Yeast Mating and Image-Based Quantification of Spatial Pattern Formation

Abstract

Communication between cells is a ubiquitous feature of cell populations and is frequently realized by secretion and detection of signaling molecules. Direct visualization of the resulting complex gradients between secreting and receiving cells is often impossible due to the small size of diffusing molecules and because such visualization requires experimental perturbations such as attachment of fluorescent markers, which can change diffusion properties. We designed a method to estimate such extracellular concentration profiles in vivo by using spatiotemporal mathematical models derived from microscopic analysis. This method is applied to populations of thousands of haploid yeast cells during mating in order to quantify the extracellular distributions of the pheromone α-factor and the activity of the aspartyl protease Bar1. We demonstrate that Bar1 limits the range of the extracellular pheromone signal and is critical in establishing α-factor concentration gradients, which is crucial for effective mating. Moreover, haploid populations of wild type yeast cells, but not BAR1 deletion strains, create a pheromone pattern in which cells differentially grow and mate, with low pheromone regions where cells continue to bud and regions with higher pheromone levels and gradients where cells conjugate to form diploids. However, this effect seems to be exclusive to high-density cultures. Our results show a new role of Bar1 protease regulating the pheromone distribution within larger populations and not only locally inside an ascus or among few cells. As a consequence, wild type populations have not only higher mating efficiency, but also higher growth rates than mixed MAT a bar1Δ/MAT α cultures. We provide an explanation of how a rapidly diffusing molecule can be exploited by cells to provide spatial information that divides the population into different transcriptional programs and phenotypes.

Quantitative analysis of the yeast pheromone pathway

“…Therefore, Bar1 limits Fus3 phosphorylation, but not Fus3 induction, and the difference between the mutant and wild-type strains is most evident at lower doses of pheromone. Collectively, these data are consistent with the fact that Bar1 degrades α-factor and dampens the downstream signal over time (Banderas, Koltai, Anders, & Sourjik, 2016;Barkai et al, 1998;Chan & Otte, 1982b;Chen, Nie, Yi, & Chou, 2016;Diener et al, 2014;Jackson & Hartwell, 1990a;Jin et al, 2011;Segota & Franck, 2017).…”

“…By this measure, cells lacking Bar1 or expressing gpa1 G302S failed to properly track a gradient; the gpa1 G302S cells in particular displayed half the persistence of that in wild‐type cells. The tracking defect exhibited by the bar1 Δ cells is well documented (Banderas et al, ; Chan & Otte, ; Chen et al, ; Ciejek & Thorner, ; Diener et al, ; Hicks & Herskowitz, ; Jackson & Hartwell, ; Jones, Clarke, Craik, & Bennett, ; Segota & Franck, ). The properties reported here for gpa1 G302S resemble those reported previously for cells lacking SST2 (Kelley et al, ).…”

“…Both Sst2 and Bar1 are transcriptionally induced in response to pheromone and are consequently required for desensitization. Sst2 is also required for proper gradient tracking, whereas Bar1 is required for proper gradient formation (Andrews, Addy, Brent, & Arkin, 2010;Barkai, Rose, & Wingreen, 1998;Diener et al, 2014;Dixit, Kelley, Houser, Elston, & Dohlman, 2014;Jin et al, 2011;Kelley et al, 2015;Moore, Chou, Nie, Jeon, & Yi, 2008;Moore, Tanaka, Kim, Jeon, & Yi, 2013;Segall, 1993). Collectively, these mutants and measurements have helped to establish predictive models that are transforming our understanding of cell signal regulation.…”

Evolutionary Selection on Barrier Activity: Bar1 Is an Aspartyl Protease with Novel Substrate Specificity

“…Bar1 is produced by a cells and sharpens the gradient of α pheromone produced by α cells, thereby facilitating chemotropism and mating between a and α partners ( 31 – 33 ). In addition to increasing mating efficiency, Bar1 promotes higher growth rates in subpopulations of a cells exposed to α pheromone by overcoming pheromone-induced cell cycle arrest ( 34 ). Interestingly, loss of Bar1 in C. albicans results in transitioning from a heterothallic to a homothallic (selfing) mode of sexual reproduction.…”

Spatial modeling of the membrane-cytosolic interface in protein kinase signal transduction

“…The first asymmetry is a cell with a nucleus moved away from the cell center (see Figure 5). The second asymmetry is a cell with a protrusion, as occurs for instance in mating yeast Diener et al (2014). Both cellular asymmetries induce an intracellular gradient along the cell membrane as well as the nuclear membrane even if a spatially homogeneous gradient is applied on the cell surface (see Figure 5B).…”