Y04C-02-5PK MilliporeCellASIC ONIX plate for haploid yeast cells (4 chamber, 3.5-5 micron)

Nucleoside analogs are frequently used to label newly synthesized DNA. These analogs are toxic in many cells, with the exception of the budding yeast. We show that Schizosaccharomyces pombe behaves similarly to metazoans in response to analogs 5-bromo-2'-deoxyuridine (BrdU) and 5-ethynyl-2'-deoxyuridine (EdU). Incorporation causes DNA damage that activates the damage checkpoint kinase Chk1 and sensitizes cells to UV light and other DNA-damaging drugs. Replication checkpoint mutant cds1Δ shows increased DNA damage response after exposure. Finally, we demonstrate that the response to BrdU is influenced by the ribonucleotide reductase inhibitor, Spd1, suggesting that BrdU causes dNTP pool imbalance in fission yeast, as in metazoans. Consistent with this, we show that excess thymidine induces G1 arrest in wild-type fission yeast expressing thymidine kinase. Thus, fission yeast responds to nucleoside analogs similarly to mammalian cells, which has implications for their use in replication and damage research, as well as for dNTP metabolism.

Hydroxyurea (HU) treatment activates the intra-S phase checkpoint proteins Cds1 and Mrc1 to prevent replication fork collapse. We found that prolonged DNA synthesis occurs in cds1Δ and mrc1Δ checkpoint mutants in the presence of HU and continues after release. This is coincident with increased DNA damage measured by phosphorylated histone H2A in whole cells during release. High-resolution live-cell imaging shows that mutants first accumulate extensive replication protein A (RPA) foci, followed by increased Rad52. Both DNA synthesis and RPA accumulation require the MCM helicase. We propose that a replication fork “collapse point” in HU-treated cells describes the point at which accumulated DNA damage and instability at individual forks prevent further replication. After this point, cds1Δ and mrc1Δ forks cannot complete genome replication. These observations establish replication fork collapse as a dynamic process that continues after release from HU block.

Bacterial pathogens have evolved specific effector proteins that, by interfacing with host kinase signalling pathways, provide a mechanism to evade immune responses during infection1, 2. Although these effectors contribute to pathogen virulence, we realized that they might also serve as valuable synthetic biology reagents for engineering cellular behaviour. Here we exploit two effector proteins, the Shigella flexneri OspF protein3 and Yersinia pestis YopH protein4, to rewire kinase-mediated responses systematically both in yeast and mammalian immune cells. Bacterial effector proteins can be directed to inhibit specific mitogen-activated protein kinase pathways selectively in yeast by artificially targeting them to pathway-specific complexes. Moreover, we show that unique properties of the effectors generate new pathway behaviours: OspF, which irreversibly inactivates mitogen-activated protein kinases4, was used to construct a synthetic feedback circuit that shows novel frequency-dependent input filtering. Finally, we show that effectors can be used in T cells, either as feedback modulators to tune the T-cell response amplitude precisely, or as an inducible pause switch that can temporarily disable T-cell activation. These studies demonstrate how pathogens could provide a rich toolkit of parts to engineer cells for therapeutic or biotechnological applications.

In budding yeast, commitment to cell division corresponds to activating the positive feedback loop of G1 cyclins controlled by the transcription factors SBF and MBF. This pair of transcription factors has over 200 targets, implying that cell-cycle commitment coincides with genome-wide changes in transcription. Here, we find that genes within this regulon have a well-defined distribution of transcriptional activation times. Combinatorial use of SBF and MBF results in a logical OR function for gene expression and partially explains activation timing. Activation of G1 cyclin expression precedes the activation of the bulk of the G1/S regulon, ensuring that commitment to cell division occurs before large-scale changes in transcription. Furthermore, we find similar positive feedback-first regulation in the yeasts S. bayanus and S. cerevisiae, as well as human cells. The widespread use of the feedback-first motif in eukaryotic cell-cycle control, implemented by nonorthologous proteins, suggests its frequent deployment at cellular transitions.

The ability to specify and maintain discrete cell fates is essential for development. However, the dynamics underlying selection and stability of distinct cell types remain poorly understood. Here, we provide a quantitative single-cell analysis of commitment dynamics during the mating-mitosis switch in budding yeast. Commitment to division corresponds precisely to activating the G1 cyclin positive feedback loop in competition with the cyclin inhibitor Far1. Cyclin-dependent phosphorylation and inhibition of the mating pathway scaffold Ste5 are required to ensure exclusive expression of the mitotic transcriptional program after cell cycle commitment. Failure to commit exclusively results in coexpression of both cell cycle and pheromone-induced genes, and a morphologically mixed inviable cell fate. Thus, specification and maintenance of a cellular state are performed by distinct interactions, which are likely a consequence of disparate reaction rates and may be a general feature of the interlinked regulatory networks responsible for selecting cell fates

Optical sensors allow dynamic quantification of metabolite levels with subcellular resolution. Here we describe protocols for analyzing cytosolic glucose levels in yeast using genetically encoded Förster resonance energy transfer (FRET) sensors. FRET glucose sensors with different glucose affinities (Kd) covering the low nano- to mid- millimolar range can be targeted genetically to the cytosol or to subcellular compartments. The sensors detect the glucose-induced conformational change in the bacterial periplasmic glucose/galactose binding protein MglB using FRET between two fluorescent protein variants. Measurements can be performed with a single sensor or multiple sensors in parallel. In one approach, cytosolic glucose accumulation is measured in yeast cultures in a 96-well plate using a fluorimeter. Upon excitation of the cyan fluorescent protein (CFP), emission intensities of CFP and YFP (yellow fluorescent protein) are captured before and after glucose addition. FRET sensors provide temporally resolved quantitative data of glucose for the compartment of interest. In a second approach, reversible changes of cytosolic free glucose are measured in individual yeast cells trapped in a microfluidic platform, allowing perfusion of different solutions while FRET changes are monitored in a microscope setup. By using the microplate fluorimeter protocol, 96 cultures can be measured in less than 1 h; analysis of single cells of a single genotype can be completed in <2 h. FRET-based analysis has been performed with glucose, maltose, ATP and zinc sensors, and it can easily be adapted for high-throughput screening using a wide spectrum of sensors.

Precise and dynamic measurement of intracellular metabolite levels has been hampered by difficulties in differentiating between adsorbed and imported fractions and the subcellular distribution between cytosol, endomembrane compartments and mitochondria. In the present study, genetically encoded FRET (Förster resonance energy transfer)-based sensors were deployed for dynamic measurements of free cytosolic glucose and ATP with varying external supply and in glucose-transport mutants. Moreover, by using the FRET sensors in a microfluidic platform, we were able to monitor in vivo changes of intracellular free glucose in individual yeast cells. We demonstrate the suitability of the FRET sensors for gaining physiological insight by demonstrating that free intracellular glucose and ATP levels are reduced in a hxt5Ä hexose-transporter mutant compared with wild-type and other hxtÄ strains.

Spinning-disk confocal microscopy is an imaging technique that combines the out-of-focus light rejection of confocal microscopy with the high sensitivity of wide-field microscopy. Because of its unique features, it is well suited to high-resolution imaging of yeast and other small cells. Elimination of out-of-focus light significantly improves the image contrast and signal-to-noise ratio, making it easier to resolve and quantitate small, dim structures in the cell. These features make spinning-disk confocal microscopy an excellent technique for studying protein localization and dynamics in yeast. In this review, I describe the rationale behind using spinning-disk confocal imaging for yeast, hardware considerations when assembling a spinning-disk confocal scope, and methods for strain preparation and imaging. In particular, I discuss choices of objective lens and camera, choice of fluorescent proteins for tagging yeast genes, and methods for sample preparation.

Atg8 is a ubiquitin-like protein that is required, along with its lipidation system, for autophagy in all eukaryotic cells. The lipidated form of Atg8 is anchored on the autophagosomal membrane during autophagy. Here, we demonstrate a previously unknown role for Atg8 in vacuolar membrane dynamics. In the methylotrophic yeast Pichia pastoris, vacuoles were found to fuse to become a single spherical vacuole during adaptation from glucose- to methanol-containing medium. Atg8 is responsible for the vacuolar fusion in P. pastoris during this adaptation to methanol. Although vacuole fusion required processing of Atg8 at the C-terminus, it did not require lipidation of Atg8 for autophagy. This is the first report of the function of any Atg8 protein family member in a process other than autophagy that is independent of lipidation.

Glucose is the preferred carbon source for most cell types and a major determinant of cell growth. In yeast and certain mammalian cells, glucose activates the cAMP-dependent protein kinase A (PKA), but the mechanisms of PKA activation remain unknown. Here, we identify cytosolic pH as a second messenger for glucose that mediates activation of the PKA pathway in yeast. We find that cytosolic pH is rapidly and reversibly regulated by glucose metabolism and identify the vacuolar ATPase (V-ATPase), a proton pump required for the acidification of vacuoles, as a sensor of cytosolic pH. V-ATPase assembly is regulated by cytosolic pH and is required for full activation of the PKA pathway in response to glucose, suggesting that it mediates, at least in part, the pH signal to PKA. Finally, V-ATPase is also regulated by glucose in the Min6 β-cell line and contributes to PKA activation and insulin secretion. Thus, these data suggest a novel and potentially conserved glucose-sensing pathway and identify a mechanism how cytosolic pH can act as a signal to promote cell growth.