SINGLEMOLECULE FORCE SPECTROSCOPY OF MICROBIAL CELL ENVELOPE PROTEINS - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 323

15.3.2 Measuring the Spring Behaviour of Yeast Membrane
Sensors

Mechanosensors in living cells convert mechanical forces into biochemical
signals. 41 In yeast, surface stresses acting on the cell wall and plasma
membrane are detected by a group of ive membrane sensors, i.e., Wsc1,
Wsc2, Wsc3, Mid2 and Mtl2. Although much is known about the genetics
and molecular biology of the sensors, how they probe extracellular signals
remains mysterious. It is believed that these membrane proteins act as
mechanosensors, activating stress pathways in response to physical changes
in the cell wall. Yet, direct evidence for such a mechanism had never been
provided.

(a) (b)

Figure 15.7. Measuring the nanospring properties of the Wsc1 sensor. (a) Force-
distance curves were recorded on yeast cells expressing Wsc1 sensors with an extended
His-tag, using AFM tips functionalized with Ni ++ -NTA groups. (b) Representative
force-extension curve obtained upon stretching a single Wsc1 molecule. Clearly
visible is the Hookean spring behaviour (red line). Reprinted with permission from
Dupres et al. 42

Using SMFS, we measured the mechanical properties of single Wsc1 on
live cells ( Fig. 15.7 ) . Genetic manipulations could solve a major experimental

constraint: in essence AFM is a surface technique, so how can it probe sensors
that are embedded within the cell wall? Simple calculations indicate that
native Wsc1 sensors extend ~80 nm above the plasma membrane. As the cell
wall is ~110 nm thick, this means native sensors cannot reach the outermost
cell surface. To elongate the molecule, extended Wsc1 proteins were designed

42