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laurent blanchoin, manuel thery

pictogramme research

RESEARCH

Directed Cytoskeleton Self-Organization

From molecules to cell polarity

From molecules to cell polarity

The reproducible shape and spatial organization of organs imply the existence of physical rules directing the assembly of complex biological structures. Organ shape and function depend on cell architecture and polarity, which are both supported by cell cytoskeleton networks. The formation of controlled and reproducible geometrical structures relies on the self-organization properties of these networks. Our aim is to unravel the physical processes underlying cytoskeleton self-organization processes and to formulate the rules directing their spatial organization.

Actin filaments and microtubules form such complex intricate networks in cells that it is difficult to identify the principles of their self-organization. Our rationale is that these principles can manifest themselves in a reproducible, and therefore understandable, way only in response to defined geometrical cues. Thus, to study the geometrical and mechanical rules underlying cytoskeleton self-organization we used microfabrication tools in order to control and manipulate the spatial boundary conditions the cytoskeleton networks are sensitive to.

These tools allow us to analyze and quantify actin and microtubule networks in cells of controled and regular shapes. Considering that the complexity of the intra-cellular biochemical conditions may partially hinder the physical rules we want to investigate, we are also developing alternative methods to analyze cytoskeleton self-organization in controlled biochemical conditions in vitro by mixing, in defined proportions, the individual cytoskeleton components. Cells extracts and cytoplastes

allow us to bridge the gap between these two approaches. The simplification of the cellular approach, the top-down way, and the complexification of the biochemical approach, the bottom-up way, should eventually encounter and provide us a continuous experimental platform to analyze the physics of cytoskeleton networks and morphogenesis from molecules to cells.

Directed Cytoskeleton Self-Organization

Actin and Microtubule Networks Geometry

Actin networks

Actin is an asymmetric protein that can self-assemble to form polarized actin filaments. Actin filaments can interact to form actin networks. Actin networks can self-organize into two main types of structures in cells: bundles (or fibers) made of aligned long filaments and meshworks made of branched and intermingled short filaments.

Our aim is to reconstitute both and investigate : 1- how the spatial distribution of actin nucleators directs the growth and assembly of filaments to form defined network geometries in 2D and 3D, 2- how the contraction and disassembly processes regulate networks turn-over and permanent renewal.

In both cases we try to relate network dynamics to its physical properties and the eventual production of mechanical forces. A particular attention is devoted to the study of the role of actin network geometry and size.

actin array actin ring contraction actin star actin sun actin Lamellipodium Self Assembled Electrical Connections

Microtubule networks

Similar to the formation of actin filaments from the self-assembly of actin monomers, tubulin forms asymmetric dimers that can self-assemble into microtubules. Compared with actin filaments, microtubules are much more rigid, and almost straight in the dimensions of a single cell. Microtubules can sustain much higher

compression forces than actin filament but are not as numerous as actin filaments. In most animal cells, the MT network forms as an aster in which microtubules radiate from the microtubule organizing center (MTOC). As cells divide, the MTOC is duplicated and the network forms a bipolar spindle. Our aim is to investigate the

role of microtubule mechanics and microtubule-associated proteins in the establishment of the various microtubule network architectures during interphase and mitosis.

averaged microtubule asters microtubule aster microtubule aster montage microtubule asters microtubule spindle kip3

Actin-microtubule crosstalk

Actin and microtubule networks interact by many ways including common signaling pathways, specific crosslinkers or via less specific steric interactions. We currently work

on the reconstitution of such interactions and their contribution to the establishment of coherent heterogeneous cytoskeleton networks architectures.

actin mt crosstalk

Budding Yeast as a model system

The efficiency of DNA strand homologous recombination in budding yeast makes it an organism of choice to remove one or several genes of interest. In addition, yeast culture methods offer the possibility to extract cell cytoplasmic content and work with large

amounts of material. We use yeast extracts to investigate cell cytoskeleton self-organization in complex, because made of whole cellular content, but controlled, because genetically modulated, biochemical conditions. Adding more and more compounds to mixtures of

purified proteins and removing proteins from cell extracts will allow us to bridge in vitro and in vivo approaches and benefit from a broad range of biochemical conditions to investigate cytoskeleton self-organization laws.

yeast extracts

Cell Architecture and Polarity

Mechanics of Centrosome Positioning

Actin Network Mechanics

We are interested in the regulation of mechanical forces production by the actin cytoskeleton. Using traction force microscopy with micropatterned cells we can precisely quantify the forces developed on cell-matrix and on cell-cell adhesions. We investigate the role of proteins associated to cell adhesions and actin

cables as well as the effect of actin cable’s size, number and spatial organization in cells. We are interested in the mechanical changes associated to key morphogenetic event such as the epithelial to mesenchymal transition.

actin cytoskeleton cell doublet forces cell cytoskeleton crossbow

Centrosome Positioning

The centrosome got its name from its geometrical position “at the center of the soma”. It is indeed generally observed at the geometrical cell centre in culture conditions or during specific developmental stages. This centering mechanism is remarkably robust and can adapt to asymmetric external adhesive cues. However, in some specific conditions, the

centrosome can be off-centred and close to the cell periphery. It is notably the case in quiescent cells where it supports the growth of the primary cilium. Our aim is to understand the mechanisms regulating centrosome centering and off-centering. In particular we would like to characterize the force balance acting on centrosome that stem from mechanical forces

developed in both actin and microtubule networks. To that end we investigate the mechanical and geometrical changes in actin and microtubule networks during two interesting events: the ciliogenesis and the formation of cell-cell junctions, since they are both coupled to centrosome position changes.

centrosome centering cell polarity crossbow cell shape ciliogenesis cell doublet polarity

Micropatterning

Providing spatial cues to self-organization processes

The development of new methods allowing us to observe and analyze cytoskeleton networks in original conditions is one of the keystones of our experimental strategy. We are permanently improving the now “classical” glass surface micropatterning method but also work on its adaptation to other surfaces such as silicones and hygrogels.

In parallel we would like to turn the permanent and flat micropatterns into dynamic and 3D micropatterns. To that end we developed a new laser micropatterning process based on protein-repellent coating ablation with pulses of light. It allows us to 1- perform contact-less micropatterning, 2- control grafted protein density, 3- control micropattern geometry

with a sub-micrometric resolution, 4- design micropatterns in 3D, 5- micropattern multiple proteins successively and 6- perform on-the-fly patterning in the presence of living cells.

micropattern rationale cell array animation livepatterning cell shape reprograming