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Control of the Homeostasis of Actin through Time: actin homeostasis during embryonic development by means of single-molecule imaging and simulation in C. elegans

Periodic Reporting for period 1 - CHAT (Control of the Homeostasis of Actin through Time: actin homeostasis during embryonic development by means of single-molecule imaging and simulation in C. elegans)

Reporting period: 2015-04-01 to 2017-03-31

The actin cytoskeleton is a major determinant of the mechanical properties of embryonic cells and tissues. Spatial and temporal modulations of these properties play a major role in cellular transformation and important aspects of cancer such as cell division, polarity and migration.
In embryonic cells, actomyosin forms a cortical reticulated gel that turns over rapidly, is weakly organized, and is composed of a variety of structures, with distinct architectures and dynamics. These structures are nucleated and elongated by different factors, and bind to specific sets of actin-binding proteins that modulate their dynamics. For example, in C. elegans, at least two types of cytoskeletal structures simultaneously coexist in the cell: short, branched actin filaments, nucleated by arp2/3, and long actin filaments/bundles nucleated by formins. Understanding how actin is distributed between these structures is critical to understand the biology and mechanics of the actin cytoskeleton and therefore its role in processes ranging from morphogenesis and cell division to endocytosis and polarization.
We take advantage of high-resolution techniques to tease out the general rules and the critical regulatory elements that control actin dynamics.
On the long term, this project may also improve our understanding of the general mechanisms that underlie the regulation of the actin cytoskeleton machinery, and how deregulations may be responsible for the onset of specific behaviors of the actin cytoskeleton that may eventually result in the development of cancer-like cellular behaviors.
Aim1: To probe actin homeostasis during maintenance phase at the 1-cell stage, we used existing tools based on single-molecule imaging to quantitatively measure key parameters of the system. Using this approach, we were able to quantitatively characterize actin dynamics in the 1-cell stage embryo.
Aim2: In order to challenge the system, we wanted to vary protein concentration precisely. We therefore decided to implement a new technique based on optogenetics to cluster proteins in the cytoplasm, called GFP-Lariat.
Aim3: Using fluorescence imaging of regulators of actin dynamics (RhoGTPases, formin), we explored the modulation of actin dynamics during morphogenesis. Focusing on this process, we analyzed the temporal dynamics of the regulatory cascade controlling actin dynamics. Our results suggest a mechanisms strikingly different from the regulatory control at the 1-cell stage. To support our analysis of actin dynamics between different embryonic stages and cell types, we are using our numerical model of actin assembly in combination with the data collected in aim 1 and aim 2.
We expect that this work will result in a publication in a high impact journal. Moreover, we are currently initiating collaborations with several groups in the field to share the technical developments generated during this work.
Single Fluorescent Actin Monomer in a Polymer Filament