Reconstitution of RhoGTPase membrane loading

Abstract

The polarisation of membrane-bound signalling molecules that control the actin cytoskeleton, such as Rho-type GTPases and phosphoinositides is essential for the spatial organisation of cell shape. However, the ubiquitous solubilisation factor RhoGDI (Guanosine Dissociation Inhibitor) sequesters the majority of RhoGTPases in the cytosol, away from the membrane. Starting from defined complexes of RhoGTPases and RhoGDI, we investigate how RhoGTPases load onto membranes. To this end, we reconstituted the recruitment of the RhoGTPase Cdc42 to supported lipid bilayers and followed this process by multi-colour single molecule imaging. This allowed us to investigate of the effects of several suggested regulators of Cdc42 recruitment. These included regulators catalytic cycle such as activating guanosine exchange factors (GEFs), the biochemical composition and characteristics of the membrane such as charge or lipid composition, and the use of cell lysate to probe for a still unidentified GDI dissociation factor (GDF). By capturing these events as a high time resolution, it was possible to not only understand what modulates Cdc42 recruitment, but also to decipher at what point during the recruitment process GDI dissociated. Using reconstitution along with powerful TIRF microscopy and the development of single molecule tracking approaches we showed that RhoGTPase separate from their solubilisation factor RhoGDI prior binding to the membrane. Furthermore, once separated from RhoGDI, it is the composition of the membrane itself which regulates the binding frequency of RhoGTPases at the membrane. We go on to show that, on a supported lipid bilayer, the GEF ITSN is incapable of promoting recruiting of Cdc42 alone, nor the Cdc42:GDI complex suggesting that the catalytic and spatial cycles of the RhoGTPases not coupled in the process of RhoGTPase membrane recruitment. Finally, through the inclusion of cell lysate in our reconstitutions and in contrast to assumptions in the field, we observe no evidence for an essential active RhoGDI displacement factor (GDFs) that facilitates RhoGDI removal.