Cells, even those having identical genotype, exhibit variability in their response to external stimuli. This variability arises from differences in the abundance, localization, and state of cellular components. Such nongenetic differences are likely heritable between successive generations and can also be influenced by processes such as cell cycle, age, or interplay between different pathways. To address the contribution of nongenetic heritability and cell cycle in cell-to-cell variability we developed a highthroughput and fully automated microfluidic platform that allows for concurrent measurement of gene expression, cell-cycle periods, age, and lineage information under a large number of temporally changing medium conditions and using multiple strains. We apply this technology to examine the role of nongenetic inheritance in cell heterogeneity of yeast pheromone signaling. Our data demonstrate that the capacity to respond to pheromone is passed across generations and that the strength of the response correlations between related cells is affected by perturbations in the signaling pathway. We observe that a ste50Δ mutant strain exhibits highly heterogeneous response to pheromone originating from a unique asymmetry between mother and daughter response. On the other hand, fus3Δ cells were found to exhibit an unusually high correlation between mother and daughter cells that arose from a combination of extended cell-cycle periods of fus3Δ mothers, and decreased cell-cycle modulation of the pheromone pathway. Our results contribute to the understanding of the origins of cell heterogeneity and demonstrate the importance of automated platforms that generate single-cell data on several parameters.single cell | microscopy | image analysis C ells must detect biochemical cues and respond appropriately to changing environmental conditions. However, under identical chemical stimuli not all cells respond the same. Heterogeneity in "cellular decision making" exists across all levels of life, from bacteria (1, 2) to simple eukaryotes (3-5) to mammalian cells (6, 7), and may be important in cellular adaptation to quickly changing microenvironments (8), in differentiation programs during development (6), and in sensitivity to drugs (9, 10). In addition to the intrinsic stochastic nature of gene expression (1, 3), each cell has an inherent capacity to respond, which is determined by the state and abundance of cellular components. This extrinsic variability may be passed across multiple generations, producing a nongenetic but heritable source of variation. Additional sources that contribute to extrinsic variability include asynchrony and interplay between different signaling pathways (11, 12), cell-cycle effects (3, 13), asymmetry in cellular divisions (14), and cellular aging (13).Investigating these effects requires tracking the genealogy of multiple generations of cells, followed by stimulation and quantitative single-cell measurement of response, including growth kinetics and gene expression. Although improved image analysis, ...

In the budding yeast Saccharomyces cerevisiae, mother cells switch mating types between a and ␣ forms, whereas daughter cells do not. This developmental asymmetry arises because the expression of the HO endonuclease, which initiates the interconversion of a and ␣ mating type cassettes, is extinguished by the daughter-specific Ash1 transcriptional repressor. When daughters become mothers in the subsequent cell cycle, Ash1 must be eliminated to enable a new developmental state. Here, we report that the ubiquitin ligase SCF Cdc4 mediates the phosphorylation-dependent elimination of Ash1. The inactivation of SCF Cdc4 stabilizes Ash1 in vivo, and consistently, Ash1 binds to and is ubiquitinated by SCF Cdc4 in a phosphorylation-dependent manner in vitro. The mutation of a critical in vivo cyclin-dependent kinase (CDK) phosphorylation site (Thr290) on Ash1 reduces its ubiquitination and rate of degradation in vivo and decreases the frequency of mating type switching. Ash1 associates with active Cdc28 kinase in vivo and is targeted to SCF Cdc4 in a Cdc28-dependent fashion in vivo and in vitro. Ash1 recognition by Cdc4 appears to be mediated by at least three phosphorylation sites that form two redundant diphosphorylated degrons. The phosphorylation-dependent elimination of Ash1 by the ubiquitin-proteasome system thus underpins developmental asymmetry in budding yeast.The conversion of one cell type into another, whether the asymmetric division of a stem cell, differentiation of a committed precursor, or transition from one cell cycle phase to the next, requires that the proteins that define the prior state be eliminated or otherwise inactivated. While changes in the factors that determine cell fate alterations are well documented at the transcriptional level, the elimination of critical regulatory proteins that define a prior cell state is less well understood. Because biological networks are often exquisitely sensitive to the abundance of such regulatory factors, their timely elimination may be essential for developmental decisions (37). The best-understood example of the developmental elimination of cell fate determinants occurs in budding yeast, where the degradation of the mating type specificity factor ␣2 via the E3 ubiquitin ligases Doa10 and Slx5-Slx8 is required for the developmental switch between a and ␣ cell types (38,58,81). The ubiquitin-dependent elimination of cell type determinants plays an important role in metazoan development and stem cell fate specification/renewal (8,26,75).The ubiquitin-proteasome system mediates the selective intracellular degradation of proteins in all eukaryotes (30). Substrate proteins are conjugated to ubiquitin through a series of enzymatic steps mediated by E1 (ubiquitin-activating), E2 (ubiquitin-conjugating), and E3 (ubiquitin ligase) enzymes; the reiteration of this cascade results in substrate polyubiquitination, which leads to substrate recognition and rapid degradation by the 26S proteasome. The E3 enzymes are the critical factors that confer substrate specificity. Two...

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.