Dynamics of Cell Polarity Establishment – DynCellPol

Cell polarity is critical for the development and maintenance of virtually all organisms at a multitude of scales from subcellular to individual cells, tissues and organs. This evolutionarily conserved process also ensures asymmetry with respect to cellular compartments, proteins and lipids and is critical for morphogenesis. Loss of or disrupted cell polarity is a hallmark of a range of diseases and birth defects, e.g. it is a crucial event in cancer progression. Furthermore, changes in morphology are crucial for the virulence of a number of plant and human fungal pathogens. Cell polarity is highly conserved from yeast to humans with key polarity proteins, such as the small Rho GTPase Cdc42 virtually identical. Little is known about the mechanisms by which a new polarity site is initiated and maintained in an already asymmetric cell. The objective of this project is to determine quantitatively the cellular repercussions of temporal and spatial perturbation of cell polarity in two evolutionarily divergent, highly asymmetric cell types. We will focus on pear-shaped pheromone-induced shmoos of the baker’s yeast Saccharomyces cerevisiae and the filamentous hyphal cells of the human fungal pathogen Candida albicans. We will use novel light-dependent recruitment systems to probe temporal and spatial control of cell polarity in these yeasts, which are different in size and shape. Specifically, such light-dependent systems will be used to recruit an active form of Cdc42 to the plasma membrane and we will determine how both site-specific and uniform plasma membrane recruitment alters cell growth, cell shape, membrane compartments, cytoskeleton organization, polarity protein and membrane lipid distribution. We will investigate three distinct stages following light-induced plasma membrane recruitment of active Cdc42: I) initial growth site perturbation/disruption; II) absence of a growth site (dynamic stage); III) establishment of a new growth site. We will identify the proteins/lipids that are required to stabilize an existing growth site and what determines the competition between two sites. We will determine which cellular compartments reorganize, following destabilization of the initial growth site (as well as when and how). Furthermore, we will determine the order of appearance of components and mechanisms that dictate when and where a new growth site is established in an already asymmetric cell. Finally, in comparing these processes in cells with different geometry/size, we will determine their importance in polarity site disruption and compartment dynamics, following disruption of the growth site and the establishment of a new polarity site. We will use live cell fluorescence microscopy, with a range of fluorescent reporters, to quantify how these perturbations affect the intracellular organization in these two cell types. Mathematical analyses, including statistical learning and hypothesis driven modeling, will be used to extract quantitative information from the experiments, to provide an unbiased description of each stage, as well as to identify key parameters, whose importance will be subsequently tested by chemical and genetic perturbation. We expect to identify short- and long- term responses to cell polarity perturbations, as well as novel links between the cell polarity and selected cellular processes and organelles. This synergistic, interdisciplinary project is the result of extensive interactions between biologists and physicists, with complementary expertise in imaging, molecular genetics, biochemistry and mathematical modeling. It is original, timely and relevant as state-of-the-art methods for precise temporal and spatial control of proteins are now available. Our preliminary data provide proof of principle for this project, which has a high potential for uncovering basic principles of cell polarity and morphogenesis, as well as identifying novel targets for human disease and fungal infection.