Accurate, precise modeling of cell proliferation kinetics from time-lapse imaging and automated image analysis of agar yeast culture arrays

BACKGROUND: Genome-wide mutant strain collections have increased demand for high throughput cellular phenotyping (HTCP). For example, investigators use HTCP to investigate interactions between gene deletion mutations and additional chemical or genetic perturbations by assessing differences in cell proliferation among the collection of 5000 S. cerevisiae gene deletion strains. Such studies have thus far been predominantly qualitative, using agar cell arrays to subjectively score growth differences. Quantitative systems level analysis of gene interactions would be enabled by more precise HTCP methods, such as kinetic analysis of cell proliferation in liquid culture by optical density. However, requirements for processing liquid cultures make them relatively cumbersome and low throughput compared to agar. To improve HTCP performance and advance capabilities for quantifying interactions, YeastXtract software was developed for automated analysis of cell array images. RESULTS: YeastXtract software was developed for kinetic growth curve analysis of spotted agar cultures. The accuracy and precision for image analysis of agar culture arrays was comparable to OD measurements of liquid cultures. Using YeastXtract, image intensity vs. biomass of spot cultures was linearly correlated over two orders of magnitude. Thus cell proliferation could be measured over about seven generations, including four to five generations of relatively constant exponential phase growth. Spot area normalization reduced the variation in measurements of total growth efficiency. A growth model, based on the logistic function, increased precision and accuracy of maximum specific rate measurements, compared to empirical methods. The logistic function model was also more robust against data sparseness, meaning that less data was required to obtain accurate, precise, quantitative growth phenotypes. CONCLUSION: Microbial cultures spotted onto agar media are widely used for genotype-phenotype analysis, however quantitative HTCP methods capable of measuring kinetic growth rates have not been available previously. YeastXtract provides objective, automated, quantitative, image analysis of agar cell culture arrays. Fitting the resulting data to a logistic equation-based growth model yields robust, accurate growth rate information. These methods allow the incorporation of imaging and automated image analysis of cell arrays, grown on solid agar media, into HTCP-driven experimental approaches, such as global, quantitative analysis of gene interaction networks

Spotsizer: High-throughput quantitative analysis of microbial growth

Microbial colony growth can serve as a useful readout in assays for studying complex genetic interactions or the effects of chemical compounds. Although computational tools for acquiring quantitative measurements of microbial colonies have been developed, their utility can be compromised by inflexible input image requirements, non-trivial installation procedures, or complicated operation. Here, we present the Spotsizer software tool for automated colony size measurements in images of robotically arrayed microbial colonies. Spotsizer features a convenient graphical user interface (GUI), has both single-image and batch-processing capabilities, and works with multiple input image formats and different colony grid types. We demonstrate how Spotsizer can be used for high-throughput quantitative analysis of fission yeast growth. The user-friendly Spotsizer tool provides rapid, accurate, and robust quantitative analyses of microbial growth in a high-throughput format. Spotsizer is freely available at https://data.csiro.au/dap/landingpage?pid=csiro:15330 under a proprietary CSIRO license

Colonyzer: automated quantification of micro-organism growth characteristics on solid agar

<p>Abstract</p> <p>Background</p> <p>High-throughput screens comparing growth rates of arrays of distinct micro-organism cultures on solid agar are useful, rapid methods of quantifying genetic interactions. Growth rate is an informative phenotype which can be estimated by measuring cell densities at one or more times after inoculation. Precise estimates can be made by inoculating cultures onto agar and capturing cell density frequently by plate-scanning or photography, especially throughout the exponential growth phase, and summarising growth with a simple dynamic model (e.g. the logistic growth model). In order to parametrize such a model, a robust image analysis tool capable of capturing a wide range of cell densities from plate photographs is required.</p> <p>Results</p> <p>Colonyzer is a collection of image analysis algorithms for automatic quantification of the size, granularity, colour and location of micro-organism cultures grown on solid agar. Colonyzer is uniquely sensitive to extremely low cell densities photographed after dilute liquid culture inoculation (spotting) due to image segmentation using a mixed Gaussian model for plate-wide thresholding based on pixel intensity. Colonyzer is robust to slight experimental imperfections and corrects for lighting gradients which would otherwise introduce spatial bias to cell density estimates without the need for imaging dummy plates. Colonyzer is general enough to quantify cultures growing in any rectangular array format, either growing after pinning with a dense inoculum or growing with the irregular morphology characteristic of spotted cultures. Colonyzer was developed using the open source packages: Python, RPy and the Python Imaging Library and its source code and documentation are available on SourceForge under GNU General Public License. Colonyzer is adaptable to suit specific requirements: e.g. automatic detection of cultures at irregular locations on streaked plates for robotic picking, or decreasing analysis time by disabling components such as lighting correction or colour measures.</p> <p>Conclusion</p> <p>Colonyzer can automatically quantify culture growth from large batches of captured images of microbial cultures grown during genome-wide scans over the wide range of cell densities observable after highly dilute liquid spot inoculation, as well as after more concentrated pinning inoculation. Colonyzer is open-source, allowing users to assess it, adapt it to particular research requirements and to contribute to its development.</p

Merging microfluidics and micro-array concepts: from molecular to nematode-based bioassays

Essential in biomedical research is the necessity of gathering statistically relevant data about large populations of specific biological entities, e.g. organisms, cells or molecules, while preserving detailed information about each single entity under investigation. This thesis deals with this need and proposes the combination of microfluidics and micro-arraying techniques in developing technological tools to conceive bio-assays at single molecule/cell/organism resolution. First, we propose an on-chip immunoassay technique, through which we demonstrated detection of the biomarker tumor necrosis factor alpha in serum down to concentrations in the attomolar range (10-18 M). In particular, we provide a comprehensive predictive model of the assay, which employs micro-arrays of superparamagnetic beads. We introduce the concept of magnetic particle-scanning, as a method for building immunoassays with extremely low limit of detection, down to the single-molecule level. Afterwards, we modified our bead micro-arraying technique, to make it suitable for the immobilization of particles and cells of various sizes and properties. Specifically, we present a method for the electrostatic self-assembly of dielectric microspheres in well templates, as a technique for fast and versatile fabrication of microlens arrays. By combining these arrays with microfluidics, we created a new tool for single-nanoparticle detection in flowing media, able to detect moving objects of sub-diffraction size through conventional low-magnification microscopes. An analogous micro-arraying method was then developed to seed large populations of non-adherent cells in isolated micro-compartments. In combination with an electrowetting-on-dielectric microfluidic platform, this technique allows implementing high-throughput cytotoxicity assays on yeast cells, at single-cell resolution. Subsequently, we conceived technological solutions for the automated analysis of Caenorhabditis elegans, one of the most employed model organisms in biomedical research. First, we developed a microfluidic platform for on-chip nematode culture and creation of synchronized C. elegans embryo micro-arrays. Long-term multi-dimensional imaging in our device allows systematic phenotyping studies at single-embryo resolution. We could discriminate embryonic development variations with unprecedented accuracy and we successfully analyzed the impact of perturbations of the mitochondrial functions on the embryogenesis. A second generation prototype of the device is then presented, enabling long-term automated studies on C. elegans at single-nematode resolution and over the whole organism development, from early embryogenesis to adulthood. Finally, we introduce a third generation prototype, which features: (i) a new microfluidic design tailored for the isolation of larvae at a desired developmental stage and for their successive culture and treatment; (ii) a method for reversible immobilization of nematodes, enabling long-term high-resolution imaging. We successfully employed this platform to analyze protein aggregation in a C. elegans model for human amyotrophic lateral sclerosis (ALS). The device allows precisely localizing protein aggregates within the nematode tissues, as well as monitoring the evolution of single aggregates over consecutive days at sub-cellular level

Pyphe, a python toolbox for assessing microbial growth and cell viability in high-throughput colony screens

Microbial fitness screens are a key technique in functional genomics. We present an allin-one solution, pyphe, for automating and improving data analysis pipelines associated with largescale fitness screens, including image acquisition and quantification, data normalisation, and statistical analysis. Pyphe is versatile and processes fitness data from colony sizes, viability scores from phloxine B staining or colony growth curves, all obtained with inexpensive transilluminating flatbed scanners. We apply pyphe to show that the fitness information contained in late endpoint measurements of colony sizes is similar to maximum growth slopes from time series. We phenotype gene-deletion strains of fission yeast in 59,350 individual fitness assays in 70 conditions, revealing that colony size and viability provide complementary, independent information. Viability scores obtained from quantifying the redness of phloxine-stained colonies accurately reflect the fraction of live cells within colonies. Pyphe is user-friendly, open-source and fully documented, illustrated by applications to diverse fitness analysis scenarios

Single-Cell Analysis of Microbial Production Strains in Microfluidic Bioreactors

Grünberger A. Single-Cell Analysis of Microbial Production Strains in Microfluidic Bioreactors. Schriften des Forschungszentrums Jülich. Reihe Schlüsseltechnologien / Key Technologies. Vol 114. Jülich: RWTH Aachen; 2015

Quantitative Fitness Analysis Shows That NMD Proteins and Many Other Protein Complexes Suppress or Enhance Distinct Telomere Cap Defects

To better understand telomere biology in budding yeast, we have performed systematic suppressor/enhancer analyses on yeast strains containing a point mutation in the essential telomere capping gene CDC13 (cdc13-1) or containing a null mutation in the DNA damage response and telomere capping gene YKU70 (yku70Δ). We performed Quantitative Fitness Analysis (QFA) on thousands of yeast strains containing mutations affecting telomere-capping proteins in combination with a library of systematic gene deletion mutations. To perform QFA, we typically inoculate 384 separate cultures onto solid agar plates and monitor growth of each culture by photography over time. The data are fitted to a logistic population growth model; and growth parameters, such as maximum growth rate and maximum doubling potential, are deduced. QFA reveals that as many as 5% of systematic gene deletions, affecting numerous functional classes, strongly interact with telomere capping defects. We show that, while Cdc13 and Yku70 perform complementary roles in telomere capping, their genetic interaction profiles differ significantly. At least 19 different classes of functionally or physically related proteins can be identified as interacting with cdc13-1, yku70Δ, or both. Each specific genetic interaction informs the roles of individual gene products in telomere biology. One striking example is with genes of the nonsense-mediated RNA decay (NMD) pathway which, when disabled, suppress the conditional cdc13-1 mutation but enhance the null yku70Δ mutation. We show that the suppressing/enhancing role of the NMD pathway at uncapped telomeres is mediated through the levels of Stn1, an essential telomere capping protein, which interacts with Cdc13 and recruitment of telomerase to telomeres. We show that increased Stn1 levels affect growth of cells with telomere capping defects due to cdc13-1 and yku70Δ. QFA is a sensitive, high-throughput method that will also be useful to understand other aspects of microbial cell biology

Characterization of Schizosaccharomyces pombe chromosome condensation factor Zas1

Pro Sekunde teilen sich mehr als 3 Millionen Zellen im menschlichen Körper (Notta et al., 2016). Bei jeder dieser Zellteilungen muss die korrekte Verteilung der Chromosomen - die Träger der genetischen Information - gewährleistet werden. Ist die Verteilung der genetischen Information ungleichmäßig, z. B. aufgrund eines fehlenden oder überschüssigen Chromosoms, kommt es zum Tod der betroffenen Tochterzelle oder Krebs. So hat sich eine faszinierend zuverlässige Machinerie entwickelt, die in der Mitose die etwa 2 Meter menschlicher DNA zuerst eng verpackt und dann an die Zellpole transportiert, sodass sich die etwa 20 Mikrometer große Zelle durchschnüren kann. Die Kompaktierung des Chromatins - genannt Chromosomenkondensation - ist eines der am wenigsten verstandenden Prozesse der Zellteilung. Um die Chromosomenkondensation besser untersuchen zu können, ist ein Mikroskopie basiertes Chromosomenkondensationsmessverfahren in der Spalthefe S. pombe entwickelt worden, welches auf spezifischer Fluo- reszenzmarkierung zweier Loci beruht. Mittels dieses Messverfahrens wurden aus einer Kollektion zufälliger, wärmeempfindlicher Mutanten drei Allele des zuvor kaum charakterisierten Gens zas1 identifiziert, bei denen die Chromosomenkondensation beeinträchtigt ist (Petrova, 2012). In der vorliegenden Arbeit charakterisiere ich zas1 und zeige, das seine Funktion von seinen Zinkfinger Domänen und einer kurzen, E2F-ähnlichen Peptidsequenz, welche zuvor noch nie in Einzellern beschrieben wurde, abhängt. Ich stelle fest, dass Zas1 die Transkription der Condensin-Untereinheit Cnd1 reguliert, was den Kondensationsdefekt in zas1 Mutanten erklärt. Damit wird zum ersten Mal ein Transkriptionsfaktor für eine Condensin-Untereinheit in S. pombe beschrieben. Im zweiten Teil der Arbeit verbessere ich das Chromosomenkondensationsmessverfahren, indem ich die rechnergestützte Bildverarbeitung und Datenanalyse weitestgehend automatisiere. Diese Optimierungen ermöglichen es, die Variabilität zwischen Experimenten zu messen. Gleichzeitig offenbart sich ein positiver Zusammenhang von Kondensationsrate mit der Distanz zwischen den Fluoreszenzmarkierungen. Diese Korrelation zeigt, dass die Kondensationsaktivität entlang des Chromosomenarms verteilt ist. Weitere Optimierung der Mikroskopkonfiguration und die Verbesserungen in der Datenverarbeitung erlauben es, die longitudinale Verkürzung der Chromosomen auf Einzelzellebene aufzulösen. Dies führt zur Beobachtung von longitudinalen Oszillationen und offenbart, dass die Kondensation linear verläuft. Schließlich etabliere ich eine verbesserte Version der Chromatin Markierung, indem ich nicht-rekombinierbare Tetracyclin Operator Wiederholungssequenzen für S. pombe adaptiere

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin