Development control of actin nucleation in C. elegans – DeCaNu

Cell state dictates some characteristic cytoskeletal architectures and its reciprocal also holds true. Actin architectures, while driving cell morphology, mechanics or gene expression profile, feedback into cell state. For example, a decrease of actin nucleation can change cell commitment during C. elegans embryo left/right symmetry breaking, revealing an interplay between actin equilibrium and cell fate. The aim of this proposal is to reveal, how the nucleation of actin architectures is temporally and spatially controlled in the different founder cells of C. elegans early embryo and how it affects their fate.
We will characterize the specific actin architectures of each blastomere at the single cell level to define actin cytoskeleton identities throughout the early lineage. First using in vivo quantification of endogenously expressed GFP-tagged actin binding proteins, which will enable us to map molecular content and dynamics throughout the lineage. Second, using an in vitro biochemical approach, to probe actin related molecular content in a controlled environment. To do so we are developing a novel device in order to produce single cell extracts and use this content for actin in vitro polymerization assays using micropatterns. Finally, we will proceed in a series of perturbation experiments either affecting actin dynamics and quantifying how it affects cell states or vice versa, in order to assess the interplay between actin and cell identity.
This project will provide fundamentally new insights into actin biochemistry as it focuses on the acquisition of cytoskeleton specific identities arising in a natural situation while cells are undergoing commitment changes. It will be of upmost importance to verify how actin dynamics feedback into early embryo development.

The main objectives of this project are to reveal in a living model system, how the nucleation of actin architectures can be temporally and spatially controlled in the different cell types found in a developing embryo and how it affects cell states. The proposal has 3 main work packages:

WP 1. Identify actin cytoskeletal content variation throughout the early lineage pattern.
Here we will focus on the developmental control of the actin proteome, by a quantification of ABPs levels and inheritance through the lineage of the early embryo. The main work is to develop an automated single cell image quantification toolbox to integrate 4D imaging and define the proper references.

WP 2. Use cell extracts to test in vitro polymerization capacities of single cells.
Here we will probe molecular content by using single cell extracts for actin in vitro nucleation assays using micropatterns. Some technological development will be performed in order to combine extract preparation, actin in vitro polymerization and visualization on the same device. It will allow us to quantify the phenotypes linked to the different actin proteomes.

WP 3. Link actin organization, cell state and developmental control.
In this final WP, we will assess the interplay and dependency of cell states versus cytoskeletal specific states, via perturbation of either the actin nucleation capacity and observing the impact on cell identity or perturbation of the cell identity and observation of actin nucleation changes.

I propose an interdisciplinary project combining top down and bottom up approaches, relying on quantitative experiments in single cells of C. elegans early embryo. The results of this proposal will lead to a precise characterization of cell specific actin related content and actin nucleation capacities, it will also reveal particular requirements linked to cell identity acquisition in the early developing C. elegans embryo.