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

Quantitative Peptidomics of <i>Purkinje Cell Degeneration</i> Mice

<div><p>Cytosolic carboxypeptidase 1 (CCP1) is a metallopeptidase that removes C-terminal and side-chain glutamates from tubulin. The <i>Purkinje cell degeneration</i> (<i>pcd</i>) mouse lacks CCP1 due to a mutation. Previously, elevated levels of peptides derived from cytosolic and mitochondrial proteins were found in adult <i>pcd</i> mouse brain, raising the possibility that CCP1 functions in the degradation of intracellular peptides. To test this hypothesis, we used a quantitative peptidomics technique to compare peptide levels in wild-type and <i>pcd</i> mice, examining adult heart, spleen, and brain, and presymptomatic 3 week-old amygdala and cerebellum. Contrary to adult mouse brain, young <i>pcd</i> brain and adult heart and spleen did not show a large increase in levels of intracellular peptides. Unexpectedly, levels of peptides derived from secretory pathway proteins were altered in adult <i>pcd</i> mouse brain. The pattern of changes for the intracellular and secretory pathway peptides in <i>pcd</i> mice was generally similar to the pattern observed in mice lacking primary cilia. Collectively, these results suggest that intracellular peptide accumulation in adult <i>pcd</i> mouse brain is a secondary effect and is not due to a role of CCP1 in peptide turnover.</p> </div

Levels of intracellular peptides in the brain of young WT and <i>pcd</i> mice.

<p>The levels of peptides derived from intracellular proteins were studied using quantitative peptidomics. The relative levels of peptides in WT mice (black circles) and <i>pcd</i> mice (grey circles) are indicated for (<b>A</b>) amygdala of adult mice; (<b>B</b>) amygdala of 3-week old mice; and (<b>C</b>) cerebellum of 3-week old mice. Each dot in the graph shows the ratio between a peptide in one WT or <i>pcd</i> replicate versus the average level in the WT replicates. The x-axis reflects the number of peptides found in <i>pcd</i> mice. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060981#pone.0060981.s003" target="_blank">Table S1</a> for data.</p

Comparison of the number of peptides detected in adult <i>pcd</i> and WT mouse brain.

<p>Data from the analysis of peptides in whole brain (minus the cerebellum and olfactory bulb) from adult mice indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060981#pone.0060981.s003" target="_blank">Table S1</a> were used. For this study, two WT mice were compared to two <i>pcd</i> mice. Conclusively and tentatively identified peptides were included in this analysis, and the peptides were counted each time they were detected as a separate m/z ion (i.e. for each animal, and for each charge state).</p