Cortical Actomyosin Dynamics in Development and Morphogenesis (CADMO)

We work to understand how embryos change shape during development, and in particular, we are interested in the biological processes that control the mechanical properties of embryonic cells ad tissues.

The cortical actomyosin cytoskeleton is a major determinant of these mechanical properties. Yet, an integrated view linking the biochemical properties of the cortex, the structures it assembles and cell mechanics is still missing. We want to characterize how from the local biochemical dynamics of the cortex in vivo emerges a structured, active and dynamic polymer material that controls the cell mechanics and morphogenesis.

For this, we use a small worm, the nematode C. elegans, an extremely powerful model system, combining a variety of approaches from different fields: quantitative live cell imaging, genetics, optogenetics, biophysics and biochemistry.

For non-specialists

During the development of an embryo, each cell plays its part in well-orchestrated ballet that shapes the organism. But to shape the embryo, cells must tune their mechanical properties in a concerted and tightly regulated manner.

A thin layer of a polymer at the cell surface, the cortex, is responsible in a large part for defining cell mechanics, not unlike how the mechanics of a balloon is defined by the nature of the plastic that makes it. And the mechanical properties of this polymer come from the fibers it's made of, like how the fabric of a shirt or a sweater makes it stiff or stretch.

But in addition to these traditional properties, the cortex has two additional skills. First, it builds up and breaks down very rapidly: it is dynamic. If you leave a water balloon on a shelf and then decide a year later to pick it up again, the same material would be there, the same polymer fibers. Same is true for the fabric of your shirt or your sweater. In the cortex, on the other hand, a filament has a shelf life of seconds to minutes. Go take a coffee for two minutes, come back: all the filaments that make it have been replaced by new ones. This has a very important consequence the cortex is visco-elastic. At short time scale, that is if you do something quick, it will behave like a spring, we talk of an elastic material. But at long time scales it behaves like a fluid. If you pull on it slowly, it will change its shape and adjust to a new one, just like honey. The second skill of the cortex is its ability to contract: it is active. The cortex is made of filaments, like fibers in a plastic or a shirt, but also of miniature motor that can contract. This gives rise to a very interesting property of the cortex: it can actively shrink. Imagine your sweater shrinking in the back, while new fabric is being weaved in the front, or your kid's waterballoon shrinking on one end while new rubber forms on the other side. Well, that's basically what happens to some cells as they migrate.

Our aim is to understand how the assembly of the cortex, this dynamic and active polymer, controls cell mechanics. 

Vidéo

Vidéo François Robin

Previous works

Spatiotemporal patterning of actomyosin contractility plays a key role in cell and tissue morphogenesis during early development. In embryonic cells, actomyosin arrays are highly dynamic structures that remodel on a time scale of ten seconds, through a combination of turnover – local filament assembly/disassembly and motor recruitment/inactivation – and motion – rapid spatial redistribution of filaments, motors and other network elements driven by the network's contraction, and caused by myosin activity or actin polymerization. Because of these dynamic and active properties, contractility is complex and intrinsically self-organizing.

We used the C. elegans early embryo to understand how cells pattern force generation through local modulation of self-organized contractility, focusing on pulsed contractility in the C. elegans embryo.

Pulsed contraction represents a widespread mode of actomyosin contractility in which transient local accumulations of F-actin and Myosin II accompany local contractions of the cell surface. Such pulsed contractions have been described in a wide range of cells and tissues, including the C. elegans zygote and later embryonic stages in multiple settings in drosophila, in Xenopus, or in cell cultured.

We combined two-color fluorescence imaging, live single-molecule imaging, particle tracking, image analysis, and numerical modeling to tease apart the mechanisms of pulse initiation and termination. Strikingly, our results demonstrated that the mechanical component (advection) played a little role in pulse initiation or termination, and that the process was mostly governed by actin and myosin turnover. In our system, autocatalytic RhoA activation/recruitment is responsible for pulse initiation, while the delayed recruitment of a RhoA inactivator (RGA-3/4) onto actin filaments drove pulse termination.

This multidisciplinary work uncovered the regulatory mechanism of actin dynamics during pulsed contractions, demonstrating that these pulses do not arise as simple mechanism of actin and myosin mechanical properties, but instead arise as a simple consequence of the local auto-activation of the RhoA pathway.

Imaging

A large part of our work uses live imaging to observe the dynamics of cortical and membrane proteins and understand how they generate forces across cells and tissue.

We use TIRF microscopy to visualize cell surface of the embryo, and record single-molecule dynamics of cell surface proteins. We then use image analysis technique, single-particle tracking and mathematical models to extract protein mobility and turnover.

In the lab, we combine all these approaches technique to quantify spatial and temporal variations in mobility and turnover for a range of surface proteins, including polarity proteins (PAR-1, PAR-6), actin, and actin dynamics regualtors (nucleators, elongators, severing proteins and crosslinkers), the molecular motor myosin, and actomyosin dynamics regualtors. These tools give us the opportunity to address a variety of questions in a simple, versatile and minimally invasive manner, a technique that can be applied to any of the large and growing collection of transgenic strains expressing GFP-tagged fusion proteins in C. elegans.

F. B. Robin, W. M. McFadden, B. Yao, E. M. Munro, Nat Meth. 11; 677-682 (2014).

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