Final Activity Report Summary - GELCELL (Mechanics of the cytoskeleton: active polymer networks)
The mechanical properties of cells are essential for many cell functions, including cell crawling, division and mechanosensing. Cell mechanics are controlled by an elastic network of interconnected semi-flexible protein filaments known as the cytoskeleton. The cytoskeleton has a highly organised, yet dynamic, structure which is regulated by a host of accessory proteins that control the length and spatial organisation of the filaments. The most important mechanical contribution stems from the actin cortex, a meshwork of filamentous (F-) actin under the plasma membrane. The actin cortex can withstand large external forces and, at the same time, it can actively generate forces which contribute to cell movement and division. Forces are generated by active processes which convert chemical energy, from hydrolysis of Adenosine triphosphate (ATP), into mechanical work. The actin filaments actively polymerise to generate pushing forces and myosin motor proteins actively slide actin filaments past one another to generate contractile forces. This implies that the cytoskeleton is an unusual type of soft condensed matter, since it is a non-equilibrium, active complex fluid that responds to biochemical signals.
The goal of this project was to elucidate how the structure and passive or active dynamics of the cytoskeleton determined its mechanical response, how was the mechanical stiffness correlated with cross-link density and filament organisation and how did motor-generated forces change the stiffness.
I used a parallel approach, combining studies of controlled in vitro model systems of purified proteins with studies of living cells. I firstly used a high-frequency microrheological method based on laser interferometry to measure the elastic and viscous shear moduli of actin networks over a large frequency window. I was able to identify new dynamic scaling regimes, with a regime dominated by single filament bending, in frequencies above 10 kHz, with a second one dominated by longitudinal tension propagation of above 100 Hz, and with third one dominated by filament entanglements and cross-links.
I went on to study the effect of different types of cross-linker proteins, i.e. the small, rigid cross-linker scruin and the large, highly floppy cross-linker filamin A. I discovered that the elastic response to an external shear stress was dominated by actin filament stretching when the cross-linker was rigid, and became sensitive to cross-linker stretching when the cross-linker was floppy. In the latter case, the cross-linked actin network stiffened dramatically, by a factor of 100 to 1 000, before breaking at large strains. This finding explained previously observed mechanical properties of adherent cells.
Finally, I added active myosin II motor proteins to generate contractile networks which mimicked more closely the living cell. I was able to demonstrate cell-like actin contractility, elasticity and non-thermal stress fluctuations. When the actin network was not cross-linked, the motors could only generate stresses on time scales of seconds. I measured these stresses directly by observing bending fluctuations of embedded, stiff microtubules. The microtubules showed short length-scale bending consistent with myosin contractile forces of 10 to 20 pN. When I also added a cross-linker, the motors were able to generate sustained tension. This resulted either in network contraction, when the network was floating, or in network stiffening, when the network was anchored to the sample chamber walls.
The actin-myosin model systems revealed molecular mechanisms underlying active contraction, stiffening and non-thermal fluctuations in cells. I measured the frequency and magnitude of non-thermal, motor-driven fluctuations in adherent Cos7 cells by employing endogenous, fluorescently labelled microtubules as microrheological probes. The microtubules in vivo showed similar bending as the microtubules in biomimetic actin-myosin networks, with similar forces, ranging from 10 to 20 pN, and time scales of seconds. Microtubules could thus be used as intracellular force probes.
In summary, I collected precise, quantitative measurements of mechanical properties of cytoskeletal protein networks which led to a fundamental understanding of the physical mechanics underlying cell mechanics.
The goal of this project was to elucidate how the structure and passive or active dynamics of the cytoskeleton determined its mechanical response, how was the mechanical stiffness correlated with cross-link density and filament organisation and how did motor-generated forces change the stiffness.
I used a parallel approach, combining studies of controlled in vitro model systems of purified proteins with studies of living cells. I firstly used a high-frequency microrheological method based on laser interferometry to measure the elastic and viscous shear moduli of actin networks over a large frequency window. I was able to identify new dynamic scaling regimes, with a regime dominated by single filament bending, in frequencies above 10 kHz, with a second one dominated by longitudinal tension propagation of above 100 Hz, and with third one dominated by filament entanglements and cross-links.
I went on to study the effect of different types of cross-linker proteins, i.e. the small, rigid cross-linker scruin and the large, highly floppy cross-linker filamin A. I discovered that the elastic response to an external shear stress was dominated by actin filament stretching when the cross-linker was rigid, and became sensitive to cross-linker stretching when the cross-linker was floppy. In the latter case, the cross-linked actin network stiffened dramatically, by a factor of 100 to 1 000, before breaking at large strains. This finding explained previously observed mechanical properties of adherent cells.
Finally, I added active myosin II motor proteins to generate contractile networks which mimicked more closely the living cell. I was able to demonstrate cell-like actin contractility, elasticity and non-thermal stress fluctuations. When the actin network was not cross-linked, the motors could only generate stresses on time scales of seconds. I measured these stresses directly by observing bending fluctuations of embedded, stiff microtubules. The microtubules showed short length-scale bending consistent with myosin contractile forces of 10 to 20 pN. When I also added a cross-linker, the motors were able to generate sustained tension. This resulted either in network contraction, when the network was floating, or in network stiffening, when the network was anchored to the sample chamber walls.
The actin-myosin model systems revealed molecular mechanisms underlying active contraction, stiffening and non-thermal fluctuations in cells. I measured the frequency and magnitude of non-thermal, motor-driven fluctuations in adherent Cos7 cells by employing endogenous, fluorescently labelled microtubules as microrheological probes. The microtubules in vivo showed similar bending as the microtubules in biomimetic actin-myosin networks, with similar forces, ranging from 10 to 20 pN, and time scales of seconds. Microtubules could thus be used as intracellular force probes.
In summary, I collected precise, quantitative measurements of mechanical properties of cytoskeletal protein networks which led to a fundamental understanding of the physical mechanics underlying cell mechanics.