotted blue and green lines). For both conditions, the characteristic shape of the force curves confirmed they reflected the detection of single His-tagged sensors. For Fig. 2b, similar data were obtained when deionized water was quickly exchanged by a buffered solution. Here again, we define a cluster as a group of sensors containing at least 10 molecules (bright pixels) either in direct contact with each other or separated by no more than one dark pixel. For each condition, cells were treated for 15 min prior to AFM measurements. The data shown are representative of results obtained on 6 different cells using 6 different tips. (c) Surface density histograms showing the number of sensors per µm² measured (from left to right): for wild-type Wsc1 in buffer at 25°C (n = 16 maps containing 1024 data points each), for wild-type Wsc1 in buffer at 37°C (n = 8 maps) and in deionized water at 25°C (n = 12 maps), as well as for Wsc1_C4,5A (n = 7 maps), Wsc1_C6,7A (n = 7 maps), and Wsc1_C8A mutants (n = 12 maps) in buffer at 25°C. Darker and lighter colors represent the surface density of clustered and isolated sensors.

Figure 3. The conserved CRD domain of Wsc1 is essential for proper sensor function.

(a) Alignment of the deduced amino acid sequences of selected proteins carrying a CRD domain. The eight conserved cysteine residues are numbered consecutively and highlighted in red. Other conserved amino acid residues identical in all proteins are highlighted in blue. Numbers before and after each sequence refer to the position of the first and last amino acid relative to the N-terminal end (assuming that the starting methionine is not processed). For alignment, the CloneManager Suite programm version 9 was used at standard settings. Wsc-sensor sequences are from Sc  =  Saccharomyces cerevisiae and Kl  =  Kluyveromyces lactis. ThCRD2  =  homologous sequence from β-1-3 exoglucanase of the fungus Trichoderma harzianum. HsPKD1  =  homologous sequence from the human polycystin. For sequence references consult , . (b) Serial dilution drop assays. Yeast strains with the indicated alleles were grown overnight into late logarithmic phase, adjusted to OD₆₀₀  = 0.1, and 3 µl each of ten-fold serial dilutions were dropped onto rich medium (YEPD) under stress conditions as indicated. 1 M sorbitol was added for the non-stressed control. Congo red was applied at a concentration of 0.1 mg/ml, caffeine at 7.5 mM. Plates were incubated for 3 days at 25°C, except for the indicated heat stress at 37°C. Scanned images in each column were taken from the same plate, adjusted for brightness and contrast with the CorelDraw photoshop programm. None of the cysteine mutants displayed significant growth under stress conditions as compared to the control strain in the second lane.

Figure 4. Clustering of Wsc1 is achieved through the CRD domain.

Adhesion force maps (1 µm×1 µm) recorded on the surface of mutants Wsc1_C4,5A (a) Wsc1_C6,7A (b) and Wsc1_C8A (c), with a Ni⁺⁺-NTA-tip in buffer solution at 25°C. While Wsc1 mutants showed a surface density similar to that of the wild-type, they were evenly distributed thus no longer clustered (see also Fig. 2c). The data shown are representative of results obtained on 17 different cells using 13 different tips.

Figure 5. Fluorescence microscopy shows vacuolar accumulation of non-clustering sensors.

(a) Elongated versions of Wsc1 and its mutant derivative Wsc1_C4,5A fused to GFP were expressed from a centromeric vector under the control of their native promoters in a wsc1 deletion strain. A representative number of cells in different growth stages was examined by differential interference contrast (DIC) microscopy (upper row) and fluorescence microscopy (lower row). Wsc1-GFP signals are shown in green. The vacuolar membrane was stained with FM4-64 and is shown in red. Scale bar: 10 µm. (b) Larger image of cells in a late stage of cell division, expressing GFP fusions of either the wild-type Wsc1 or the Wsc1_C4,5A construct. Imaging conditions are as above, with the additional display of the separated signals from GFP and FM4-64 in the lower row. Scale bar: 5 µm.

Figure 6. Biological significance of CRD-mediated Wsc1 clustering.

(a) Non-stressing conditions. The CRD domains of Wsc1 sensors interact with cell wall glucans. Two representative glucan chains of the cell wall (CW, shaded in grey) are depicted as interconnected orange dots. Binding of the CRD domain should be transient to allow for lateral movement of sensors in the cell wall and plasma membrane. The CRD domain is followed by a Ser/Thr-rich (STR) region, with blue lines indicating mannosylation. The single transmembrane domain (TMD) of Wsc1 is shown as a red cylinder spanning the plasma membrane (PM). The cytoplasmic tail is curled and not competent for signalling to the thus inactive downstream components Rom2 and Rho1 (light blue and yellow, respectively). (b) As a first response to cell wall stress, the glucan chains are stretched, exerting a force on the Wsc1-STR nanospring through their coupling to CRD. The induced conformational change in the cytoplasmic domain of the sensor, designated by the straight red bar, allows it to interact with and activate Rom2 (dark blue), which in turn promotes activation (exchange of GDP for GTP) of Rho1 (bright yellow), and signalling to the downstream components of the CWI pathway. Concomitantly, the CRD domain adapts an interaction-competent surface structure. (c) Intermolecular interactions of the CRD domains promote sensor clustering, with a concomitant increase of the downstream-signalling components at the internal side of the plasma membrane. This local accumulation enhances the stress signal and the cellular response. We propose to call this signalling complex a sensosome.