Control of cell movement, chemotaxis and the actin cytoskeleton by Scar/WAVE: A genetic analysis using Dictyostelium

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The movement of most mammalian cells is driven by structures made of actin such as lamellipodia. However, it is still unclear how signals from inside and outside the cell control the formation of new actin structures. We and others have shown that signals can control actin through a pathway that includes the actin-nucleating Arp2/3 complex and the signalling adapters WASP and Scar/WAVE. Recent work suggests that Scar/WAVE is particularly important, but the mechanics by which its activity is controlled are still unclear. We and others have shown that a protein complex including the Rac-binding protein PIR121 holds Scar inactive. Signals, for example through Rac, cause the complex to dissociate and release active Scar/WAVE, which in turn activates actin polymerization. The signal appears to be turned off by rapid breakdown of Scar/WAVE, perhaps by proteolysis.
We will now perform a thorough analysis of the mechanisms which control Scar/WAVE activity using Dictyostelium molecular genetics. Dictyostelium is ideal for this work - it has single genes encoding Scar and PIR121, which can each be disrupted to give cells with opposite strong phenotypes. We will follow four complementary but independent paths:
(a) we will investigate the motility defects in cell lacking Scar and PIR121 using a combination of optical and electron microscopy. This will enable us to define the molecular architecture of Scar-induced lamellipods, and their normal physiological role.
(b) we will elucidate the roles of the other members of the inactive complex. It seems extraordinary that four cofactors are needed to couple Rac to Scar - we will find if they perform identical roles, using a combination of gene knockouts and RNAi.
(c) we will analyze the upstream pathways which control Scar, to show what activates it and when during normal motility. We will devise and optimize different assays for Scar activation, using both biochemical separations and FRET between Scar and inhibitory subunits. We will then investigate which signalling pathways couple to Scar and where within the cell they act.
(d) we will elucidate the mechanism by which Scar is broken down in detail.
Together these results will provide an important window on how signalling controls actin polymerization. This will be a