Die Systembiochemie der polarisierten Zellformgebung
Biophysik
Zellbiologie
Zusammenfassung der Projektergebnisse
To understand how eukaryotic cells generate polarized signaling at the plasma membrane, we focused on the master spatial regulators of the actin cytoskeleton: RhoGTPases. These peripheral membrane proteins form local activity patterns in the plasma membrane to selectively target key actin nucleators to distinct cellular locations. While numerous proteins are known to control the activity and membrane binding of Rho-type GTPases, we do not know how their collective action can result in membrane-associated patterns. Comprehensive biochemical understanding requires the full reconstitution of the spatial activity cycle of Rho GTPases, which has not been realized until now. In the course of this project, we succeeded in reconstituting all elementary biochemical reactions in the catalytic and membrane-binding transitions of Rho-family proteins. Specifically, we could show that the ubiquitous solubilization factor RhoGDI potently and directly extracts both active and inactive states of RhoGTPases from membranes, albeit the latter more efficiently. This adds a new, unanticipated branch to the spatial GTPase cycle, whose functional importance we established in a system of single cell wound healing. From a mechanistic perspective, our results explain how coexisting membranebound/active and soluble/inactive GTPase pools can be established from a limited set of regulators. We had also uncovered the mechanism by which RhoGTPases are recruited to cellular membranes and shed their RhoGDI solubilization factor in this process. Starting from heterodimeric RhoGTPase:RhoGDI complexes we reconstituted the recruitment of RhoGTPases to model membranes. 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). Using multi-color TIRF microscopy we showed that RhoGTPase separate from their solubilisation factor RhoGDI prior binding to 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. In contrast to assumptions in the field, we observe no evidence for an essential active RhoGDI displacement factor (GDFs) that facilitates RhoGDI removal. RhoGTPases dissociate from GDI prior to and not upon membrane association. Hence, contrary to the textbook picture, membrane recruitment does not involve a dedicated GDI displacement factor. These results show that the spatial and catalytic cycles of RhoGTPases are only indirectly coupled via i) a preferential destabilization of inactive GTPases via GDI-mediated extraction and, more importantly ii) selective stabilization of active GTPases by interacting with oligomeric of membrane-bound effector proteins. We are currently finalizing a manuscript that contains a large fraction of these findings. The results obtained up to this point pave the way for the reconstitution of self-organized RhoGTPase polarity, which we will pursue in the near future.
Projektbezogene Publikationen (Auswahl)
- Extraction of active RhoGTPases by RhoGDI regulates spatiotemporal patterning of RhoGTPases. eLife, 2019 Oct 24;8:e50471
Golding AE, Visco I, Bieling P, Bement WM
(Siehe online unter https://doi.org/10.7554/elife.50471)
