Tropomyosins and differentiation of actin filament networks – TropActin

Within a single cell, the actin cytoskeleton forms diverse networks of filaments, implicated in many fundamental functions. These actin networks are assembled from a common pool of actin and regulatory proteins, but how network differentiation is regulated has remained a very challenging question for decades. Recently, a family of actin binding proteins emerged as a key to elucidate this question: Tropomyosins (Tpm).

In every cell, multiple Tpm isoforms are coexpressed and have been shown to spatially segregate to specific networks. Tpm has been proposed to give filaments an identity by regulating the recruitment and activity of the other cytoskeletal proteins. However, the mechanisms controlling Tpm localization are still unknown. The major goal of the TropActin project is to investigate the biochemical and biomechanical regulation of Tpm, using new powerful approaches, in order to shed light on actin network differentiation.

The project main hypothesis is that each Tpm isoform recognizes specific biochemical and biomechanical signatures/cues, that determine to which actin network they get recruited. Such signatures include actin isoform, post-translational modifications, other actin binding proteins, tension, curvature and twist, network density and dynamics, etc.

Experiments will be performed in vitro, on purified and fluorescently labelled proteins. However, reconstituting actin filaments bearing one or more of such signatures remains very challenging. To overcome this limitation, I will develop new methods, based on a state-of-the-art microfluidic setup, to assemble single actin filaments and networks, in tightly controlled conditions. I will quantity the affinity of Tpm isoforms to actin in order to identify the biochemical and biomechanical conditions that promote the recruitment of Tpm.

The project will be strengthened by two collaborations. First, Pekka Lappalainen (University of Helsinki) will bring insights from the molecular scale, using cryoEM to resolve details of the interactions between Tpm isoforms and actin filaments. I will also initiate a collaboration with the team of François Robin (Institut de Biologie Paris Seine) who will perform in vivo experiments on his model system, C. elegans embryo. These collaborations will support results obtained in vitro and, together, will bring a comprehensive understanding of Tpm recruitment, from the molecular to the cellular scale, to advance our comprehension of actin network differentiation.