All cells can mechanosense – the ability to perceive and respond to internally or externally derived mechanical stress. Cells have developed feedback loops by combining force-generating machinery with the ability to sense mechanical stress, a process which likely evolved from the need of simple cells to respond to changing environmental cues. These mechanical feedback loops have been appropriated for a number of complex processes involving a range of length- and time-scales, including, but not limited to hearing, blood pressure regulation, and muscle contraction. During cell division, a mother cell must separate appropriately into two daughter cells under a variety of external environments. Therefore, a mechanical feedback loop must have evolved to respond to these internally derived forces, allowing for crosstalk across the heterogeneous cytoskeletal network to occur. Two observations in Dictyostelium
supported this original hypothesis: (1) Dividing cells that exhibit a cellular asymmetry recruit myosin II to correct that asymmetry before proceeding through cytokinesis [57
] and (2) dividing cells that are overlaid with an agarose sheet, respond to that compression with a higher accumulation of myosin II at the cleavage furrow [60
To explore whether or not such a feedback system exists in cells, micropipette aspiration (MPA) was used to apply stress to the surface of dividing cells equivalent to that experienced at the cleavage furrow cortex (0.1–1 nN/μm2
] (). This MPA-induced external stress leads to a deformation of the cortex over ~10–30 μm2
of surface area, which is on the scale of the deformation that the cleavage cortex imposes during furrow ingression. For comparison, the surface area of the cleavage furrow cortex at the beginning of cell division is ~200 μm2
, which reduces to 5–10 μm2
near the end of division. The applied stress leads to the accumulation of myosin II, as well as the actin crosslinking protein cortexillin I, at the cell cortex inside the micropipette tip. The augmentation of myosin II and cortexillin I at the site of deformation is interdependent – neither protein accumulates in the absence of the other. Additionally, the recruitment of myosin and cortexillin to the micropipette tip is independent of the global crosslinking proteins fimbrin, dynacortin, and enlazin, suggesting that myosin II and cortexillin I are unique in delineating a mechanosensory module. In this module, cortexillin I would allow myosin II to generate force and experience tension by anchoring the actin network, locking the motor into its isometric state. Additionally, the full bipolar thick filament assembly dynamics are required as either inhibition of BTF assembly or disassembly kills the mechanosensitive localization of myosin II. Taken together, these data imply that the myosin II-cortexillin I mechanosensory module has three elements required for its cooperative and coordinated response to mechanical stress: (1) mechanosensitive myosin II mechanochemistry, (2) myosin II BTF dynamics, and (3) cortexillin I-mediated actin-filament stabilization.
Mechanical stress directs myosin II and cortexillin accumulation
To determine if myosin II was the active element of the sensor, the pressure-dependencies of the mechanosensory response of wild type myosin II and mutant myosin II proteins with altered lever-arm lengths were measured and compared (). To this end, three mutants were generated: the 2xELC mutant with an extra essential light chain, 13 nm lever arm and 4 μm/s unloaded actin sliding velocity; the ΔBLCBS mutant with both light chain binding sites deleted, containing a 2 nm lever arm and 0.6 μm/s unloaded actin sliding velocity; and the S456L mutant with a wild type lever arm and ATPase activity but a 10-fold slower unloaded actin filament sliding velocity. For comparison, wild type myosin II has a 9 nm lever arm and a 3 μm/s unloaded actin sliding velocity. The two lever-arm mutants have full actin-activated ATPase activity, imparting fully phosphorylated wild type myosin II characteristics. The longer lever arm mutant 2xELC was more responsive than wild type, while the shorter lever arm mutant ΔBLCBS was less responsive, requiring much higher pressures to generate the mechanosensitive accumulation of myosin II and cortexillin I. The S456L lever arm mutant showed a near wild type mechanosensitive response. These data clearly imply that myosin II’s mechanochemistry is the active element of the myosin II-cortexillin I mechanosensitive module, given that myosin’s Fmax is inversely related to the lever arm length. The implication of this lever arm-length dependency is that as myosin II produces force, it also experiences tension by pulling against an anchored actin filament. Consequently, the motor stalls at the isometric state, which further causes the myosin II motor to dwell tightly bound to the actin filament ().
Structure-function analysis revealed that the BTF assembly/disassembly dynamics were essential for mechanosensitive localization. Therefore, to determine where the sensitive step in the assembly pathway might exist, kinetic simulations were conducted using measured kinetic or equilibrium constants for each step of the assembly pathway [58
]. These simulations revealed two interesting properties. First, BTF sizes will be dispersed exponentially, with the minimal BTF (BTF3
where the BTF contains three dimers) the most frequent size. This distribution occurs if the population of 80% free assembly-incompetent (heavy chain phosphorylated) monomer (M0
) is maintained. Second, the major rate-limiting step is the conversion of M0
to the assembly-competent monomer (M). These characteristics suggest that one of three possible assembly mechanisms occurs during the mechanosensitive response. In the first mechanism, assembly is initiated upon the activation of a heavy chain phosphatase. In the second possibility, assembly is driven by the local inactivation of MHCK. In the third scenario, assembly is promoted by cooperative interactions of smaller BTFs with the actin polymer. Here, the mini-BTFs are stabilized by force locking the motor in the isometric state, which leads to the local accumulation of more myosin II monomers, promoting their insertion into the pre-existing BTF.
The final element of the mechanosensory module is the interaction between cortexillin I and myosin II, leading to their stabilization on the actin polymer network. This interaction likely occurs through cooperative binding to the actin filament. Myosin II motors alone cooperatively bind to actin filaments and this cooperativity appears to depend on the isometric state of the myosin motor, the heterologous proteins associated with the actin polymer, or the structure of the actin filament itself [62
]. In a complex living system, all three modes of cooperative interactions between myosin II motors may contribute to mechanosensitive localization. The isometric state-dependent mode is strongly suggested by the lever-arm length dependency of the mechanosensory response. For myosin II to be stabilized in the isometric state, the actin filament must be anchored so that when the motor pulls on the filament, tension can be generated and experienced by the motor. To determine if cortexillin I has such characteristics as a stable actin anchor under the influence of force, cortexillin I-actin-binding lifetimes were measured using single molecule analyses [58
]. Over a force range of −2 to 2 pN, cortexillin I has an actin-binding life-time of 550 ms, significantly longer than myosin II in either the unloaded or loaded strongly bound state. Thus, cortexillin I remains bound to actin filaments long enough for myosin to generate tension on the filament.