Actin network in cells looks highly complex. It is composed of thousands of filaments which appear more or less regularly organized depending on the specific structures they belong to. Actin filament organisation seems highly ordered in stress fibers at the cell rear and rather randomly arranged in the lamellipodium at the cell front. The rules governing the geometrical arrangement of actin filaments are quite mysterious and difficult to study in cells where hundreds of protein of dozens of signaling pathways work together to regulate network dynamics and architecture. In vitro reconstitution of actin assembly with controled mixtures of purified proteins constitutes a good alternative to circumvent this complexity. Micropatterning techniques can be used to control the location of actin filament nucleation sites. It is thus possible to control the geometry and biochemical composition during actin filament growth and thus identify the exact rules governing the spatial arrangement of actin filaments during network self-assembly.
Cell adhesions are the first structures whom assembly and localisation depend on the geometrical configuration of cell microenvironment. These large transmembrane super-complexes constitute the structural basis on which the entire cytoskeleton will be constructed. They are fine mechanical sensors and transducers, a function which is directly related to their localisation at the junction between extracellular fibers and intra-cellular actin filaments. Their assembly is also regulated by mechanical and biochemical signals from the cell cytoskeleton. Their physical state is interesting to investigate since it results from the equilibrium between extra-cellular physical and geometrical constraints and intra-cellular architecture.
The specific astral configuration of the microtule networks gives it the property to collect, gather and integrate signals of all kinds throughout the entire cytoplasm. The centrosome has a central position from where it regulates the network by nucleating and sometimes anchoring the microtubules.It has a private connection with the nucleus. It is linked to the Golgi apparatus and thereby is strongly implicated in intra-cellular traffic. In our mind, it plays a key role in the spatial integration of signals that are transported along microtubules. Its position with respect to other organelles reflects a mechanical and functionnal equilibrium. It varies along the cell cycle. The investigation of the parameters governing its positioning should bring much light on the regulation of cell polarity and the way it is set in coherence to external spatial informations.
Organism morphogenesis is mainly based on cell division. It is a dramatic transformation, called mitosis, by which one cell gives rise to two daughter cells. The cell architecture undergoes a complete reorganisation during which dynamics and spatial arrangements of the cytoskeleton networks are profondly changed. The orientation of the division with respect to external cues and the final position of the two daughter cells participate to proper organ formation and renewal. It is an intrinsically asymmetric process since all cell components have to be duplicated prior to mitosis and segregated between the two cells which will receive either the original or the duplicate. This asymmetry is more or less determinant depending on cell state, the level of asymmetry and the way how it is coupled to cell microenvironment. In vivo, many factors of diverse origin affect the regulation and orientation of cell division. It is difficult to identify the respective role of each. Cell culture conditions are highly artefactual and one could wonder what remains from the physiological event. The mechanism and the cellular structures involved are still present. But, the variability of the conditions in which they orchestrate the division hide some clear rules that can be identified using micro-engineered environments.
Micropatterns are particularly usefull for quantitative image analysis. Since all cells have the exact same shape, pictures from numerous distinct cells can be aligned and projected on each other with various mathematical operator. One can obtained the averaged spatial distribution of a given cell labelling. We call this map the "averaged cell". It is also possible to quantify the variability of signal intensity anywhere in the cell. These cell maps can highlight small and significative local variations of a labelling.
The reproducibility of internal cell organisation and cell behavior on defined micropattern geometries can be used for two types of applications. First reproducibility can be used to identify and describe the physical rules inducing this reproducibility (first part of the website), it also can be used to detect any abnormal cell behavior. when all cells behave reproducibly it becomes much easier to detect the cells which don't respect the rules. Numerous movies of cell behavior or protein mapping using quantitative image analysis with the "averaged cell" are powerful tools to detect even tiny differences between normal and abnormal cell state. We used these approaches to analyse the effect of drug treatment or gene inactivation in normal cells and to study the behavior of KnockOut cells or cancer cells. This method could easilty be transfered to a larger diagnostic tool to quantitatively and accurately compare and detect normal and abnormal cells in biopsies.
We don't have a long experience but experiments are the main component of our every day work. Most of our protocols have been inspired by or copied from the work of renowned lab. Repeated pratices and sustained efforts on specific steps to improve and facilitate the experiments give us a sort of know-how which we thought could be usefull for incomers in the field. Feel free to use our protocols and don't hesitate to share your comments on them.
World Cell Race
To have fun, and to collect interesting data on cell migration properties, we organize the World First Cell Race !! Cells from the whole world will compete and run on tracks to become the fastest cell on earth !
Conférences grand public en français