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Adaptive Actin Architectures

Periodic Reporting for period 2 - AAA (Adaptive Actin Architectures)

Reporting period: 2019-03-01 to 2020-08-31

Although we have a good knowledge of many important processes in cell biology, including knowledge of many molecules involved and how they interact with each other, we still do not understand most of the dynamical features that are the essence of living systems. This is particularly true for the actin cytoskeleton a major component of the internal architecture of eukaryotic cells. Our aim is to reconstitute actin dynamic in vitro and study its contribution to the unique ability of living systems to permanently sense and adapt to external changes.

This should allow us to answer three main questions:

1- How are different actin architectures built from a common set of components and how do they coexist in the same environment (neighborhood)?
2- What are the conditions ensuring the establishment of dynamic steady-states?
3- How do actin dynamics confer cytoskeleton adaptability to drive cellular responses?

Answering these questions should provide a comprehensive understanding of the interplay between molecular elements and macroscopic properties to achieve a dynamic process fundamental in defining a biological function.

At the core on this proposal is the development and establishment of a next generation of reconstituted systems that would be closer to physiological conditions in order to solve the complexity of actin cytoskeleton dynamics. Using our unique and powerful toolbox, we propose to explore a large set of parameters involved in dynamic network renewal. Quantitative comparisons between in vitro and in vivo networks will allow us to establish the first biophysical model of networks renewal and adaptation.
We have been able to reconstitue actin dynamics in cell-sized confinement (see attached image). We are currently investigated how the biochemical composition, architecture and competition between the different actin networks affect the dynamic steady state . We have some very excited data on how a limited amount of components limits the growth of actin network under confinement.These data have been presented at the Cell Phys 2019 meeting in Saarbrücken, Germany.

We have studied the role of actin filament crosslinking in the spatial integration of mechanical forces that ensures the adaptation of intracellular symmetry axes in accordance with the geometry of extracellular cues.(Senger et al., J. Cell Science, 2019).

In a recent study, we used a unique combination of micropatterning, local photoablation of contractile elements, global-force measurements, and theoretical modeling to describe finely the mechanics of the subcellular actin networks. We demonstrated that actin fibers were fully embedded along their entire length in a continuous and contractile network of cortical filaments. Therefore, the propagation of the contraction of these bundles throughout the entire cell was dependent on this embedding. In addition, these bundles appeared to originate from the alignment and coalescence of thin and unattached cortical actin filaments from the surrounding mesh. (Publication in revision).

We have also studied how the actin architecture affects cargo positioning.We showed that the precision of cargo positioning is set by the gradient of net actin polarity in the network and by the run length of the cargo in an attached state. (Richard et al., PNAS, 2019).

We have also developed a method that allowed contactless and mask-free photo-micropatterning of electron microscopy grids for site-specific deposition of extracellular matrix-related proteins. Micropatterns generated predictable intracellular organization, allowing direct correlation between cell architecture and in-cell three-dimensional structural characterization of the underlying molecular machinery. (Toro-Nahuelpan et al., Nat. Methods, 2020).

Finally, we have developed new tools (lipid patterning) to study the respective contribution of external and internal friction on the acto-myosin response. We are able to demonstrate how external friction can drive the symmetry of the contractile response (Publication in preparation).
The reconstitution of actin dynamic steady state under cell-sized confinement will be a major achievement of this proposal. We hope to be able to understand how actin dynamics derives from the complex interplay between its biochemical, structural and mechanical properties. We will combine confinement with our unique capacity to perform dynamic micropatterning, to add or remove actin nucleation sites in real time, in order to investigate the ability of dynamic networks to adapt to changes and the role of coupled network dynamics in this emergent property.