Fig. 2: Bleb induced by laser ablation in a detached L929 fibroblast. 0s: <strong>of</strong>fset <strong>of</strong> ablation. Bar, <strong>10</strong>µm (author: Julia Roensch). In the long term, this approach will help defining the minimal ingredients necessary for cortex contractility, paving the way for a biomimetic system <strong>of</strong> the cell cortex. 2. Mechanisms <strong>of</strong> formation <strong>of</strong> blebs The growth <strong>of</strong> blebs depends on myosin activity and is commonly believed to directly result from intracellular pressure, however this hypothesis has not been tested and the mechanisms <strong>of</strong> bleb growth remain elusive. We have shown that laser ablation <strong>of</strong> the cell cortex leads to the formation <strong>of</strong> a bleb, supporting the view that bleb expansion is a direct, mechanical, result <strong>of</strong> intracellular pressure (Fig. 2). Moreover, multiple ablations <strong>of</strong> the same cell at different locations indicate that the growth <strong>of</strong> a bleb considerably reduces pressure. We have then induced blebs on cells with different tensions and shown that the size <strong>of</strong> a bleb directly depends on tension. This dependence can be fitted with a theoretical model <strong>of</strong> the actomyosin cortex and allows us to estimate elastic parameters <strong>of</strong> the cortex and <strong>of</strong> the cytoplasm, and to accurately predict bleb shape (collaboration with the group <strong>of</strong> J.F. Joanny, Institut Curie, Paris). We now plan to further analyze the dynamics <strong>of</strong> bleb expansion in cells with different tensions. Coupled to theoretical modeling, this will allow us to elucidate which dissipation source is the major limiting factor for bleb growth mechanics. This is particularly important because the type <strong>of</strong> protrusion formed by a cell is likely to modify its migration pattern (cf point 4) – bellow. Modifying the magnitude <strong>of</strong> dissipation linked to bleb growth can be used by cells to favor or reduce the formation <strong>of</strong> blebs versus other protrusion types such as lamellipodia. 3. Role <strong>of</strong> cortex tension and blebs during cytokinesis We have discovered that ablation <strong>of</strong> the actin cortex during cytokinesis leads to oscillations <strong>of</strong> the cleavage furrow and results in division failure (Fig. 3). Similar furrow oscillations can be observed after depletion <strong>of</strong> different actin binding 70 Annual Report 2008 proteins. Strikingly, small oscillations <strong>of</strong> the furrow can also sometimes be observed in control divisions, although their amplitude remains limited, allowing for division to proceed. Based on our observations, we have proposed that the cortex controls its own contractility during furrow ingression and prevents the built-up <strong>of</strong> an imbalance in contractile forces by self-disassembling and forming blebs above a threshold tension. When this control is removed, like for example after laser ablation, the cleavage becomes unstable and cytokinesis fails. We are currently testing this interpretation using both biophysical and molecular techniques. 4. Protrusion formation during migration in 3D-environments In 3D-environments, bleb-based migration is a widespread alternative to lamellipodial migration, and is commonly used by cancer cells and during development. It is not known why cells form one or the other type <strong>of</strong> protrusion, and how the cells can switch between protrusion types is poorly understood. Strikingly, certain cell types, e.g. mesendodermal cells in Danio rerio (zebrafish) embryos, are able to form both lamellipodia and blebs at the same time. We have initiated a study <strong>of</strong> the mechanisms <strong>of</strong> formation <strong>of</strong> these protrusions and <strong>of</strong> their respective contributions to cell migration in the zebrafish embryo (collaboration with the lab <strong>of</strong> C.P. Heisenberg, MPI-CBG). We have characterized wild type migration and have shown that the protrusions formed by mesendodermal progenitors consist <strong>of</strong> blebs (50%), lamellipodia (35%) and filopodia (15%). We have also shown that the expression <strong>of</strong> dominant negative (resp. constitutively active) ezrin (a protein linking the actin cortex to the membrane) shifts this distribution and leads to the formation <strong>of</strong> more (resp. fewer) blebs. We are currently investigating the effects <strong>of</strong> these treatments on migration. The long term perpective is to better understand the mechanisms <strong>of</strong> protrusion formation and the role <strong>of</strong> blebs during migration in vivo and in 3D environments.
Fig. 3: Oscillations induced by laser ablation <strong>of</strong> the cortex in L929 fibroblast during cytokinesis. Left: DIC. Right: Myosin regulatory light chain-GFP (author: Jakub Sedziński). Laboratory <strong>of</strong> Cell Cortex Mechanics 71
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