Is there anyone out there?—Single-molecule atomic force microscopy meets yeast genetics to study sensor functions

Abstract

The ability to react to environmental stress is a key feature of microbial cells, which frequently involves the fortification of their cell wall as a primary step. In the model yeast Saccharomyces cerevisiae the biosynthesis of the cell wall is regulated by the so-called cell wall integrity signal transduction pathway, which starts with the detection of cell surface stress by a small family of five membrane-spanning sensors (Wsc1-Wsc3, Mid2, Mtl1). Although genetic evidence indicated that these proteins act as mechanosensors, direct in vivo evidence for their function remained scarce. Here, we review a new approach integrating the tools and concepts of genetics with those of nanotechnology. We show how atomic force microscopy can be combined with advanced protein design by yeast genetics, to study the function and the mechanical properties of yeast sensors in living cells down to the single molecule level. We anticipate that this novel integrated technology will enable a paradigm shift in cell biology, so that pertinent questions can be addressed, such as the nanomechanics of single sensors and receptors, and how they distribute across the cell surface when they respond to extracellular stress.

Deciphering cell wall integrity signalling in Aspergillus fumigatus: identification and functional characterization of cell wall stress sensors and relevant Rho GTPases

“…On the one hand, this finding is not surprising since Wsc2 is lacking a cytosolic tail and it was previously shown that the cytosolic tail of ScWsc1 is essential for signal transduction (Lodder et al ., 1999; Straede and Heinisch, 2007). On the other hand, GFP‐tagged Wsc2 localizes to the plasma membrane and it was suggested that the WSC domains might mediate specific protein–protein interaction, thereby supporting, for example, the function of other CWI sensors (Heinisch and Dufrêne, 2010). Thus, a contribution to CWI signalling appeared conceivable but experimentally not provable.…”

Up against the Wall: Is Yeast Cell Wall Integrity Ensured by Mechanosensing in Plasma Membrane Microdomains?

“…It should also be noted that similar AFM approaches could be applied to study any cell surface proteins that can be elongated to penetrate the yeast cell wall (e.g., the pheromone receptors) and also for the investigation of basically any cell surface protein in other organisms (33).…”

“…This leads to conformational changes in the CRD, stabilized by disulfide bridges, and triggers both sensor clustering and conformational changes in the cytoplasmic domain, which then interacts with the downstream CWI components. The assembly of sensors in plasma membrane microdomains that recruit intracellular signaling components thus leads to the formation of a Wsc1 sensosome and enhances CWI signaling capacity (33). Wsc1 is depicted as a plasma membrane-spanning sensor that clusters upon the application of cell surface stress.…”

Together we are strong—cell wall integrity sensors in yeasts

“…Therefore, the control mechanisms for its remodelling during growth and upon extracellular stress have drawn considerable attention in the past two decades, as reviewed in [26,28,29]. The signal transduction pathway (called the CWI pathway, for cell wall integrity) involved in this process has also been extensively reviewed [16,17,29]. A simplified scheme of its central components is presented in Figure 1.…”