Partner Search Strategy and Mechanisms of Cell Polarization during Fission Yeast Mating

Abstract In the living world there are many kinds of self-organized biological patterns at different length scales from molecules to cells to animal communities. Cell polarity is a process of self-organization of cellular components into a highly asymmetric structure which leads to asymmetry of cell shape, structure or function. Cell polarization during fission yeast mating, the subject of this thesis, is part of a broader topic of cell polarization which is fundamental in biophysics and cell biology. I used computational modeling to address this topic in mating fission yeast cells.Mating fission yeast cells use diffusion-based molecular communication to find the closest potential opposite mating partner. Each mating type secretes its own specific pheromone peptide to make a pheromone concertation field in its vicinity to communicate with the neighboring opposite mating type cells. Fission yeast cells sense the pheromone gradient by binding of opposite mating type pheromones to their cognate receptors on the cell surface (Merlini et al, 2013). Initiation of cell polarization toward a mating partner in fission yeast cells involves accumulation of signaling proteins, mainly small GTPases such as Cdc42 and Ras1, into a polarity zone close to the mating partner (Park & Bi, 2007). This eventually results in directional growth of a mating projection (shmoo) from both partners toward one another and fusing with each other. Recently Bendezu et al showed that the establishment of the polarity zone in fission yeast is independent of gradient sensing (Bendezu & Martin, 2013). They demonstrated that prior to shmoo formation Cdc42, the main regulator of cell growth, accumulates into a dynamic polarity zone that explores the cell periphery in discrete jumps and stabilizes close to the opposite mating partner which is also a location with high pheromone concentration. Besides it has been previously shown that establishment of the polarity axis, accumulation of signaling lipids (such as PIP3) and signaling proteins (such as Rac and Rho) in the direction of migration, in larger motile eukaryotic model organisms like neutrophils and Dictyostelium discoideum amoebae which can migrate toward the chemical gradients is also independent of gradient sensing mechanism (Insall, 2010). In this thesis, I focus on studying the role of the polarity patch as well as the underlying mechanism for the patch formation, exploration and stabilization in mating fission yeast cells.First, we studied the role of the polarity patch in the mating selection mechanism in fission yeast cells. By developing 2D simulations mimicking a mating experiment consisting of a field of opposite mating type cells on a thin agarose pad, the effect of range of the pheromone gradient on the efficiency of the final number of paired cells was studied. The shape of a pheromone gradient field from neighboring cells depends on the diffusion coefficient of each pheromone type, the sites of pheromone secretion and the concentration profiles of the secreted proteases around the cell that degrade the pheromones (Arkowitz, 2009). We found that the combination of a local secretion and local sensing of pheromones from the polarity sites, short decay length of pheromone and pheromone-concentration-dependent scaling of the polarity patch lifetime results in the maximal number of paired cells. This study provided evidence that fission yeast applies a temporal averaging sensing strategy by employing the randomly exploring polarity patch that biases it random walk towards the opposite mating partner. These results were tested experimentally by our collaborator Dr. Laura Merlini from the Martin laboratory at the University of Lausanne.Second, we looked into the underlying mechanism of polarity patch formation, exploration and stabilization in mating fission yeast cells. Particularly, we studied the dynamic regulation of Ras1, the only Ras GTPase homolog in fission yeast, through positive and negative feedbacks. Ras1 is an upstream regulator of Cdc42 and is essential for polarity establishment and mating (Merlini et al, 2013). Like other GTPases it exists in inactive form of guanosine diphosphate (GDP) and active form of guanosine triphosphate (GTP) states. We developed a 3D reaction-diffusion model taking into account the diffusion and the interactions between Ras1-GDP, Ras1-GTP and Gap1, the only GTPase-activating protein (GAP) for Ras1, on the curved geometry of the cell membrane. By implementing an autocatalytic positive feedback and a negative feedback through the Ras1-GTP recruited GAP, Gap1, the model captured the appearance and disappearance behavior of the patch at random locations on the cell cortex. To estimate the diffusion coefficients and membrane dissociation rates of each component, we analyzed and used 3D simulations to fit the data from the Fluoresce Recovery After Photo bleaching (FRAP) experiments performed by Dr. Laura Merlini. Furthermore, we investigated the switch from exploration to stabilization of the Ras1 patch upon sensing of higher concentrations of pheromone in its vicinity. The patch in this model becomes stabilized at positions with higher rate of positive feedback, which may result from higher pheromone concentrations in its vicinity. The model predicts that the patch size and number can be regulated through positive and negative feedback rates. In simulations an increase in the negative feedback rates results in narrower patches and an increase in positive feedback results in multiple simultaneous patches. These results were then tested and supported experimentally by our collaborator Dr. Laura Merlini.