Assay for Adhesion and Agar Invasion in S. cerevisiae

Summary

We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation.

Abstract

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8 The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar. Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.

Protocol

1. Put 200ul of growing cultures of interest on synthetic media plates with the required starvation conditions (SC with 2% glucose versus SC with 0.2% glucose, for example) If the density of cultures are too different from each other, adjust cell count per 200ul culture so each drop has roughly the same amount of cells.
2. Make sure to keep records of which drop on the plates is which culture.
3. Keep the plate lid ajar and leave either at room temperature or at 30°C until drops are dry.
4. Seal plates with parafilm and plastic wrap (optional) and leave in 30°C for 3-7 days.
5. Document the growth of cells on plates (by scanning, taking ....

Discussion

Yeast cells display various differentiation modes according to nutrient availability and environmental conditions, including spore formation under starvation and stress conditions, filamentation under various nutrient stresses, and flocculation. Various yeasts, including S. cerevisiae and C. albicans, can also be found in biofilms formed by a diverse set of microorganisms. Though there is some correlation with filamentation and invasive behavior, it is not clear exactly how filamentation might cause invasion and coloniza.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Moticam 350	Camera	Motic	discontinued (new model: Moticam 352)	A relatively cheap camera that attaches to eye pieces of microscopes and captures digital images for PC or Mac.