Engineering topics

Wiley, H S

Publications and source records attributed to Wiley, H S.

13 records

DOE OSTI2012

Integrated Experimental and Model-based Analysis Reveals the Spatial Aspects of EGFR Activation Dynamics

The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases, and controls a diverse set of cellular responses relevant to development and tumorigenesis. ErbB activation is a complex process involving receptor-ligand binding, receptor dimerization, phosphorylation, and trafficking (internalization, recycling and degradation), which together dictate the spatio-temporal distribution of active receptors within the cell. The ability to predict this distribution, and elucidation of the factors regulating it, would help to establish a mechanistic link between ErbB expression levels and the cellular response. Towards this end, we constructed mathematical models for deconvolving the contributions of receptor dimerization and phosphorylation to EGFR activation, and to examine the dependence of these processes on sub-cellular location. We collected experimental datasets for EGFR activation dynamics in human mammary epithelial cells, with the specific goal of model parameterization, and used the data to estimate parameters for several alternate models. Model-based analysis indicated that: 1) signal termination via receptor dephosphorylation in late endosomes, prior to degradation, is an important component of the response, 2) less than 40% of the receptors in the cell are phosphorylated at any given time, even at saturating ligand doses, and 3) receptor dephosphorylation rates at the cell surface and early endosomes are comparable. We validated the last finding by measuring EGFR dephosphorylation rates at various times following ligand addition both in whole cells, and in endosomes using ELISAs and fluorescent imaging. Overall, our results provide important information on how EGFR phosphorylation levels are regulated within cells. Further, the mathematical model described here can be extended to determine receptor dimer abundances in cells co-expressing various levels of ErbB receptors. This study demonstrates that an iterative cycle of experiments and modeling can be used to gain mechanistic insight regarding complex cell signaling networks.

kinetic modeling↗

DOE OSTI2012

Network Analysis of Epidermal Growth Factor Signaling using Integrated Genomic, Proteomic and Phosphorylation Data

To understand how integration of multiple data types can help decipher cellular responses at the systems level, we analyzed the mitogenic response of human mammary epithelial cells to epidermal growth factor (EGF) using whole genome microarrays, mass spectrometry-based proteomics and large-scale western blots with over 1000 antibodies. A time course analysis revealed significant differences in the expression of 3172 genes and 596 proteins, including protein phosphorylation changes measured by western blot. Integration of these disparate data types showed that each contributed qualitatively different components to the observed cell response to EGF and that varying degrees of concordance in gene expression and protein abundance measurements could be linked to specific biological processes. Networks inferred from individual data types were relatively limited, whereas networks derived from the integrated data recapitulated the known major cellular responses to EGF and exhibited more highly connected signaling nodes than networks derived from any individual dataset. While cell cycle regulatory pathways were altered as anticipated, we found the most robust response to mitogenic concentrations of EGF was induction of matrix metalloprotease cascades, highlighting the importance of the EGFR system as a regulator of the extracellular environment. These results demonstrate the value of integrating multiple levels of biological information to more accurately reconstruct networks of cellular response.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2011

EMSL Geochemistry, Biogeochemistry and Subsurface Science-Science Theme Advisory Panel Meeting

This report covers the topics of discussion and the recommendations of the panel members. On December 8 and 9, 2010, the Geochemistry, Biogeochemistry, and Subsurface Science (GBSS) Science Theme Advisory Panel (STAP) convened for a more in-depth exploration of the five Science Theme focus areas developed at a similar meeting held in 2009. The goal for the fiscal year (FY) 2011 meeting was to identify potential topical areas for science campaigns, necessary experimental development needs, and scientific members for potential research teams. After a review of the current science in each of the five focus areas, the 2010 STAP discussions successfully led to the identification of one well focused campaign idea in pore-scale modeling and five longer-term potential research campaign ideas that would likely require additional workshops to identify specific research thrusts. These five campaign areas can be grouped into two categories: (1) the application of advanced high-resolution, high mass accuracy experimental techniques to elucidate the interplay between geochemistry and microbial communities in terrestrial ecosystems and (2) coupled computation/experimental investigations of the electron transfer reactions either between mineral surfaces and outer membranes of microbial cells or between the outer and inner membranes of microbial cells.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2011

Characterization and Improvement of RNA-Seq Precision in Quantitative Transcript Expression Profiling

Measurement precision determines the power of any analysis to reliably identify significant signals, such as in screens for differential expression, independent of whether the experimental design incorporates replicates or not. With the compilation of large scale RNA-Seq data sets with technical replicate samples, however, we can now, for the first time, perform a systematic analysis of the precision of expression level estimates from massively parallel sequencing technology. This then allows considerations for its improvement by computational or experimental means. Results: We report on a comprehensive study of target coverage and measurement precision, including their dependence on transcript expression levels, read depth and other parameters. In particular, an impressive target coverage of 84% of the estimated true transcript population could be achieved with 331 million 50 bp reads, with diminishing returns from longer read lengths and even less gains from increased sequencing depths. Most of the measurement power (75%) is spent on only 7% of the known transcriptome, however, making less strongly expressed transcripts harder to measure. Consequently, less than 30% of all transcripts could be quantified reliably with a relative error < 20%. Based on established tools, we then introduce a new approach for mapping and analyzing sequencing reads that yields substantially improved performance in gene expression profiling, increasing the number of transcripts that can reliably be quantified to over 40%. Extrapolations to higher sequencing depths highlight the need for efficient complementary steps. In discussion we outline possible experimental and computational strategies for further improvements in quantification precision.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2011

Integrating Multiple Types of Data for Signaling Research: Challenges and Opportunities

New technologies promise to provide unprecedented amounts of information that can provide a foundation for creating predictive models of cell signaling pathways. To be useful, however, this information must be integrated into a coherent framework. In addition, the sheer volume of data gathered from the new technologies requires computational approaches for its analysis. Unfortunately, there are many barriers to data integration and analysis, mostly because of a lack of adequate data standards and their inconsistent use by scientists. However, solving the fundamental issues of data sharing will enable the investigation of entirely new areas of cell signaling research.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2010

Oscillatory Dynamics of the Extracellular Signal-regulated Kinase Pathway

The extracellular signal-regulated kinase (ERK) pathway is a central signaling pathway in development and disease and is regulated by multiple negative and positive feedback loops. Recent studies have shown negative feedback from ERK to upstream regulators can give rise to biochemical oscillations with a periodicity of between 15-30 minutes. Feedback due to the stimulated transcription of negative regulators of the ERK pathway can also give rise to transcriptional oscillations with a periodicity of 1-2h. The biological significance of these oscillations is not clear, but recent evidence suggests that transcriptional oscillations participate in developmental processes, such as somite formation. Biochemical oscillations are more enigmatic, but could provide a mechanism for encoding different types of inputs into a common signaling pathway.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2010

One of the Good Guys

I was talking with some younger colleagues at a meeting last month when the subject of career goals came up. These colleagues were successful in that they had recently received tenure at top research universities and had some grants and good students. Thus, the early career pressure to simply survive was gone. So now what motivated them? Solving challenging and significant scientific problems was at the top of their lists. Interestingly, they were also motivated by a desire to become one of the “good guys” in science. The fact that being an important contributor to the scientific community can be fulfilling should not come as a surprise to anyone. However, what I do consider surprising is how rarely this seems to be discussed with students and postdocs. What we do discuss are either those issues that are fundamental aspects of the job (get a grant, get tenure, do research in an important field) or those that are important to our institutions. Knowing how to do our jobs well is indeed essential for any kind of professional success. However, achieving the right balance in our ambitions is also important for our happiness.

99 GENERAL AND MISCELLANEOUS↗

DOE OSTI2010

The heparin-binding domain of HB-EGF mediates localization to sites of cell-cell contact and prevents HB-EGF proteolytic release

Heparin-binding EGF-like growth factor (HB-EGF) is a ligand for EGF receptor (EGFR) and possesses the ability to signal in juxtacrine, autocrine and/or paracrine mode, with these alternatives being governed by the degree of proteolytic release of the ligand. Although the spatial range of diffusion of released HB-EGF is restricted by binding heparan-sulfate proteoglycans (HSPGs) in the extracellular matrix and/or cellular glycocalyx, ascertaining mechanisms governing non-released HB-EGF localization is also important for understanding its effects. We have employed a new method for independently tracking the localization of the extracellular EGFlike domain of HB-EGF and the cytoplasmic C-terminus. A striking observation was the absence of the HB-EGF transmembrane proform from the leading edge of COS-7 cells in a wound-closure assay; instead, this protein localized in regions of cell-cell contact. A battery of detailed experiments found that this localization derives from a trans interaction between extracellular HSPGs and the HBEGF heparin-binding domain, and that disruption of this interaction leads to increased release of soluble ligand and a switch in cell phenotype from juxtacrine-induced growth inhibition to autocrine-induced proliferation. Our results indicate that extracellular HSPGs serve to sequester the transmembrane pro-form of HB-EGF at the point of cell-cell contact, and that this plays a role in governing the balance between juxtacrine versus autocrine and paracrine signaling.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2010

Scientists vs. Engineers

In the past, I have heard there was conflict between the “two cultures” of science and the humanities. I don’t see a lot of evidence for that type of conflict today, mostly because my scientific friends all are big fans of the arts and literature. However, the two cultures that I do see a great deal of conflict between are those of science and engineering.

99 GENERAL AND MISCELLANEOUS↗

DOE OSTI2010

Think Outside the Bio-Box

After listening to my friends for many years (and reading numerous online comments in The Scientist), I was surprised that being a biologist was considered a low stress job with a bright future. Grousing about our jobs has always been a favorite pastime for biologists, but I suspect that in our hearts, we know how good we really have it. Even though research funding is tight and faculty positions are scarce, biologists still have one of the most interesting and exciting jobs around. I certainly don’t think that our current situation is worse than in the past. The real reason why the job outlook in biology seems dim is that research professors are training students to be university research scientists instead of preparing them for the broader job market. The result is that many new PhDs are frustrated to find that the specific positions they are trained for either don’t exist or have 300 applicants.

99 GENERAL AND MISCELLANEOUS↗

DOE OSTI2010

Structure of the EGF receptor transactivation circuit integrates multiple signals with cell context

Transactivation of the epidermal growth factor receptor (EGFR) has been proposed to be a mechanism by which a variety of cellular inputs can be integrated into a single signaling pathway, but the regulatory topology of this important system is unclear. To understand the transactivation circuit, we first created a “non-binding” reporter for ligand shedding. We then quantitatively defined how signals from multiple agonists were integrated both upstream and downstream of the EGFR into the extracellular signal regulated kinase (ERK) cascade in human mammary epithelial cells. We found that transactivation is mediated by a recursive autocrine circuit where ligand shedding drives EGFR-stimulated ERK that in turn drives further ligand shedding. The time from shedding to ERK activation is fast (<5 min) whereas the recursive feedback is slow (>15 min). Simulations showed that this delay in positive feedback greatly enhanced system stability and robustness. Our results indicate that the transactivation circuit is constructed so that the magnitude of ERK signaling is governed by the sum of multiple direct inputs, while recursive, autocrine ligand shedding controls signal duration.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2010

Basic Fibroblast Growth Factor Regulates Persistent ERK Osciliations in Premaligant but not Malignant JB6 Cells

basic fibroblast growth factor (bFGF or FGF2) plays an important role in epidermal wound healing in vivo and is associated with a persistent increased in the extracellular signal-regulated kinase (ERK) pathway in vitro. Here we have examined whether bFGF induces the closure of an experimental scratch wound in JB6 mouse epidermal cells and have explored the regulation of the ERK pathway by bFGF in the context of kinase oscillations. bFGF stimulation is associated with increases in cellular phospho-ERK and phospho-c-Jun levels. In addition, bFGF increases cell proliferation and a change in cell morphology (stellate appearance) in a dose-dependent fashion (0.1 – 100 ng/ml). bFGF treatment also promoted the closure of an experimental scratch wound in vitro. JB6 cells were stably transfected with an ERK1-GFP chimera to follow temporal ERK subcellular distribution patterns. We observe a persistent upregulation of the ERK pathway, as evidenced by a significant increase in nuclear ERK1-GFP levels at time points up to 24 hr after bFGF treatment. Interestingly, at the single cell level, ERK is observed to oscillate between nuclear and cytosolic compartments in response to bFGF treatment. Because this oscillatory behavior is asynchronous in the cell population, it is only clearly resolved at the single cell level. Collectively, data presented here are consistent with an important role for bFGF in wound healing and suggest a more complex regulation of the ERK pathway by bFGF than has previously been appreciated.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2010

Keep it Simple

Creating a story for a particular audience is one of the most difficult tasks for anyone to learn. This is true for scientists and writers as well as any creative artist who tries to understand the complexity of the world and explain it to other people. Telling a good story always takes skill. Telling a popular story, however, requires simplification.

59 BASIC BIOLOGICAL SCIENCES↗