High-throughput single-cell analysis for the proteomic dynamics study of the yeast osmotic stress response

Motorized fluorescence microscopy combined with high-throughput microfluidic chips is a powerful method to obtain information about different biological processes in cell biology studies. Generally, to observe different strains under different environments, high-throughput microfluidic chips require complex preparatory work. In this study, we designed a novel and easily operated high-throughput microfluidic system to observe 96 different GFP-tagged yeast strains in one switchable culture condition or 24 different GFP-tagged yeast strains in four parallel switchable culture conditions. A multi-pipette is the only additional equipment required for high-throughput patterning of cells in the chip. Only eight connections are needed to control 96 conditions. Using these devices, the proteomic dynamics of the yeast stress response pathway were carefully studied based on single-cell data. A new method to characterize the proteomic dynamics using a single cell’s data is proposed and compared to previous methods, and the new technique should be useful for studying underlying control networks. Our method provides an easy and systematic way to study signaling pathways at the single-cell level.

protein abundance increases in response to DNA replication stress.

FIG4
Phosphatidylinositol 3,5-bisphosphate (PtdIns[3,5]P) phosphatase; required for efficient mating and response to osmotic shock; physically associates with and regulated by Vac14p; contains a SAC1-like domain; homologous to human FIG4, which is associated with CMT4J, a form of Charcot-Marie-Tooth disorde .

PPZ1
Serine/threonine protein phosphatase Z, isoform of Ppz2p; involved in regulation of potassium transport, which affects osmotic stability, cell cycle progression, and halotolerance.

GPH1
Glycogen phosphorylase required for the mobilization of glycogen; non-essential; regulated by cyclic AMP-mediated phosphorylation; expression is regulated by stress-response elements and by the HOG MAP kinase pathway.

GLC3
Glycogen branching enzyme, involved in glycogen accumulation; green fluorescent protein (GFP)-fusion protein localizes to the cytoplasm in a punctate pattern; relocalizes from nucleus to cytoplasmic foci upon DNA replication stress; glycogen accumulation defect of the null mutant is functionally complemented by human GBE1, which is associated with glycogen storage disease.

TPS2
Phosphatase subunit of the trehalose-6-P synthase/phosphatase complex; involved in synthesis of the storage carbohydrate trehalose; expression is induced by stress conditions and repressed by the Ras-cAMP pathway; protein abundance increases in response to DNA replication stress.

RCK2
Protein kinase involved in response to oxidative and osmotic stress; identified as suppressor of S. pombe cell cycle checkpoint mutations; similar to CaM (calmodulin) kinases; RCK2 has a paralog, RCK1, that arose from the whole genome duplication.

PGM2
Phosphoglucomutase; catalyzes the conversion from glucose-1-phosphate to glucose-6-phosphate, which is a key step in hexose metabolism; functions as the acceptor for a Glc-phosphotransferase; protein abundance increases in response to DNA replication stress; PGM2 has a paralog, PGM1, that arose from the whole genome duplication.

CTT1
Cytosolic catalase T; has a role in protection from oxidative damage by hydrogen peroxide 3.

RVS167
Actin-associated protein with roles in endocytosis and exocytosis; N-BAR domain protein that interacts with Rvs161p to regulate actin cytoskeleton, endocytosis, and viability following starvation or osmotic stress; recruited to bud tips by Gyl1p and Gyp5p during polarized growth; homolog of mammalian amphiphysin.

MSC1
Protein of unknown function; mutant is defective in directing meiotic recombination events to homologous chromatids; the authentic, non-tagged protein is detected in highly purified mitochondria and is phosphorylated.

ALD4
Mitochondrial aldehyde dehydrogenase; required for growth on ethanol and conversion of acetaldehyde to acetate; phosphorylated; activity is K+ dependent; utilizes NADP+ or NAD+ equally as coenzymes; expression is glucose repressed; can substitute for cytosolic NADP-dependent aldehyde dehydrogenase when directed to the cytosol; human homolog ALDH2 can complement yeast ald4 mutant.

COS8
Endosomal protein involved in turnover of plasma membrane proteins; member of the DUP380 subfamily of conserved, often subtelomeric COS genes; required for the multivesicular vesicle body sorting pathway that internalizes plasma membrane proteins for degradation; Cos proteins provide ubiquitin in trans for nonubiquitinated cargo proteins.

YML131W
Protein of unknown function; similar to medium chain dehydrogenase/reductases; expression induced by stresses including osmotic shock, DNA damaging agents, and other chemicals; GFP-fusion protein localizes to the cytoplasm; protein abundance increases in response to DNA replication stress.

YPR1
NADPH-dependent aldo-keto reductase; reduces multiple substrates including 2-methylbutyraldehyde and D,L-glyceraldehyde, expression is induced by osmotic and oxidative stress; functionally redundant with other aldo-keto reductases; protein abundance increases in response to DNA replication stress; YPR1 has a paralog, GCY1, that arose from the whole genome duplication; human homolog AKR1B1 can complement yeast null mutant.

GRE2
3-methylbutanal reductase and NADPH-dependent methylglyoxal reductase; stress induced (osmotic, ionic, oxidative, heat shock and heavy metals); regulated by the HOG pathway; restores resistance to glycolaldehyde by coupling reduction of glycolaldehyde to ethylene glycol and oxidation of NADPH to NADP+; protein abundance increases in response to DNA replication stress; methylglyoxal reductase (NADPH-dependent) is also known as D-lactaldehyde dehydrogenase.

GRE3
Aldose reductase; involved in methylglyoxal, d-xylose, arabinose, and galactose metabolism; stress induced (osmotic, ionic, oxidative, heat shock, starvation and heavy metals); regulated by the HOG pathway; protein abundance increases in response to DNA replication stress.

GLO1
Monomeric glyoxalase I; catalyzes the detoxification of methylglyoxal (a by-product of glycolysis) via condensation with glutathione to produce S-D-lactoylglutathione; expression regulated by methylglyoxal levels and osmotic stress.

TDH1
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), isozyme 1; involved in glycolysis and gluconeogenesis; tetramer that catalyzes the reaction of glyceraldehyde-3-phosphate to 1,3 bis-phosphoglycerate; detected in the cytoplasm and cell wall; protein abundance increases in response to DNA replication stress; GAPDH-derived antimicrobial peptides secreted by S.
cerevisiae are active against a wide variety of wine-related yeasts and bateria 1.

GPP1
Constitutively expressed DL-glycerol-3-phosphate phosphatase; also known as glycerol-1-phosphatase; involved in glycerol biosynthesis, induced in response to both anaerobic and osmotic stress; GPP1 has a paralog, GPP2, that arose from the whole genome duplication.

RVS161
Amphiphysin-like lipid raft protein; N-BAR domain protein that interacts with Rvs167p and regulates polarization of the actin cytoskeleton, endocytosis, cell polarity, cell fusion and viability following starvation or osmotic stress.

DOG2
2-deoxyglucose-6-phosphate phosphatase; member of a family of low molecular weight phosphatases, induced by oxidative and osmotic stress, confers 2-deoxyglucose resistance when overexpressed; DOG2 has a paralog, DOG1, that arose from a single-locus duplication; the last half of DOG1 and DOG2 are subject to gene conversions among S. cerevisiae, S. paradoxus, and S. mikatae.

HSP26
Small heat shock protein (sHSP) with chaperone activity; forms hollow, sphere-shaped oligomers that suppress unfolded proteins aggregation; long-lived protein that is preferentially retained in mother cells and forms cytoplasmic foci; oligomer activation requires heat-induced conformational change; also has mRNA binding activity 3.

YAR1
Ankyrin-repeat containing, nucleocytoplasmic shuttling chaperone; prevents aggregation of Rps3p in the cytoplasm, associates with nascent Rps3p during its translation in the cytoplasm and delivers it to the 90S in the nucleus; required for 40S ribosomal subunit export, biogenesis and adaptation to osmotic and oxidative stress; expression repressed by heat shock.

YPD1
Osmotic stress-responsive phosphorelay intermediate sensor protein; phosphorylated by the plasma membrane sensor Sln1p in response to osmotic stress and then in turn phosphorylates the response regulators Ssk1p in the cytosol and Skn7p in the nucleus.

PAI3
Cytoplasmic proteinase A (Pep4p) inhibitor; dependent on Pbs2p and Hog1p protein kinases for osmotic induction; intrinsically unstructured, N-terminal half becomes ordered in the active site of proteinase A upon contact.

YJL144W
Cytoplasmic hydrophilin essential in desiccation-rehydration process; expression induced by osmotic stress, starvation and during stationary phase; protein abundance increases in response to DNA replication stress 1.

SIP18
Phospholipid-binding hydrophilin; essential to overcome desiccation-rehydration process; expression is induced by osmotic stress; SIP18 has a paralog, GRE1, that arose from the whole genome duplication.

MPC3
Highly conserved subunit of the mitochondrial pyruvate carrier (MPC); expressed during growth on nonfermentable carbon sources, and heterodimerizes with Mpc1p to form the respiratory isoform of MPC; MPC localizes to the mitochondrial inner membrane and mediates pyruvate uptake; MPC3 paralog, MPC2, heterodimerizes with Mpc1p to form the fermentative MPC isoform; protein abundance increases in response to DNA replication stress.

TOS6
Glycosylphosphatidylinositol-dependent cell wall protein; expression is periodic and decreases in respone to ergosterol perturbation or upon entry into stationary phase; depletion increases resistance to lactic acid.

HSP12
Plasma membrane protein involved in maintaining membrane organization; involved in maintaining organization during stress conditions; induced by heat shock, oxidative stress, osmostress, stationary phase, glucose depletion, oleate and alcohol; protein abundance increased in response to DNA replication stress and dietary restriction; regulated by the HOG and Ras-Pka pathways; required for dietary restriction-induced lifespan extension.

PAN1
Part of actin cytoskeleton-regulatory complex Pan1p-Sla1p-End3p; associates with actin patches on cell cortex; promotes protein-protein interactions essential for endocytosis; binds to and activates Arp2/3 complex in vitro; phosphorylation of Thr-1225 is regulated by MAPK Hog1p in response to osmotic stress; previously thought to be a subunit of poly(A) ribonuclease.

ANP1
Subunit of the alpha-1,6 mannosyltransferase complex; type II membrane protein; has a role in retention of glycosyltransferases in the Golgi; involved in osmotic sensitivity and resistance to aminonitrophenyl propanediol.

HXT6
High-affinity glucose transporter; member of the major facilitator superfamily, nearly identical to Hxt7p, expressed at high basal levels relative to other HXTs, repression of expression by high glucose requires SNF3; HXT6 has a paralog, HXT1, that arose from the whole genome duplication.

GSP2
GTP binding protein (mammalian Ranp homolog); involved in the maintenance of nuclear organization, RNA processing and transport; interacts with Kap121p, Kap123p and Pdr6p (karyophilin betas); not required for viability; protein abundance increases in response to DNA replication stress; GSP2 has a paralog, GSP1, that arose from the whole genome duplication 1.

POR1
Mitochondrial porin (voltage-dependent anion channel); outer membrane protein required for maintenance of mitochondrial osmotic stability and mitochondrial membrane permeability; couples the glutathione pools of the intermembrane space (IMS) and the cytosol; interacts with Om45 and Om14 in the outer membrane; phosphorylated; protein abundance increases in response to DNA replication stress.

Supplementary Table S2
The functions of 40 proteins from the primary selected proteins