CORDIS - EU research results

Adaptive Actin Architectures

Periodic Reporting for period 4 - AAA (Adaptive Actin Architectures)

Reporting period: 2022-03-01 to 2023-08-31

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. We reveals the balancing mechanism for long-term network dynamics with a limited amount of building blocks (EMBO J. 2023). This work has been presented at several meeting (10) including a Keynote Lecture at the European Cytoskeleton meeting 2022. Alexandra Colin the first author of this publication obtained a PI permanent position at the CNRS in France in 2023. We were recently invited to write a review on this topic in Nature Cell Biology (review in press).

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.( 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. (Nature Materials 2021).

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).

Active cytoskeletal materials in vitro demonstrate self-organizing properties similar to those observed in their counterparts in cells. We have combined two networks (actin filaments and microtubules)in a dynamic system. In this composite, actin filaments can act as structural memory and, depending on the concentration of the components, microtubules either write this memory or get guided by it. The system is sensitive to external stimuli, suggesting possible autoregulatory behavior in changing mechanochemical environments. We thus establish an artificial active actin-microtubule composite as a system demonstrating architectural stability and plasticity (PNAS 2023).Ondre Kucera the first author of this publication obtained a lecturer position at the SETU in Ireland.

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 (PNAS 2023).
0ne major challenge in the reconstitution of actin dynamic assembly and turnover coupled to contractility is related to the number of available active molecular constituents over time. Until this project most of In vitro experiments used biologically-relevant concentrations of the components but they are typically performed in relatively large volumes (order of magnitude bigger than a cell). Therefore, in such large compartments, the reservoir of components necessary for actin turnover will be unlimited. The depletion of certain components, a critical effect in small volumes such as a cell, cannot be observed, shifting chemical kinetics to a non-biological regime in which the lifespan of an autonomous system is not limited by its ability to recycle its components.

The reconstruction of the dynamic steady state of actin under cell-size confinement was one of the main achievements of this proposal. We have shown how actin dynamics result from the complex interplay between its biochemical and structural properties. These results now open up the possibility of using this living material to generate more complex behaviors, such as the movement of an artificial cell based on its intra-"cellular" organization. It also opens up the possibility of generating in vitro reactive systems capable of sensing and adapting to their environment. It therefore opens up a wide field of possibilities in the emerging field of synthetic (artificial) cells.