According to the World Health Organization, cancer is the second most important cause of death and morbidity in Europe. Understanding the mechanisms by which cancer cells proliferate and disseminate will identify new treatments.
Aberrant regulation and activation of Ras-family GTPases, a network of proteins that control cell proliferation and migration, has been linked to tumorogenesis in diverse cancers.
This study focused on one family member, Cdc42. Recent work demonstrated that Cdc42 activation by oncogenic Ras is crucial for Ras-mediated tumorogenesis. Thus, understanding how Cdc42 is activated may provide drug targets aimed at blocking oncogenic Ras signaling (1, 2).
The project's objective was to understand how Cdc42 activation is controlled. Recent work highlights the importance of protein diffusion on membranes and the organization of proteins in discrete, nanometer-scale signaling hubs on the plasma membrane (PM) that serve as reaction centers. The best-studied example of this nanoclustering, is that of Ras-family proteins. Ras forms nanoclusters in cell membranes that contain the active GTPase, hence, mutants affecting nanoclustering also perturb Ras signaling (3). At the outset of the project, it was unknown if Cdc42 was also organized in nanoclusters, what function this serves, and what may regulate Cdc42 nanoclustering at the PM.
Studying the nanoclustering and diffusion of proteins on the PM presents formidable technical challenges due to the short spatial and temporal scales at which these processes occur. We developed super-resolution microscopy technology, combining very high-speed imaging of fluorescently-tagged Cdc42 with powerful tracking software to facilitate single molecule localization of Cdc42. This enabled us to measure Cdc42 diffusion in live cells and nanoclustering in fixed cells. The experiments were performed in budding yeast, a model organism used to understand fundamental mechanisms of signaling.
In budding yeast, Cdc42 is concentrated and activated at a unique site on the PM, the pole, which is the site used to establish a polarity axis for cell growth and division during the cell cycle. Cdc42 activation at the pole of the cell is controlled by its activating GDP-GTP Exchange Factor (GEF), Cdc24, and the associated scaffold protein Bem1. Cdc42 activation and localization have also been shown to be influenced by the lipid composition of the PM, particularly the negatively charged lipid phosphatidylserine (PS), which is also enriched at the cell pole relative to the non-pole of the cell. The objectives of this project were to understand how the diffusion of Cdc42 relates to its activation; whether Cdc42 is organized in nanoclusters; and whether the lipid environment plays a role in this regulation. Collectively, the results would provide insight into the organization and activation of a critical polarity protein and, more generally, how signaling at the PM is controlled by Ras-family members.
