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Cytoskeletal Mechanics

Cells can respond to mechanical forces, thanks to sophisticated measuring systems. The cell cytoskeleton rearranges accordingly to external mechanical stimuli, ultimately affecting cell growth and differentiation. Many different proteins cooperate to hold together the cytoskeleton and to connect it to the cell membrane and organelles, but the detailed molecular mechanisms of force transduction are largely unexplored.

We aim to elucidate the force transmission between proteins at the single molecule level. Single-molecule force-spectroscopy (SMFS) techniques have emerged as powerful tools to manipulate isolated components of various biological systems. We explored a number of protein complexes using Atomic Force Spectroscopy (AFM) and optical tweezers, mainly focusing on three topics.

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Mechanosensing

Filamin connects the cytoskeleton to transmembrane complexes such as integrins or the von Willebrand receptor glycoprotein Ib. Using a dual-beam optical tweezers setup, we showed that the Ig domain pair 20–21 of human filamin A acts as a force-activated mechanosensor. The native autoinhibiting interaction between the domain pair can be disrupted by the cytoskeletal forces (actin binding), enabling the binding of transmembrane proteins.

Multivalency of parallel bonds

Several protein/protein interactions with important structural roles have surprisingly low affinities. 

The single components of the cytoskeleton exchange very rapidly, while the overall architecture has long-term stability. An example is the bond between alpha-actinin and titin, which is the one of the major candidates for the anchoring of titin in the sarcomeric Z-disk. Up to seven similar titin regions, called Z-repeats, can simultaneously bind parallel alpha-actinin layers. We showed that three Z-repeats bind alpha-actinin, even if the bond detaches on a second timescale. We used an avidity argument to show how the concerted action of multiple parallel bonds can extend the anchoring time of titin to the scale of hours, even in presence of force. This model explains how the fast turnover of single alpha-actinins (< 1 minute) is compatible with stable anchoring of titin in the Z-disk. Multivalency can also explain how filamin firmly binds to the cell membrane.

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