Natural Genetic Variation for Growth and Development Revealed by High-Throughput Phenotyping in Arabidopsis thaliana

Leaf growth and development determines a plant’s capacity for photosynthesis and carbon fixation. These morphological traits are the integration of genetic and environmental factors through time. Yet fine dissection of the developmental genetic basis of leaf expansion throughout a growing season is difficult, due to the complexity of the trait and the need for real time measurement. In this study, we developed a time-lapse image analysis approach, which traces leaf expansion under seasonal light variation. Three growth traits, rosette leaf area, circular area, and their ratio as compactness, were measured and normalized on a linear timescale to control for developmental heterogeneity. We found high heritability for all growth traits that changed over time. Our study highlights a cost-effective, high-throughput phenotyping approach that facilitates the dissection of genetic basis of plant shoot growth and development under dynamic environmental conditions

Genetic Variants Contributing to Colistin Cytotoxicity: Identification of TGIF1 and HOXD10 Using a Population Genomics Approach

Colistin sulfate (polymixin E) is an antibiotic prescribed with increasing frequency for severe Gram-negative bacterial infections. As nephrotoxicity is a common side effect, the discovery of pharmacogenomic markers associated with toxicity would benefit the utility of this drug. Our objective was to identify genetic markers of colistin cytotoxicity that were also associated with expression of key proteins using an unbiased, whole genome approach and further evaluate the functional significance in renal cell lines. To this end, we employed International HapMap lymphoblastoid cell lines (LCLs) of Yoruban ancestry with known genetic information to perform a genome-wide association study (GWAS) with cellular sensitivity to colistin. Further association studies revealed that single nucleotide polymorphisms (SNPs) associated with gene expression and protein expression were significantly enriched in SNPs associated with cytotoxicity (p ≤ 0.001 for gene and p = 0.015 for protein expression). The most highly associated SNP, chr18:3417240 (p = 6.49 × 10−8), was nominally a cis-expression quantitative trait locus (eQTL) of the gene TGIF1 (transforming growth factor β (TGFβ)-induced factor-1; p = 0.021) and was associated with expression of the protein HOXD10 (homeobox protein D10; p = 7.17 × 10−5). To demonstrate functional relevance in a murine colistin nephrotoxicity model, HOXD10 immunohistochemistry revealed upregulated protein expression independent of mRNA expression in response to colistin administration. Knockdown of TGIF1 resulted in decreased protein expression of HOXD10 and increased resistance to colistin cytotoxicity. Furthermore, knockdown of HOXD10 in renal cells also resulted in increased resistance to colistin cytotoxicity, supporting the physiological relevance of the initial genomic associations

Comprehensive Binary Interaction Mapping of SH2 Domains via Fluorescence Polarization Reveals Novel Functional Diversification of ErbB Receptors

First-generation interaction maps of Src homology 2 (SH2) domains with receptor tyrosine kinase (RTK) phosphosites have previously been generated using protein microarray (PM) technologies. Here, we developed a large-scale fluorescence polarization (FP) methodology that was able to characterize interactions between SH2 domains and ErbB receptor phosphosites with higher fidelity and sensitivity than was previously achieved with PMs. We used the FP assay to query the interaction of synthetic phosphopeptides corresponding to 89 ErbB receptor intracellular tyrosine sites against 93 human SH2 domains and 2 phosphotyrosine binding (PTB) domains. From 358,944 polarization measurements, the affinities for 1,405 unique biological interactions were determined, 83% of which are novel. In contrast to data from previous reports, our analyses suggested that ErbB2 was not more promiscuous than the other ErbB receptors. Our results showed that each receptor displays unique preferences in the affinity and location of recruited SH2 domains that may contribute to differences in downstream signaling potential. ErbB1 was enriched versus the other receptors for recruitment of domains from RAS GEFs whereas ErbB2 was enriched for recruitment of domains from tyrosine and phosphatidyl inositol phosphatases. ErbB3, the kinase inactive ErbB receptor family member, was predictably enriched for recruitment of domains from phosphatidyl inositol kinases and surprisingly, was enriched for recruitment of domains from tyrosine kinases, cytoskeletal regulatory proteins, and RHO GEFs but depleted for recruitment of domains from phosphatidyl inositol phosphatases. Many novel interactions were also observed with phosphopeptides corresponding to ErbB receptor tyrosines not previously reported to be phosphorylated by mass spectrometry, suggesting the existence of many biologically relevant RTK sites that may be phosphorylated but below the detection threshold of standard mass spectrometry procedures. This dataset represents a rich source of testable hypotheses regarding the biological mechanisms of ErbB receptors.</p

Comprehensive Binary Interaction Mapping of SH2 Domains via Fluorescence Polarization Reveals Novel Functional Diversification of ErbB Receptors

<div><p>First-generation interaction maps of Src homology 2 (SH2) domains with receptor tyrosine kinase (RTK) phosphosites have previously been generated using protein microarray (PM) technologies. Here, we developed a large-scale fluorescence polarization (FP) methodology that was able to characterize interactions between SH2 domains and ErbB receptor phosphosites with higher fidelity and sensitivity than was previously achieved with PMs. We used the FP assay to query the interaction of synthetic phosphopeptides corresponding to 89 ErbB receptor intracellular tyrosine sites against 93 human SH2 domains and 2 phosphotyrosine binding (PTB) domains. From 358,944 polarization measurements, the affinities for 1,405 unique biological interactions were determined, 83% of which are novel. In contrast to data from previous reports, our analyses suggested that ErbB2 was not more promiscuous than the other ErbB receptors. Our results showed that each receptor displays unique preferences in the affinity and location of recruited SH2 domains that may contribute to differences in downstream signaling potential. ErbB1 was enriched versus the other receptors for recruitment of domains from RAS GEFs whereas ErbB2 was enriched for recruitment of domains from tyrosine and phosphatidyl inositol phosphatases. ErbB3, the kinase inactive ErbB receptor family member, was predictably enriched for recruitment of domains from phosphatidyl inositol kinases and surprisingly, was enriched for recruitment of domains from tyrosine kinases, cytoskeletal regulatory proteins, and RHO GEFs but depleted for recruitment of domains from phosphatidyl inositol phosphatases. Many novel interactions were also observed with phosphopeptides corresponding to ErbB receptor tyrosines not previously reported to be phosphorylated by mass spectrometry, suggesting the existence of many biologically relevant RTK sites that may be phosphorylated but below the detection threshold of standard mass spectrometry procedures. This dataset represents a rich source of testable hypotheses regarding the biological mechanisms of ErbB receptors.</p> </div

Comparison of the affinity with which each ErbB family member recruits proteins representing several molecular function categories.

<p>Relative binding free energies of interactions described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044471#pone-0044471-g005" target="_blank">Figure 5C</a> were summed across all domains contained in a particular functional category <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044471#pone.0044471-Liu2" target="_blank">[55]</a> in (<b>A</b>) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044471#pone.0044471.s032" target="_blank">Table S16</a>) and divided by the number of SH2 domains represented in each class in (<b>B</b>) to determine an average recruitment potential for SH2s from each functional class. (<b>C</b>) ErbB receptor enrichment and depletion for binding sites for functional groups depicted by Z-score transformation of the raw data as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044471#pone-0044471-g005" target="_blank">Figure 5D</a>.</p

Competitive inhibition binding curves of protein-peptide interactions detected by FP.

<p>Nine ErbB phosphotyrosine sites were queried against five proteins: (<b>A</b>) RASA1-N, (<b>B</b>) SRC, (<b>C</b>) GRB7, (<b>D</b>) GRB2, and (<b>E</b>) PTK6. The predicted binding affinities of competitor peptide curves are color-coded as follows: red (<i>K_D<</i>1), purple (1≤ <i>K_D<</i>5), blue (5≤ <i>K_D<</i>20), and black (<i>K_D</i> ≥20). “OP” refers to the original rhodamine-labeled peptides and “CP” to the unlabeled competitor peptides, which have been numbered in the figure with sequences.</p

Comprehensive SH2 domain recruitment potential of the ErbB family as determined by high-throughput fluorescence polarization (HT-FP).

<p>Color-coded heat maps represent apparent dissociation constants (K_Ds) for FP interactions between SH2/PTB domains and phosphopeptides representing all potential ErbB1, ErbB2, ErbB3, and ErbB4 phosphotyrosine sites; black boxes indicate interactions that are too weak to be detected by the assay. Homologous ErbB peptides with identical amino acid residues from +1 to the +4 position relative to the phosphotyrosine (X) are indicated with an asterisk followed by the number (in order of occurrence) of the homologous receptor. Sequences of peptides used are indicated for each homologous receptor site, in which a small “d” denotes the pre-charged aspartic acid (Asp) residue on the peptide synthesis resin and not a naturally occurring Asp. NS refers to peptides that were unable to be synthesized, while NI refers to synthesized peptides that produced no positive hits in the study; therefore we cannot confirm nor deny interactions at these sites with our assay. Rows of the heatmaps for these peptides are grayed out to indicate that our FP assay could neither confirm nor deny positive or negative interactions from these peptides.</p

Characterization of unique and overlapping SH2 domain recruitment patterns by individual ErbB receptors.

<p>(<b>A</b>) SH2 recruitment potential of ErbB1 family members at different affinity thresholds. The total number of unique SH2 and PTB domains recruited over a range of affinity thresholds are depicted for each receptor. (<b>B</b>) Four-way Venn diagram (not to scale) depicts SH2 domain interactions shared by or exclusive to ErbB1, ErbB2, ErbB3, and ErbB4. (<b>C</b>) Relative binding free energies of interactions described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044471#pone-0044471-g002" target="_blank">Figure 2</a> are summed for each ErbB receptor. (<b>D</b>) Relative enrichment and depletion of binding sites for recruitment of each SH2 domain by each ErbB receptor, depicted by Z-score transforming the observed number of binding sites each receptor had for a particular domain relative to the average number of sites that bound that domain across all ErbB receptors. Domains recruited by fewer than four independent pY sites were excluded from this analysis.</p

Methodological cross-comparison of SH2 interactions determined by fluorescence polarization or in published protein microarray data for the ErbB family.

<p>(<b>A</b>) Venn diagram comparison of overall SH2 recruitment profiles revealed by FP and PMs for the ErbB family of RTKs for only peptides and proteins tested by both platforms. The red circle represents protein-peptide interactions observed by FP; the green circle represents protein-peptide interactions previously observed by PMs; and the yellow overlap represents interactions observed by both methods. (<b>B</b>) SH2 and ErbB interactions quantified over a range of binding affinity thresholds as determined previously by PMs and in this study by FP data. The red line represents interactions characterized exclusively by FP; the green line represents interactions characterized exclusively by PMs; the blue line represents interactions observed by both methods.</p