Polarizing cells undergo widespread molecular rearrangements in a spatially and temporally concerted manner, reorganizing cellular components along the primary cellular polarized growth axis. Polarization mechanisms are important for both basic cell division and specialized growth processes, such as formation of neuronal processes,(1
) cell motility(2
) and asymmetric stem cell differentiation.(3
) The resultant coordinated asymmetry affects major developmental, cell division, and metabolic programs in the cell. The budding yeast, Saccharomyces cerevisiae
, has proved an excellent model system for studying polarized growth, as it restructures the cell differentially in vegetative growth, mating and filamentous growth.(4
) Each of these developmental fates is characterized by a distinct morphology, and the stimulus and outcomes of each differentiation pathway are unique. For example, internal bud cues from the previous bud site drive the cycle of bud emergence and cytokinesis. External cues such as mating pheromone and nutrient starvation trigger signal transduction pathways that induce formation of a mating projection or the filamentous ‘foraging’ phenotype, respectively. These changes in cellular morphology are accompanied by dynamic changes in the cellular proteome that function in setting up the polarization axis and orchestrating specific activities required for the appropriate growth response.
Wild-type yeast cells, upon treatment with pheromone from the opposite mating type, undergo a differentiation program typified by the formation of a mating projection (the ‘shmoo’) accompanied by cell cycle arrest in the G1 phase (reviewed in refs (4
)). Many proteins are redistributed to different subcellular locations culminating in the formation of a highly structured polarization axis oriented toward the shmoo tip. Although similar polarization mechanisms dominate the formation of a bud and a mating projection, there are obvious differences underlying the two processes. While budding is preordained by location of the previous budding landmark, mating projection formation appears to be a dynamic process that relies in the wild on a directional pheromone gradient. Mating-competent cells can therefore continuously sense pheromone gradients and alter the site of projection formation accordingly, and in some instances, even sequentially form multiple ‘shmoos’.(4
) Cells subjected in the laboratory to uniform pheromone concentrations form randomly oriented mating projections. The shape and structure of mating projections also differ from buds, with the conspicuous constriction that marks buds being largely absent at the base of the ‘shmoos’. This region contains the septins, chitin, and pheromone induced proteins such as Afr1p that interact with the septins. It is therefore likely that an altered set of protein interactions during the mating process determines the shape and morphology of the projection and distinguishes it from bud formation during vegetative growth. This is more evident when one analyzes finer details such as the growth dynamics of a bud relative to a shmoo. Bud growth becomes isotropic after the apical growth phase, while a shmoo displays more unidirectional growth dynamics, possibly because of differential rates of recruitment of proteins that deposit cell wall constituents. These differences arise due to the distinct functional goals that each process seeks to achieve—nuclear segregation to the daughter and cytokinesis during vegetative growth, as opposed to cell fusion and karyogamy during mating.(4
) Systematic identification of the proteins associated with each process will therefore begin to shed light on their mechanistic differences.
Significant strides have been made in technologies for increasing the throughput of imaging protein spatial localization in mammalian cells (e.g., see refs (6
)), but the power of yeast genetics has already resulted in proteome-wide imaging and epitope tagging strategies being successfully employed in this model organism. Cellular imaging of protein localization has been conducted on transposon-tagged(8
) as well as recombination-based tagged open reading frame (ORF) libraries.(9
) Immunofluorescence and live-cell imaging of the tagged strains in S. cerevisiae
has identified the subcellular localization of most of the proteome under standard laboratory conditions and these data are now accessible through the TRIPLES, GFP/UCSF, and other databases.
We previously reported the development of spotted cell microarrays(10
) (cell chips) for measuring cell morphology and morphology defects across collections of thousands of yeast strains, more recently applied to measure a bacterial protein’s localization in thousands of differing genetic backgrounds.(11
) Briefly, spotted cell microarrays allow for cells of different genetic backgrounds to be robotically arrayed onto coated glass slides at high density, then each strain imaged in turn using automated microscopy. The cell microarray approach is readily adapted to measure eukaryotic protein subcellular localization by taking advantage of the availability of epitope-tagged strain collections, such as the green fluorescent protein (GFP)-tagged strain collection.(9
) In this strain set, each of the ~4200 S. cerevisiae
strains carries a genomic copy of the Aequoria victoria
GFP (S65T) gene fused to the carboxy-terminus of a different open reading frame. Arraying this strain set on spotted cell microarrays and imaging the entire set of strains thus measures the subcellular localizations of ~4200 proteins in parallel, providing a measure of each tagged protein’s localization under the assayed conditions. This approach might logically be combined with immunofluorescence experiments, as a major advantage of the cell chips is the minimal use of expensive reagents on the chips, achieved by limiting the use of antibodies and dyes to single microscope slides. Imaging entire libraries on chips also results in reduced imaging times in comparison to, for example, imaging the 50 96-well plates required for the complete GFP tagged collection.
In this study, we have attempted to map the changes in localization of the yeast proteome upon formation of a mating projection. Although individual proteins that localize to the shmoo tip have been characterized (e.g., the shmoo tip marker Fus1(12
)), proteome-wide screens have not been performed to measure such localization changes due to their expensive and cumbersome nature. We developed and implemented a cell microarray-based imaging assay for measuring the spatial redistribution of a large fraction of the yeast proteome, and applied this assay to identify proteins localized along the mating projection following pheromone treatment. By further incorporating information about known yeast gene associations and about protein localization during vegetative growth, we trained a machine learning algorithm to refine the cell imaging screen, resulting in a total of 74 proteins identified that specifically localize to the mating projection. Functional analysis of these proteins, coupled with analyses of individual organelle movements during shmoo formation, suggests a model in which the basic machinery for cell polarization is generally conserved between processes forming the bud and the shmoo, with a distinct subset of proteins used only for shmoo formation. The net effect is a defined ordering of major organelles along the polarization axis, with specific proteins implicated at the proximal growth tip.