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MINICELL Report Summary

Project ID: 335672
Funded under: FP7-IDEAS-ERC
Country: Netherlands

Mid-Term Report Summary - MINICELL (Building minimal cells to understand active cell shape control)

The cells in our body constantly change shape to perform vital functions such as cell growth, division, and movement. Understanding how cells achieve precise and reproducible control of their shape is therefore a central scientific goal. This also has important biomedical implications since abnormal shape changes can result in life-threatening diseases such as cancer and developmental defects. The main determinants of cell shape in mammalian cells are the cell membrane and a tightly anchored polymer gel beneath it that is composed of actin filaments and myosin motors. The motors can actively drive cell shape changes by generating contractile forces that can round up or invaginate the membrane. Our goal is to understand the physical basis of cell shape control through the balance of these active forces with passive forces arising from cortex-membrane adhesion and elasticity. We address this question by building simplified cell models that combine a model biomembrane with a controlled lipid composition with a reconstituted cortex made of purified actin and myosin cortex. We focus on septins as linker proteins since septin is known to be essential for many cellular processes requiring shape changes. Our focus initially has been to set up the experimental model systems and techniques to probe self-organization, dynamics and mechanics of the minimal cells. We established two complementary model systems that allow us to investigate different aspects of cell shape control: flat supported lipid bilayers with an adhered thin cortical actin-myosin layer and cell-sized liposomes with an encapsulated actin-myosin cytoskeleton. We constructed a dedicated microscope equipped with micropipette aspiration, optical tweezers, and confocal fluorescence imaging that will allow us to measure the mechanical properties of minimal cells. We moreover added a UV laser that allows us to locally sever the cortex or locally activate myosin motors. We have so far found that septins bind to lipid membranes through anionic lipids and are able to form dense filamentous meshworks that strongly influence lipid mobility. Septins can thus contribute to the formation of diffusion barriers in the plasma membrane, even independent of the actin cortex. We found that septins also bind actin filaments and generate curved actin bundles. We now plan to study the interplay of actin and septins in model cortices attached to lipid bilayer membranes. We thus far studied the mechanical properties of actin networks by quantitative rheology measurements and found that the stiffness and rupture strength of these networks sensitively depend on the load-dependent binding kinetics of the crosslinker. We successfully developed thin actin-myosin cortical networks on flat lipid bilayers that mimic the contractile properties of the cell cortex. By systematically varying the crosslink density of the cortex and the density of cortex-membrane anchors, we found that contraction occurs only in an optimal connectivity window. Taking advantage of the high spatiotemporal resolution of TIRF microscopy we furthermore discovered that the mechanism by which myosin remodels and contracts the actin meshwork strongly depends on the type of crosslinker present.

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