Single Yeast Cells Research Articles

Droplet-based microfluidic high-throughput screening of heterologous enzymes secreted by the yeast Yarrowia lipolytica

BackgroundDroplet-based microfluidics is becoming an increasingly attractive alternative to microtiter plate techniques for enzymatic high-throughput screening (HTS), especially for exploring large diversities with lower time and cost footprint. In this case, the assayed enzyme has to be accessible to the substrate within the water-in-oil droplet by being ideally extracellular or displayed at the cell surface. However, most of the enzymes screened to date are expressed within the cytoplasm of Escherichia coli cells, which means that a lysis step must take place inside the droplets for enzyme activity to be assayed. Here, we take advantage of the excellent secretion abilities of the yeast Yarrowia lipolytica to describe a highly efficient expression system particularly suitable for the droplet-based microfluidic HTS.ResultsFive hydrolytic genes from Aspergillus niger genome were chosen and the corresponding five Yarrowia lipolytica producing strains were constructed. Each enzyme (endo-β-1,4-xylanase B and C; 1,4-β-cellobiohydrolase A; endoglucanase A; aspartic protease) was successfully overexpressed and secreted in an active form in the crude supernatant. A droplet-based microfluidic HTS system was developed to (a) encapsulate single yeast cells; (b) grow yeast in droplets; (c) inject the relevant enzymatic substrate; (d) incubate droplets on chip; (e) detect enzymatic activity; and (f) sort droplets based on enzymatic activity. Combining this integrated microfluidic platform with gene expression in Y. lipolytica results in remarkably low variability in the enzymatic activity at the single cell level within a given monoclonal population (<5%). Xylanase, cellobiohydrolase and protease activities were successfully assayed using this system. We then used the system to screen for thermostable variants of endo-β-1,4-xylanase C in error-prone PCR libraries. Variants displaying higher thermostable xylanase activities compared to the wild-type were isolated (up to 4.7-fold improvement).Conclusions Yarrowia lipolytica was used to express fungal genes encoding hydrolytic enzymes of interest. We developed a successful droplet-based microfluidic platform for the high-throughput screening (105 strains/h) of Y. lipolytica based on enzyme secretion and activity. This approach provides highly efficient tools for the HTS of recombinant enzymatic activities. This should be extremely useful for discovering new biocatalysts via directed evolution or protein engineering approaches and should lead to major advances in microbial cell factory development.

Pan de Muerto

Pan de Muerto Maria Nieto (bio) Melodic coos reached Gloria’s bed carried on the tail of a damp breeze. Pajarito Travieso Hermoso Mi Amor was awake and eager for a pre-dawn flight. The wayward homing pigeon that captured Gloria’s heart nearly three years ago was too much bird to bear a single name; Gloria decided on several to express how much she loved her mischievous, beautiful feathered friend. Today the pigeon’s coos captured more than Gloria’s heart, they captured her dream, transporting her back in time to a college campus demonstration spearheaded by a fraternity. The brothers of Upsilon Upsilon Upsilon were demanding mass extermination of the local pigeon population, yelling “Death to flying rats. Kill the pigeons now.” They were convinced pigeon poop was to blame for a recent outbreak of flu-like symptoms on fraternity row. Gloria’s student organization, Animal Lovers Unite, believed the pigeons were being scapegoated to steer attention away from a rash of recent violent pigeon murders, possibly perpetrated by the brothers themselves. Her group held strong against the hateful rhetoric, holding signs that read, A pigeon’s coo says I love you, Fertilize the natural way, pigeon poop is here to stay, and Equal rights for all our avian friends. Just when it looked like the demonstration was going to get physical, Gloria was pulled out of her dream by a loud shrill. It was Pajarito Travieso Hermoso Mi Amor. He had grown impatient and left his cage to walk on Gloria’s head. “Ay, Pajarito. Okay, okay, I’m up.” Gloria reached over to open her window and the bird immediately took to the sky. ________ No timekeeper was needed. When Felix Galván started to knead his first mound of dough the short arm of the clock clicked into place; it was 4 a.m. Felix [End Page 191] dug the palms of his large muscular hands into the mass and stretched it across a floured tabletop. He then tucked the dough into a round before leveling it again. He repeated the process to create a smooth, elastic texture off the backs of life – hordes of single-celled yeast. The microorganisms fed off the flour’s starch and gave in return their waste, carbon dioxide gas forcing the dough to rise. After loading the last tray of Mexican sweet bread into the display case it was 7:00am and time for Felix to open for business. Conchas whose shapes and sugary coats rivaled the rarest, colored seashells, flakey orejas that looked like the ears of Goliath, and empanadas de manzana that would win the envy of Johnny Appleseed, filled the panadería with a smell of golden caramel awash with the sentimentality of a home you never wanted to leave. The early morning aroma used to lure a steady flow of people, but over the last couple of years business was slow for Pan Del Corazón, Felix’s 24th Street shop where each baked good reflected the store-front’s name, Bread from the Heart. People like Socorro Prieta, Rudy Garcia, and countless others no longer came; they no longer lived in the neighborhood Felix had called home his whole life. The Mission District of San Francisco was changing. New residents now occupied many of the Victorian flats and ornate Queen Anne buildings dotting the historic landscape; their palates demanded the avant-garde in flavor - donuts infused with lavender and hibiscus oils and iced with lemon chiffon, ice cream boasting large chunks of imported dark chocolate and flavored with orange cardamom, and bite-sized green tea cupcakes layered with mint frosting. Price was no factor for the newcomers. In fact, the higher the cost, the more people clamored for the chance to eat what they could easily afford and damn well thought they deserved. For all the customers Felix had lost over the years, he still had a few loyal patrons like Gloria Muñoz. Nearly every morning she made her way to the panadería to share space and time with Felix over a cup of coffee and pan. Felix was sure she’d be arriving soon because today was special, it was...

Exploring Catalase and Invertase Activity Using Sodium Alginate-Encapsulated Yeast (Yeast Spheres).

Finding the right enzyme experiment can be problematic, depending what one is trying to show, what supplies and equipment are available, and the time one can devote to the topic. I’ve developed simple and inexpensive labs for looking at catalase and invertase activity using yeast encapsulated in sodium alginate. Single-celled yeast, Saccharomyces cerevisiae, are encapsulated in sodium alginate, a readily available extract from brown algae that, when it comes in contact with calcium chloride (CaCl2), forms a sphere or “bead.” These spheres may then be put into a solution containing substrate to test for enzyme activity. The spheres are easy to manipulate, one doesn’t have the variability and mess of a yeast solution, and since there are no cells in solution, there is nothing to interfere with the various assay methods one might want to use to test for product. The graduated cylinder method for testing catalase activity introduced here is especially good for collecting large amounts of data that enable students to use statistics and, unlike similar yeast catalase experiments using paper disks and a yeast solution, the yeast spheres are easy to manipulate and there is very little variability. I have used this procedure with students in class and with teachers in workshops with positive results and comments.

The Natural Variation in Lifespans of Single Yeast Cells Is Related to Variation in Cell Size, Ribosomal Protein, and Division Time

There is a large variability in lifespans of individuals even if they are genetically identical and raised under the same environmental conditions. Our recent system wide study of replicative aging in baker’s yeast predicts that protein biogenesis is a driver of aging. Here, we address how the natural variation in replicative lifespan within wild-type populations of yeast cells correlates to three biogenesis-related parameters, namely cell size, ribosomal protein Rpl13A-GFP levels, and division times. Imaging wild type yeast cells in microfluidic devices we observe that in all cells and at all ages, the division times as well as the increase in cell size that single yeast undergo while aging negatively correlate to their lifespan. In the longer-lived cells Rpl13A-GFP levels also negatively correlate to lifespan. Interestingly however, at young ages in the population, ribosome concentration was lowest in the cells that increased the most in size and had shorter lifespans. The correlations between these molecular and cellular properties related to biogenesis and lifespan explain a small portion of the variation in lifespans of individual cells, consistent with the highly individual and multifactorial nature of aging.

Large-Scale Single Particle and Cell Trapping based on Rotating Electric Field Induced-Charge Electroosmosis.

We propose a simple, inexpensive microfluidic chip for large-scale trapping of single particles and cells based on induced-charge electroosmosis in a rotating electric field (ROT-ICEO). A central floating electrode array, was placed in the center of the gap between four driving electrodes with a quadrature configuration and used to immobilize single particles or cells. Cells were trapped on the electrode array by the interaction between ROT-ICEO flow and buoyancy flow. We experimentally optimized the efficiency of trapping single particles by investigating important parameters like particle or cell density and electric potential. Experimental and numerical results showed good agreement. The operation of the chip was verified by trapping single polystyrene (PS) microspheres with diameters of 5 and 20 μm and single yeast cells. The highest single particle occupancy of 73% was obtained using a floating electrode array with a diameter of 20 μm with an amplitude voltage of 5 V and frequency of 10 kHz for PS microbeads with a 5-μm diameter and density of 800 particles/μL. The ROT-ICEO flow could hold cells against fluid flows with a rate of less than 0.45 μL/min. This novel, simple, robust method to trap single cells has enormous potential in genetic and metabolic engineering.

Fungal Sex: The Ascomycota.

This article provides an overview of sexual reproduction in the ascomycetes, a phylum of fungi that is named after the specialized sacs or "asci" that hold the sexual spores. They have therefore also been referred to as the Sac Fungi due to these characteristic structures that typically contain four to eight ascospores. Ascomycetes are morphologically diverse and include single-celled yeasts, filamentous fungi, and more complex cup fungi. The sexual cycles of many species, including those of the model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe and the filamentous saprobes Neurospora crassa, Aspergillus nidulans, and Podospora anserina, have been examined in depth. In addition, sexual or parasexual cycles have been uncovered in important human pathogens such as Candida albicans and Aspergillus fumigatus, as well as in plant pathogens such as Fusarium graminearum and Cochliobolus heterostrophus. We summarize what is known about sexual fecundity in ascomycetes, examine how structural changes at the mating-type locus dictate sexual behavior, and discuss recent studies that reveal that pheromone signaling pathways can be repurposed to serve cellular roles unrelated to sex.

Noise reduction facilitated by dosage compensation in gene networks.

Genetic noise together with genome duplication and volume changes during cell cycle are significant contributors to cell-to-cell heterogeneity. How can cells buffer the effects of these unavoidable epigenetic and genetic variations on phenotypes that are sensitive to such variations? Here we show that a simple network motif that is essential for network-dosage compensation can reduce the effects of extrinsic noise on the network output. Using natural and synthetic gene networks with and without the network motif, we measure gene network activity in single yeast cells and find that the activity of the compensated network is significantly lower in noise compared with the non-compensated network. A mathematical analysis provides intuitive insights into these results and a novel stochastic model tracking cell-volume and cell-cycle predicts the experimental results. Our work implies that noise is a selectable trait tunable by evolution.

Probing RNA recognition by human ADAR2 using a high-throughput mutagenesis method.

Adenosine deamination is one of the most prevalent post-transcriptional modifications in mRNA. In humans, ADAR1 and ADAR2 catalyze this modification and their malfunction correlates with disease. Recently our laboratory reported crystal structures of the human ADAR2 deaminase domain bound to duplex RNA revealing a protein loop that binds the RNA on the 5′ side of the modification site. This 5′ binding loop appears to be one contributor to substrate specificity differences between ADAR family members. In this study, we endeavored to reveal detailed structure–activity relationships in this loop to advance our understanding of RNA recognition by ADAR2. To achieve this goal, we established a high-throughput mutagenesis approach which allows rapid screening of ADAR variants in single yeast cells and provides quantitative evaluation for enzymatic activity. Using this approach, we determined the importance of specific amino acids at 19 different positions in the ADAR2 5′ binding loop and revealed six residues that provide essential structural elements supporting the fold of the loop and key RNA-binding functional groups. This work provided new insight into RNA recognition by ADAR2 and established a new tool for defining structure–function relationships in ADAR reactions.

Characterization of Single Yeast Cell Phenotypes Using Microfluidic Impedance Cytometry and Optical Imaging

Single-cell impedance cytometry is a noninvasive method for characterizing the dielectric properties of individual cells. The method has been used for counting and sizing of cells and particles, as well as for discriminating between different cell types in a label-free manner. However, finding a relationship between a specific cell phenotype and the corresponding measured impedance remains a challenging problem. This paper reports on a platform that combines a microfluidic impedance cytometer with a high-speed camera in order to allow for characterizing the physical morphology of single cells in parallel with the dielectric properties. This enables the extraction of signatures in the impedance data that can be associated with specific morphological phenotypes. We demonstrate the functionality of the platform by developing such signatures for discriminating between single and budding yeast cells.

The Mould-specific M46 gene is not essential for yeast-mould dimorphism in the pathogenic fungus Histoplasma capsulatum.

Histoplasma capsulatum (Hc) is the causative agent for the respiratory infection histoplasmosis. The fungus exists in the environment as a saprophytic multi-cellular mould. Spores are inhaled by mammals whereupon the organism will convert into the single-celled yeast morphotype resulting in infection. The shift to the yeast morphotype is required for pathogenesis. Most studies on dimorphism have examined yeast-phase-specific genes and few mould-phase-specific genes have been investigated. It is likely, that some mould-phase-specific genes must be downregulated for the yeast to form or upregulated for the mould to form. We isolated a strongly expressed mould-specific gene, M46, from an expression library enriched for mould upregulated genes in Hc strain G186AS. To determine if M46 is involved in dimorphism, M46 was ectopically expressed in yeast phase growing temperature, and an m46 knockout strain was created via allelic replacement. Ectopically expressing M46 in yeast, did not induce filamentous growth. Genomic disruption of M46 by allelic replacement did not alter the morphology of the mould as seen in bright field microscopy, scanning electron microscopy, and transmission electron microscopy. A growth curve study, revealed that M46 is not involved in maintaining the growth rate of cells. These findings indicate that the mould specific M46 gene is not necessary nor essential for dimorphism, maintaining the normal mould morphology, and growth rate of Histoplasma capsulatum.

Fungi and diabetes

Fungi and diabetes

Extended longevity and survivorship during amino-acid starvation in a Drosophila Sir2 mutant heterozygote.

The regulation of energy homeostasis is pivotal to survive periods of inadequate nutrition. A combination of intricate pathways and proteins are responsible for maximizing longevity during such conditions. The sirtuin deacetylase Sir2 is well conserved from single-celled yeast to mammals, and it controls a number of downstream targets that are active during periods of extreme stress. Overexpression of Sir2 has been established to enhance survival of a number of model organisms undergoing calorie restriction, during which insulin receptor signalling (IRS) is reduced, a condition that itself can enhance survivorship during starvation. Increased Sir2 expression and reduced IRS result in an increase in the activity of the transcription factor foxo, an advantageous activation during stress but lethal when overly active. We have found that a lowered gene dosage of Sir2, in mutant heterozygotes, can extend normal longevity and greatly augment survivorship during amino-acid starvation in Drosophila. Additionally, these mutants, in either heterozygous or homozygous form, do not appear to have any disadvantageous effects upon development or cell growth of the organism unlike IRS mutants. These results may advance the understanding of the biological response to starvation and allow for the development of a model organism to mimic the ability of individuals to tolerate nutrient deprivation.

Phenotypic diversity and correlation between white-opaque switching and the CAI microsatellite locus in Candida albicans.

Candida albicans is a commensal fungal pathogen that is often found as part of the human microbial flora. The aim of the present study was to establish a relationship between diverse genotypes and phenotypes of clinical isolates of C. albicans. Totally 231 clinical isolates were collected and used for genotyping and phenotypic switching analysis. Based on the microsatellite locus (CAI) genotyping assay, 65 different genotypes were identified, and some dominant types were found in certain human niches. For example, the genotypes of 30-44 and 30-45 were enriched in vaginal infection samples. C. albicans has a number of morphological forms including the single-celled yeasts, multicellular filaments, white, and opaque cell types. The relationship between the CAI genotype and the ability to undergo phenotypic switching was examined in the clinical isolates. We found that the strains with longer CAA/G repeats in both alleles of the CAI locus were more opaque competent. We also discovered that some MTL heterozygous (a/alpha) isolates could undergo white-opaque switching when grown on regular culture medium (containing glucose as the sole carbon source). Our study establishes a link between phenotypic switching and genotypes of the CAI microsatellite locus in clinical isolates of C. albicans.

Total reflection x-ray fluorescence spectroscopy as a tool for evaluation of iron concentration in ferrofluids and yeast samples

In this study, total reflection x-ray fluorescent (TXRF) spectrometry was applied for the evaluation of iron concentration in ferrofluids and biological samples containing iron oxide magnetic nanoparticles obtained by the laser target evaporation technique. Suspensions of maghemite nanoparticles of different concentrations were used to estimate the limitation of the method for the evaluation of nanoparticle concentration in the range of 1–5000ppm in absence of organic matrix. Samples of single-cell yeasts grown in the nutrient media containing maghemite nanoparticles were used to study the nanoparticle absorption mechanism. The obtained results were analyzed in terms of applicability of TXRF for quantitative analysis in a wide range of iron oxide nanoparticle concentrations for biological samples and ferrofluids with a simple established protocol of specimen preparation.

Accurate concentration control of mitochondria and nucleoids.

All cellular materials are partitioned between daughters at cell division, but by various mechanisms and with different accuracy. In the yeast Schizosaccharomyces pombe, the mitochondria are pushed to the cell poles by the spindle. We found that mitochondria spatially reequilibrate just before division, and that the mitochondrial volume and DNA-containing nucleoids instead segregate in proportion to the cytoplasm inherited by each daughter. However, nucleoid partitioning errors are suppressed by control at two levels: Mitochondrial volume is actively distributed throughout a cell, and nucleoids are spaced out in semiregular arrays within mitochondria. During the cell cycle, both mitochondria and nucleoids appear to be produced without feedback, creating a net control of fluctuations that is just accurate enough to avoid substantial growth defects.

Mitochondria migration during mitosis

Cell Biology Every time a cell divides, it is faced with the problem of ensuring adequate distribution of all components to the daughter cells. This includes components that are present in relatively small numbers, like the mitochondria and their DNA-containing nucleoids. Jajoo et al. used quantitative time-lapse fluorescence microscopy of single yeast cells to explore how this works. Unlike chromosomes or macromolecules, mitochondria and nucleoids are distributed in proportion to the volume of cytoplasm received by each daughter cell. Partitioning errors are kept low by even distribution of the mitochondrial volume and roughly equal spacing of the nucleoids before they are distributed. Science , this issue p. [169][1] [1]: /lookup/doi/10.1126/science.aaa8714

Monitoring of chromosome dynamics of single yeast cells in a microfluidic platform with aperture cell traps.

Chromosome movement plays important roles in DNA replication, repair, genetic recombination, and epigenetic phenomena during mitosis and meiosis. In particular, chromosome movement in the nuclear space is essential for the reorganization of the nucleus. However, conventional methods for analyzing the chromosome movements in vivo have been limited by technical constraints of cell trapping, cell cultivation, oxygenation, and in situ imaging. Here, we present a simple microfluidic platform with aperture-based cell trapping arrays to monitor the chromosome dynamics in single living cells for a desired period of time. Under the optimized conditions, our microfluidic platform shows a single-cell trapping efficiency of 57%. This microfluidic approach enables in situ imaging of intracellular dynamics in living cells responding to variable input stimuli under the well-controlled microenvironment. As a validation of this microfluidic platform, we investigate the fundamental features of the dynamic cellular response of the individual cells treated with different stimuli and drug. We prove the basis for dynamic chromosome movement in single yeast cells to be the telomere and nuclear envelope ensembles that attach to and move in concert with nuclear actin cables. Therefore, these results illustrate the monitoring of cellular functions and obtaining of dynamic information at a high spatiotemporal resolution through the integration of a simple microfluidic platform.

Single Cell Mass Measurement Using Drag Force Inside Lab-on-Chip Microfluidics System.

Single cell mass (SCM) is an intrinsic property of single cell, it arouses a great interest among scientists as cell mass depends on the synthesis of proteins, DNA replication, cell wall stiffness, cell cytoplasm density, cell growth, ribosome, and other analogous of organisms. To date, several great strides have been taken to the advancements of SCM measurement techniques. Nevertheless, more works are required to enable the technology to push frontier in deep analysis of SCM measurement, hence to elucidate intracellular properties. In this paper, we present a lab-on-chip microfluidics system for SCM measurement, related with the force required to drag a single cell and Newton's law of motion inside microfluidics channel. Drag force on the cell was generated by a pressure driven syringe micropump and the motion of the cell was measured using optical observation under an inverted microscope. This approach of measuring SCM was calibrated using known mass (77.3 pg) of a polystyrene particle of 5.2 μm diameter. Furthermore, we used Saccharomyces cerevisiae baker's yeast cells of different sizes ([Formula: see text] diameter) for SCM measurement. Mass of 4.4 μm diameter of single yeast cell was measured as 2.12 pg which is in the range of previously reported single yeast cell mass (2-3 pg). In addition, we also studied the relation between SCM and single cell size. Results showed that single yeast cell mass increases exponentially with the increasing of single cell size.

Chromatin

Chromatin is a complex of proteins, RNA and DNA that constitutes the physiological state of the genome. Its basic structure is essentially the same in nearly all eukaryotes, from single-celled yeasts to the most complex multicellular organisms (exceptions include the chromatin of dinoflagellates and vertebrate sperm). Its fundamental role is to package the genome in a sufficiently compact form that allows comparatively very large molecules of DNA to fit inside the cell's nucleus. In human cells, the contour length of the DNA molecules comprising the largest chromosomes is nearly 10,000 times the diameter of the nucleus (typically on the order of 5-10 microns). How is this compaction accomplished? Through multiple layers of folding.

Histone deacetylase-mediated morphological transition in Candida albicans.

Candida albicans is the most common opportunistic fungal pathogen, which switches its morphology from single-cell yeast to filament through the various signaling pathways responding to diverse environmental cues. Various transcriptional factors such as Nrg1, Efg1, Brg1, Ssn6, and Tup1 are the key components of these signaling pathways. Since C. albicans can regulate its transcriptional gene expressions using common eukaryotic regulatory systems, its morphological transition by these signaling pathways could be linked to the epigenetic regulation by chromatin structure modifiers. Histone proteins, which are critical components of eukaryotic chromatin structure, can regulate the eukaryotic chromatin structure through their own modifications such as acetylation, methylation, phosphorylation and ubiquitylation. Recent studies revealed that various histone modifications, especially histone acetylation and deacetylation, participate in morphological transition of C. albicans collaborating with well-known transcription factors in the signaling pathways. Here, we review recent studies about chromatin-mediated morphological transition of C. albicans focusing on the interaction between transcription factors in the signaling pathways and histone deacetylases.