High-throughput Growth Measurements of Yeast Exposed to Visible Light.

Light is a double-edged sword: it is essential for life on the planet but also causes cellular damage and death. Consequently, organisms have evolved systems not only for harvesting and converting light energy into chemical energy but also for countering its toxic effects. Despite the omnipresence and importance of such light-dependent effects, there are very few unbiased genetic screens, if any, investigating the mechanistic consequences that visible light has on cells. Baker's yeast, Saccharomyces cerevisiae, is one of the best annotated organisms thanks to several easily available mutant collections and its amenability to high-throughput genetic screening. However, until recently this yeast was thought to lack receptors for visible light, therefore its response to visible light was poorly understood. Nevertheless, a couple of years ago it was discovered that yeast senses light via a novel and unconventional pathway involving a peroxisomal oxidase, hydrogen peroxide, and a particular type of antioxidant protein, called peroxiredoxin. Here, we describe in detail a protocol for scoring yeast genes involved in the resistance to visible light (400-700 nm) on a genome-wide scale. Because cells in dense cultures shield each other from light exposure, resulting in apparent light resistance, our method involves adaptations to reduce inoculum size under conditions amenable to high-throughput screens, to properly be able to identify light-sensitive mutants. We also describe how to measure growth in the presence of light, including two follow-up validation tests. In this way, this method makes it possible to score light-sensitivity on a genome-wide scale with high confidence. Graphic abstract: Overview of strategy for high-throughput determination of yeast growth upon visible light stress.

Protein kinase A controls yeast growth in visible light.

BACKGROUND: A wide variety of photosynthetic and non-photosynthetic species sense and respond to light, having developed protective mechanisms to adapt to damaging effects on DNA and proteins. While the biology of UV light-induced damage has been well studied, cellular responses to stress from visible light (400-700 nm) remain poorly understood despite being a regular part of the life cycle of many organisms. Here, we developed a high-throughput method for measuring growth under visible light stress and used it to screen for light sensitivity in the yeast gene deletion collection. RESULTS: We found genes involved in HOG pathway signaling, RNA polymerase II transcription, translation, diphthamide modifications of the translational elongation factor eEF2, and the oxidative stress response to be required for light resistance. Reduced nuclear localization of the transcription factor Msn2 and lower glycogen accumulation indicated higher protein kinase A (cAMP-dependent protein kinase, PKA) activity in many light-sensitive gene deletion strains. We therefore used an ectopic fluorescent PKA reporter and mutants with constitutively altered PKA activity to show that repression of PKA is essential for resistance to visible light. CONCLUSION: We conclude that yeast photobiology is multifaceted and that protein kinase A plays a key role in the ability of cells to grow upon visible light exposure. We propose that visible light impacts on the biology and evolution of many non-photosynthetic organisms and have practical implications for how organisms are studied in the laboratory, with or without illumination.

Computational high-throughput screening of fluid permeability in heterogeneous fiber materials.

We explore computational high-throughput screening as a design strategy for heterogeneous, isotropic fiber materials. Fluid permeability, a key property in the design of soft porous materials, is systematically studied using a multi-scale lattice Boltzmann framework. After characterizing microscopic permeability as a function of solid volume fraction in the microstructure, we perform high-throughput computational screening of in excess of 35 000 macrostructures consisting of a continuous bulk interrupted by spherical/elliptical domains with either lower or higher microscopic permeability (hence with two distinct microscopic solid volume fractions and therefore two distinct microscopic permeabilities) to assess which parameters determine macroscopic permeability for a fixed average solid volume fraction. We conclude that the fractions of bulk and domains and the distribution of solid volume fraction between them are the primary determinants of macroscopic permeability, and that a substantial increase in permeability compared to the corresponding homogenous material is attainable.

Continuous light exposure causes cumulative stress that affects the localization oscillation dynamics of the transcription factor Msn2p.

Light exposure is a potentially powerful stress factor during in vivo optical microscopy studies. In yeast, the general transcription factor Msn2p translocates from the cytoplasm to the nucleus in response to illumination. However, previous time-lapse fluorescence microscopy studies of Msn2p have utilized a variety of discrete exposure settings, which makes it difficult to correlate stress levels and illumination parameters. We here investigate how continuous illumination with blue light, corresponding to GFP excitation wavelengths, affects the localization pattern of Msn2p-GFP in budding yeast. The localization pattern was analyzed using a novel approach that combines wavelet decomposition and change point analysis. It was found that the Msn2p nucleocytoplasmic localization trajectories for individual cells exhibit up to three distinct and successive states; i) Msn2p localizes to the cytoplasm; ii) Msn2p rapidly shuttles between the cytoplasm and the nucleus; iii) Msn2p localizes to the nucleus. Many cells pass through all states consecutively at high light intensities, while at lower light intensities most cells only reach states i) or ii). This behaviour strongly indicates that continuous light exposure gradually increases the stress level over time, presumably through continuous accumulation of toxic photoproducts, thereby forcing the cell through a bistable region corresponding to nucleocytoplasmic oscillations. We also show that the localization patterns are dependent on protein kinase A (PKA) activity, i.e. yeast cells with constantly low PKA activity showed a stronger stress response. In particular, the nucleocytoplasmic oscillation frequency was found to be significantly higher for cells with low PKA activity for all light intensities.

Sulfate assimilation mediates tellurite reduction and toxicity in Saccharomyces cerevisiae.

Despite a century of research and increasing environmental and human health concerns, the mechanistic basis of the toxicity of derivatives of the metalloid tellurium, Te, in particular the oxyanion tellurite, Te(IV), remains unsolved. Here, we provide an unbiased view of the mechanisms of tellurium metabolism in the yeast Saccharomyces cerevisiae by measuring deviations in Te-related traits of a complete collection of gene knockout mutants. Reduction of Te(IV) and intracellular accumulation as metallic tellurium strongly correlated with loss of cellular fitness, suggesting that Te(IV) reduction and toxicity are causally linked. The sulfate assimilation pathway upstream of Met17, in particular, the sulfite reductase and its cofactor siroheme, was shown to be central to tellurite toxicity and its reduction to elemental tellurium. Gene knockout mutants with altered Te(IV) tolerance also showed a similar deviation in tolerance to both selenite and, interestingly, selenomethionine, suggesting that the toxicity of these agents stems from a common mechanism. We also show that Te(IV) reduction and toxicity in yeast is partially mediated via a mitochondrial respiratory mechanism that does not encompass the generation of substantial oxidative stress. The results reported here represent a robust base from which to attack the mechanistic details of Te(IV) toxicity and reduction in a eukaryotic organism.

Investigations on light-induced stress in fluorescence microscopy using nuclear localization of the transcription factor Msn2p as a reporter.

We utilized the nuclear localization of a stress-sensitive transcription factor, Msn2p, to study light-induced stress caused by time-lapse fluorescence imaging of green fluorescent protein (GFP) in budding yeast Saccharomyces cerevisiae. A range of exposure times, light intensities and intervals between exposures were tested in order to provide guidelines for noninvasive imaging. We found that the cellular response, revealed as an enhanced nuclear shuttling of Msn2p-GFP, is induced at significantly lower light exposures than those causing observable changes in cell morphology or cell growth. However, no stress induction was observed if the accumulated photon energy per area unit used to obtain an image was maintained at 0.16 J cm(-2) or below. Above this 'safe' level, the stress response is determined by both the intensity and the exposure time. In particular, for a given accumulated photon energy per area unit, a high intensity applied during a short exposure causes more stress than vice versa. Interestingly, no correlation was found between the degree of stress and the absolute fluorescence signal, indicating that light-induced cellular stress in the studied system is not specifically related to GFP excitation.

Image analysis algorithms for cell contour recognition in budding yeast.

Quantification of protein abundance and subcellular localization dynamics from fluorescence microscopy images is of high contemporary interest in cell and molecular biology. For large-scale studies of cell populations and for time-lapse studies, such quantitative analysis can not be performed effectively without some kind of automated image analysis tool. Here, we present fast algorithms for automatic cell contour recognition in bright field images, optimized to the model organism budding yeast (Saccharomyces cerevisiae). The cell contours can be used to effectively quantify cell morphology parameters as well as protein abundance and subcellular localization from overlaid fluorescence data.

The sodium pump Ena1p provides mechanistic insight into the salt sensitivity of vacuolar protein sorting mutants.

The vacuolar/endosomal network has an important but as yet undefined role in the cellular tolerance to salt stress. We hypothesized that the mechanistic basis for the importance of vacuolar protein sorting (vps) components in salt tolerance is the targeting of the crucial sodium exporter Ena1p to the plasma membrane. The link between Ena1p and the vps components was established by the observation that overexpression of Ena1p could suppress the salt sensitivity of the ESCRT knockouts vps20Delta, snf7/vps32Delta and snf8/vps22Delta. To further investigate this functional interaction, fluorescence microscopy was utilized to monitor localization of GFP-tagged Ena1p. For all analyzed vps mutants, Ena1p seemed properly localized to the plasma membrane, even during saline growth. However, quantitative differences in plasma membrane localized Ena1p were recorded; e.g. the highly salt sensitive pep12Delta mutant exhibited substantially enhanced Ena1p levels. In addition, the kinetics of Ena1p localization to the plasma membrane was severely delayed in several vps mutants, and this delay correlated to the salt specific growth defect. This paper discusses potential mechanistic hypotheses, like Ena1p transporter activity or localization kinetics, or ESCRT component's influence on signaling, for linking endosomal sorting functions to cellular salt sensitivity.

A microfluidic system enabling Raman measurements of the oxygenation cycle in single optically trapped red blood cells.

Using a lab-on-a-chip approach we demonstrate the possibility of selecting a single cell with certain properties and following its dynamics after an environmental stimulation in real time using Raman spectroscopy. This is accomplished by combining a micro Raman set-up with optical tweezers and a microfluidic system. The latter gives full control over the media surrounding the cell, and it consists of a pattern of channels and reservoirs defined by electron beam lithography that is moulded into rubber silicon (PDMS). Different buffers can be transported through the channels using electro-osmotic flow, while the resonance Raman response of an optically trapped red blood cell (RBC) is simultaneously registered. This makes it possible to monitor the oxygenation cycle of the cell in real time and to investigate effects like photo-induced chemistry caused by the illumination. The experimental set-up has high potential for in vivo monitoring of cellular drug response using a variety of spectroscopic probes.

Resonance Raman spectroscopy of optically trapped functional erythrocytes.

We introduce a novel setup combining a micro-Raman spectrometer with external optical tweezers, suitable for resonance Raman studies of single functional trapped cells. The system differs from earlier setups in that two separate laser beams used for trapping and Raman excitation are combined in a double-microscope configuration. This has the advantage that the wavelength and power of the trapping and probe beam can be adjusted individually to optimize the functionality of the setup and to enable the recording of resonance Raman profiles from a single trapped cell. Trapping is achieved by tightly focusing infrared (IR) diode laser radiation (830 nm) through an inverted oil-immersion objective, and resonance Raman scattering is excited by the lines of an argon:krypton ion laser. The functionality of the system is demonstrated by measurements of trapped single functional erythrocytes using different excitation lines (488.0, 514.5, and 568.2 nm) in resonance with the heme moiety and by studying spectral evolution during illumination. We found that great care has to be taken in order to avoid photodamage caused by the visible Raman excitation, whereas the IR trapping irradiation does not seem to harm the cells or alter the hemoglobin Raman spectra. Stronger photodamage is induced by Raman excitation using 488.0- and 514.5-nm irradiation, compared with excitation with the 568.2-nm line.