Making Epistasis Fun

With some basic ingredients – including common yeast, a few test tubes, and notebooks – Dr. Maitreya Dunham’s broader impacts project has not only created research experiences for high school students – the work has also yielded new findings on specific interactions between genes (epistasis) that influence yeast resistance to azoles. Azoles are a class of synthetic anti-fungal compounds that inhibit the growth of yeasts and fungi, including those that affect foods and health.

The student-run experiments are a component of a collaborative project between Dunham and co-investigator Dr. Paul Rowley (“Collaborative Research: Eukaryotic virus-host interaction and evolution in Saccharomyces yeasts” (NSF award #1817816)). The students grow common yeast (S. cerevisiae) in a media containing an azole known to inhibit yeast growth. Successive generations of the most successful yeast are transferred to media with increasingly greater levels of azole. Students track the progression and return the final yeast cultures to Dr. Dunham’s lab for genetic sequencing. After the yeast’s genomes are sequenced, Dunham and her team return the results to the school and students research the mutations as part of their classwork.

The research enables students to observe how mutations in specific genes interact, and how correlated mutations lead to different changes in azole resistance. When the interactions are not additive, but are either greater or less than expected, it’s known as epistasis.

Pigmented yeast makes the research competitive and the experiments more visually exciting. In “yeast fights,” students observe the growth of differently colored yeasts to track which strains are more drug resistant. The colored yeasts come from the lab of Dr. Jef Boeke, also an MCB-funded researcher, and are developed by research assistants “playing” with yeast in their spare time. Some high school students call these colonies their “yeast babies” and, Dunham says, the students are excited to learn what genetic mutations are present in the final yeast colony.

The project itself has evolved, enabling the experiments to persist despite school closings caused by the COVID-19 pandemic. includes a step-by-step protocol and a form for requesting more information on how to participate.

Other collaborators include Dr. Ryan Skophammer, a biology teacher at Westridge School for Girls in Pasadena, CA, who initiated the idea by asking Dunham for a “real” science project for his class; Dr. Bryce Taylor, a yeast geneticist in Dunham’s lab who provides the genetic sequencing; and three undergraduate students. Rowley, the project’s co-investigator, runs yEvo labs in schools local to him in Idaho. “I feel like this is what a real scientist does,” wrote one student in response to a survey question. And indeed, it is just what real scientists do.

SHARING MCB SCIENCE: Cellular Polarization in Yeast

Many cellular processes are regulated by the ability of a cell to transmit and receive signaling molecules such as hormones, cytokines, and neurotransmitters. The binding of a signaling molecule to the extracellular portion of a receptor protein embedded in a cell’s membrane starts a signaling cascade inside the cell, which activates intracellular proteins and changes cellular responses. G proteins constitute a large family of intracellular proteins that act as molecular switches inside the cell upon coupling with G protein receptors in the membrane.

The mating process in Saccharomyces cerevisiae (budding yeast) requires the activation of a heterotrimeric G protein switch in response to the yeast cell’s G protein receptors binding pheromone signaling molecules released by a potential mate. This activation results in the growth of a mating projection–or shmoo–extending in the direction of the pheromone source. Cellular growth in response to a chemical stimulus is called chemotropism. The location and accumulation of G protein receptors across the cell membrane plays a critical role in the cell’s ability to detect the pheromone source and directional growth towards a mating partner. However, some of the molecular mechanisms underlying shmoo growth and G protein receptor accumulation have not been well understood until recently. A discovery by Dr. David Stone, a Professor in the Department of Biological Sciences at the University of Illinois at Chicago, and his research team has led to advancements in this area.

As mentioned previously, G protein receptors that bind pheromones are not uniformly distributed in the cell’s membrane. Instead, they cluster wherever the pheromone concentration is the highest and a shmoo grows in the direction of the cell’s suitor. The interaction between the pheromone and G protein receptor causes intracellular signaling, resulting in the delivery of secretory vesicles and cytoskeletal rearrangement. The delivery of the secretory vesicles is actin cable-directed. These processes ultimately lead to yeast cell fusion and reproduction. The mechanism that Dr. Stone and his team wanted to understand was how G protein receptors polarize towards the source of the pheromone gradient and determine the position of the shmoo growth site prior to the delivery of secretory vesicles and cytoskeletal rearrangement.

In an article published in Science Signaling, the Stone lab showed that the interaction of an enzyme called yeast casein kinase 1 (Yck1) with a G protein (Gβ) at the cell membrane plays an important role in the establishment of polarity by inhibiting phosphorylation (addition of a phosphoryl group) and internalization of the G protein receptor. The authors further showed differential phosphorylation of the receptor is essential to pheromone gradient sensing and generates receptor polarity independently of actin-cable nucleation and vesicle delivery. Dr. Stone and his team also proposed that differences in G protein receptor occupancy modulate the chemotropic growth site in budding yeast through signal amplification produced by two positive feedback loops that the authors describe in the publication.

When asked about the broader impacts of his research, Dr. Stone responded:

“My lab has contributed to an emerging paradigm in G-protein coupled receptor (GPCR) signaling which is of broad interest to the cell signaling community: GPCRs and their associated heterotrimeric G proteins are spatiotemporally regulated, and in turn, regulate spatiotemporal processes. We were among the first groups to show that heterotrimeric G proteins serve to recruit effector molecules to specific regions of the plasma membrane at specific times.

My NSF-sponsored research program has involved scientists in training at all levels — from middle school and high school students to undergraduate students, graduate students and postdoctoral fellows. Most notably, I worked closely with a group of 6th graders from a local middle school to formulate a proposed experiment to be carried out on the International Space Station. NASA picked our experiment from a national pool of applicants, and the experiment was performed by astronauts on the International Space Station. The students set up the experiment and analyzed the results in my lab. The launch of their experiment was covered by local media. Over the years, my research groups have included a significant number of women and underrepresented minorities (>50%). The training of my graduate students has been highly enriched by their visits to the labs of our numerous collaborators, who have provided them with experience in a range of approaches—e.g., proteomics, advanced imaging, microfluidics, and mathematical modeling.”

This work is partially funded by the Division of Molecular and Cellular Biosciences, Award #MCB-1415589.