Architecture of developing multicellular yeast colony: spatio‐temporal expression of Ato1p ammonium exporter

Abstract

Yeasts, when growing on solid surfaces, form organized multicellular structures, colonies, in which cells differentiate and thus possess different functions and undergo dissimilar fate. Understanding the principles involved in the formation of these structures requires new approaches that allow the study of individual cells directly in situ without needing to remove them from the microbial community. Here we introduced a new approach to the analysis of whole yeast microcolonies either containing specific proteins labelled by fluorescent proteins or stained with specific dyes, by two-photon excitation confocal microscopy. It revealed that the colonies are covered with a thin protective skin-like surface cell layer which blocks penetration of harmful compounds. The cells forming the layer are tightly connected via cell walls, the presence of which is essential for keeping of protective layer function. Viewing the colonies from different angles allowed us to reconstruct a three-dimensional profile of the cells producing ammonium exporter Ato1p within developing microcolonies growing either as individuals or within a group of microcolonies. We show that neighbouring microcolonies coordinate production of Ato1p-GFP. Ato1p itself appears synchronously in cells, which do not originate from the same ancestor, but occupy specific position within the colony.

Cell Signals, Cell Contacts, and the Organization of Yeast Communities

“…Two recent studies introduced new methods for investigating colony structure. In the first of these, the pattern of expression of a green fluorescent protein (GFP) fusion gene within colonies was monitored using two-photon excitation fluorescence microscopy (59). By this method, fusion gene expression in up to 10 to 20 cell widths can be visualized inside the colony.…”

“…Several recent studies clearly establish that different regions of yeast communities express different genes. In one striking example, spot colonies express an ATO1-GFP fusion gene (59) in different regions of the colony at different stages of colony development. ATO1 encodes a transporter that exports ammonium from the cell, and prior to extensive ammonium production, colonies express ATO1-GFP only in a narrow layer of cells at the top surface of the colony.…”

The Rim101p/PacC Pathway and Alkaline pH Regulate Pattern Formation in Yeast Colonies

“…For example, periodic ammonia production during colony maturation can subdivide colonies into regions of cell growth and cell death (Vachova and Palkova 2005), colonies can form a skin-like cell layer on their surface (Vachova et al 2009), and S. cerevisiae can form elaborate biofilm-like structures on agar surfaces (Reynolds and Fink 2001). Finally, yeast colonies preferentially contain sporulated cells at their edge .…”

Cryosectioning Yeast Communities for Examining Fluorescence Patterns

“…However, direct optical sectioning using confocal or two-photon microscopy has been only able to reach a few cell layers deep into yeast colonies. This limitation is likely because of strong scattering of light from yeast cells 4 .…”

“…Optical sectioning techniques, including confocal and two-photon microscopy, have proven useful for observing spatial organization of bacterial and archaeal communities 2,3 . A combination of confocal imaging and physical sectioning of yeast colonies has revealed internal organization of cells 4 . However, direct optical sectioning using confocal or two-photon microscopy has been only able to reach a few cell layers deep into yeast colonies.…”