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The diffusion and nanoclustering of a polarity module in the lipid environment

Periodic Reporting for period 1 - Lipid and Polarity (The diffusion and nanoclustering of a polarity module in the lipid environment)

Reporting period: 2016-04-01 to 2018-03-31

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.
We developed very high-density single particle tracking Photo-Activation Localization Microscopy (sptPALM) in budding yeast. The method was developed for very high-speed imaging (50 images per second), and provided image resolution of 48 nm. To our knowledge, this represents the highest density and highest resolution of live cell imaging reported in this organism to date. The development of these experiments was made possible through our collaboration with the team of Dr. Sibarita in Bordeaux.

1. Peripheral PM protein analysis by sptPALM.
In order to investigate the utility of sptPALM in the study of PM-associated proteins, we tagged two peripheral membrane proteins, Cdc42 and the eisosome protein Pil1, and compared their dynamics in living cells. After reconstructing thousands of single molecule tracks, the diffusion of Pil1 on the PM was observed to be much more confined than that of Cdc42. These experiments demonstrate the utility of sptPALM to measure the diffusion of yeast PM proteins displaying a wide range of mobility.

2. Cdc42 displays heterogeneous diffusion on the PM.
Having demonstrated that different PM proteins display different rates of mobility, we next addressed whether individual proteins display uniform or heterogeneous mobility on the PM, and whether this may be linked to their function. We observed that Cdc42 mobility is reduced at the pole compared to the non-pole. This is important because any mechanism that reduces Cdc42 mobility at the pole will stabilize the protein, and in doing so, stabilize the polarity axis of the cell.
The reduced diffusion of Cdc42 at the pole was dependent upon the scaffold protein Bem1, which the host lab has recently demonstrated being involved in Cdc42 activation. Reasoning that reduced Cdc42 diffusion at the pole may be linked to Cdc42 activation, we imaged a mutant of Cdc42 that is constitutively active. Consistently, we observed that the diffusion of hyperactive Cdc42 was reduced even further at the cell pole than the wild type protein.

3. Cdc42 is organized in nanoclusters on the PM.
Careful examination of single molecule tracks revealed that a cohort of Cdc42 proteins tended to cluster. Using PALM, we observed that Cdc42 was enriched in sub diffraction-limited areas of the PM that we term nanoclusters. The Cdc42 nanoclusters were larger at the pole of the cell than at the non-pole. Moreover, the requirements for these larger Cdc42 nanoclusters mirrored the requirements for reduced Cdc42 diffusion in live cells: Bem1 was required for large Cdc42 nanoclusters at the pole, and hyperactive Cdc42 was observed in even larger nanoclusters at the cell pole than the wild type protein.

4. Regulation of Cdc42 nanoclustering by the lipid environment.
The levels of PS in the PM were increased or decreased through genetic or pharmacological manipulation and the resulting effects on Cdc42 nanoclustering were monitored. Increasing PS levels either genetically or pharmacologically resulted in larger Cdc42 nanoclusters than those observed at the pole of control cells. This was linked to increased Cdc42 activation, since we observed concomitantly increased levels of an active Cdc42 bioprobe when PS levels were increased on the PM. Importantly, the organization of Cdc42 into large nanoclusters in the presence of PS was dependent upon the scaffold Bem1. These results indicate that PS results in the recruitment of Cdc42 into large nanoclusters at the pole in a Bem1-dependent fashion.
These results were presented in several scientific meetings and are currently submitted for publication.
The project provided insight into PM organization and the mechanisms underlying the spatial organization of cells. We identified the nano-clustering of Cdc42, which reflects its reduced mobility at the cell pole. These features of Cdc42 dynamics are linked to its state of activation. The innovative technology used in this work is perfectly suited to investigate the mechanisms of protein mobility and PM organization in diverse biological systems. It is also expected that once published, the project will stimulate active collaborations between the host laboratory and other research groups in Europe and beyond.

1. Arias-Romero and Chernoff. Expert Opin Ther Targets. Nov. 2013; 17(11): 1263–1273.
2. Qadir, Parveen and Ali. Chem Biol Drug Dec. 2015; 86: 432–439
3. Zhou and Hancock. Biochimica et Biophysica Acta. Apr. 2015; 1853(4): 841-849
Cdc42 nanoclusters in a yeast cell