Article about how specific cytoskeletal proteins and their interactions help to generate powerful forces within the cell to counter mounting pressures from its environment. Written for MBI.
Like any living organism, the cells in our bodies are forced to reckon with mechanical stress from their external environment. To hold out against the pressure, they internally produce their own cellular force, which is exerted back on their environment. This is known as the ‘cell traction force’, and it is essential for cell shape maintenance, mobility within tissues, growth and survival.
Creation of traction force involves the stress fibres of the cell’s actomyosin cytoskeleton, which is like a network of support beams that bolsters the cell, and focal adhesions, which anchor the beams to the cell membrane and transmit the force generated by the stress fibres to the extracellular environment.
Stress fibres are long, linear and have two main components: actin filaments and myosin filaments. Actin filaments can extend or retract by adding or removing individual actin molecules, while myosin filaments — composed of myosin II motor proteins — ‘drag’ the actin filaments, giving rise to an overall contractile motion. Being dynamic and sensitive to changes in environmental stress, stress fibres are constantly undergoing turnover, incorporating — and losing — actin molecules.
Although it is known that actomyosin turnover is involved the generation of traction force, the relationship and feedback between these two events has yet been sufficiently characterised. Thus, a team of researchers from the Bershadsky Lab and Viasnoff Lab at MBI studied the contractile mechanism of a cell by using mouse fibroblast, a cell that produces essential proteins in the extracellular matrix, to elucidate the dynamics between actomyosin turnover and the generation of traction force.
The researchers quantified the turnover of stress fibre by tracking the incorporation of fluorescent-labelled actin into actin filaments. Additionally, they measured the force exerted by stress fibres by creating a map of the force applied by the cell using micropillars and fluorescent beads as reference points for displacement. To untangle the link between them, they experimented with various regulators of actin filaments and traction force — including the use of inhibitors, application of external force through cell stretching and increasing cross-linking between actin filaments with α-actinin — to test which functions were subsequently affected and sieving out the proteins and pathways responsible.
The researchers found that actin was constantly incorporated into the stress fibres and focal adhesions of the cell and cell stretching increased the incorporation. Among the various types of inhibitors used, researchers found that actin filament inhibitors subjectively arrested actin incorporation, but did not inhibit traction force generation, whereas myosin filament inhibitors stopped both actin incorporation and traction force generation. The researchers also used the small molecule inhibitor of Formin Homology 2 domains (SMIFH2), which not only inhibited both processes, but its effect was able to be reversed by cell stretching, and the drop in traction force generation was rescued by increasing cross-linking between actin filaments.
Taken together, the process of actin incorporation into stress fibres is governed by a complex mechano-regulatory feedback system, dependent on myosin II and external forces. This was observed from how myosin filament inhibitors could fully suppress both actin incorporation into stress fibres and generation of traction force, whereas cell stretching enhanced the uptake of actin into stress fibres.
Formin also had a part to play, but it was much more complicated to dissect its specific effects. Formins typically help elongate actin filaments and can be inhibited by SMIFH2, which also inhibits myosin and actin incorporation. However, the SMIFH2-induced reduction in traction force generation could neither be due to the direct inhibition of actin incorporation, nor the inhibition of myosin. This is because the inhibition of actin incorporation does not necessarily lead to a drop in force generation, as observed with actin filament inhibitors, and the concentration of SMIFH2 used in this experiment was too low to inhibit myosin II and cause the drop, according to previous studies. These implied that force reduction may actually be due to SMIFH2’s inhibition of formin instead.
This deduction was compounded by evidence that increasing the amount of α-actinin cross-linkers managed to partially redeem traction force. Formin, like α-actinin, has been previously established to also have a cross-linking function between actin filaments. SMIFH2 could disable this formin function by detaching formin from the actin filaments, disrupting the network of actin filaments, thereby reducing traction force generation. Thus, it made sense that increasing α-actinin concentration and cross-linking between actin filaments could, to some extent, restore the connectivity of the actin network and the regeneration of traction force.