Jonathan S. Minden

Difference gel electrophoresis.

Difference gel electrophoresis (DIGE) was invented to circumvent the inherent variability of 2‐DE. This variability is a natural consequence of separating thousands of proteins over a large space, such as a 15×20 cm slab of polyacrylamide gel. The originators of 2‐DE envisioned being able to compare cancerous cells and normal cells to understand what makes these cells different. Gel‐to‐gel variability made this an extremely difficult task. We reasoned that if both samples could be run on the same gel, then the inherent variability would be obviated. Thus, we created matched sets of fluorescent dyes that allows one to compare two or three protein samples on a single gel. In the 12 years since the description of DIGE first appeared in Electrophoresis, this founding paper has been cited over 660 times. This review highlights some of the improvements and applications of DIGE. We hope these examples are illustrative of what has been done and where the field is headed.

Drosophila ventral furrow morphogenesis: a proteomic analysis.

Ventral furrow formation is a key morphogenetic event during Drosophila gastrulation that leads to the internalization of mesodermal precursors. While genetic analysis has revealed the genes involved in the specification of ventral furrow cells, few of the structural proteins that act as mediators of ventral cell behavior have been identified. A comparative proteomics approach employing difference gel electrophoresis was used to identify more than fifty proteins with altered abundance levels or isoform changes in ventralized versus lateralized embryos. Curiously, the majority of protein differences between these embryos appeared well before gastrulation, only a few protein changes coincided with gastrulation, suggesting that the ventral cells are primed for cell shape change. Three proteasome subunits were found to differ between ventralized and lateralized embryos. RNAi knockdown of these proteasome subunits and time-dependent difference-proteins caused ventral furrow defects, validating the role of these proteins in ventral furrow morphogenesis.

Comparative Proteomics and Difference Gel Electrophoresis

The goal of comparative proteomics is to analyze proteome changes in response to development, disease, or environment. This is a two-step process in which proteins within cellular extracts are first fractionated to reduce sample complexity, and then the proteins are identified by mass spectrometry. Two-dimensional electrophoresis (2DE) is the long-time standard for protein separation, but it has suffered from poor reproducibility and limited sensitivity. Difference gel electrophoresis (DIGE), in which two protein samples are separately labeled with different fluorescent dyes and then co-electrophoresed on the same 2DE gel, was developed to overcome the reproducibility and sensitivity limitations. In this essay, I discuss the principles of comparative proteomics and the development of DIGE.

Guidelines for reporting the use of gel electrophoresis in proteomics

Gibson, Frank Anderson, Leigh Babnigg, Gyorgy Baker, Mark Berth, Matthias Binz, Pierre-Alain Borthwick, Andy Cash, Phil Day, Billy W. Friedman, David B. Garland, Donita Gutstein, Howard B. Hoogland, Christine Jones, Neil A. Khan, Alamgir Klose, Joachim Lamond, Angus I. Lemkin, Peter F. Lilley, Kathryn S. Minden, Jonathan Morris, Nicholas J. Paton, Norman W. Pisano, Michael R. Prime, John E. Rabilloud, Thierry Stead, David A. Taylor, Chris F. Voshol, Hans Wipat, Anil Jones, Andrew R. 2 NATURE PUBLISHING GROUP NEW YORK 335WX

A pharmacoproteomic approach implicates eukaryotic elongation factor 2 kinase in ER stress-induced cell death

Apoptosis triggered by endoplasmic reticulum (ER) stress has been implicated in many diseases but its cellular regulation remains poorly understood. Previously, we identified salubrinal (sal), a small molecule that protects cells from ER stress-induced apoptosis by selectively activating a subset of endogenous ER stress-signaling events. Here, we use sal as a probe in a proteomic approach to discover new information about the endogenous cellular response to ER stress. We show that sal induces phosphorylation of the translation elongation factor eukaryotic translation elongation factor 2 (eEF-2), an event that depends on eEF-2 kinase (eEF-2K). ER stress itself also induces eEF-2K-dependent eEF-2 phosphorylation, and this pathway promotes translational arrest and cell death in this context, identifying eEF-2K as a hitherto unknown regulator of ER stress-induced apoptosis. Finally, we use both sal and ER stress models to show that eEF-2 phosphorylation can be activated by at least two signaling mechanisms. Our work identifies eEF-2K as a new component of the ER stress response and underlines the utility of novel small molecules in discovering new cell biology.

Caspase-independent cell engulfment mirrors cell death pattern in Drosophila embryos

Programmed cell death plays an essential role during Drosophila embryonic development. A stereotypic series of cellular changes occur during apoptosis, most of which are initiated by a caspase cascade that is triggered by a trio of proteins, RPR, HID and GRIM. The final step in apoptosis is engulfment of the cell corpse. To monitor cell engulfment in vivo, we developed a fluorogenic β-galactosidase substrate that is cleaved by an endogenous, lysosomal β-galactosidase activity. The pattern of cell engulfment in wild-type embryos correlated well with the known pattern of apoptosis. Surprisingly, the pattern of cell engulfment persisted in apoptosis-deficient embryos. We provide evidence for a caspase-independent engulfment process that affects the majority of cells expected to die in developing Drosophila embryos.

DIGE: Past and Future

This chapter provides a brief historical perspective of the development of difference gel electrophoresis, from its inception to commercialization and beyond.

TOWARDS AN IMAGE ANALYSIS TOOLBOX FOR HIGH-THROUGHPUT DROSOPHILA EMBRYO RNAI SCREENS

We build an image analysis toolbox for high-throughput Drosophila embryo RNAi screens. The goal is to tag the embryo as normal, developmentally delayed or abnormal based on the ventral furrow formation. We break the problem into two parts: in the first, we detect the developmental stage based on the progress of the ventral furrow formation, and in the second, we tag the embryo as normal/developmentally delayed/abnormal based on the stage detected and the elapsed time. The crux of the algorithm is the multiresolution classifier, and we show that, by classifying in multiresolution spaces, we obtain better results than by classifying the embryo image alone. The final 2D accuracy obtained was 93.17%, while by using 3D information, it increased to 98.35%

Proteomic analysis reveals CCT is a target of Fragile X mental retardation protein regulation in Drosophila

Fragile X mental retardation protein (FMRP) is an RNA-binding protein that is required for the translational regulation of specific target mRNAs. Loss of FMRP causes Fragile X syndrome (FXS), the most common form of inherited mental retardation in humans. Understanding the basis for FXS has been limited because few in vivo targets of FMRP have been identified and mechanisms for how FMRP regulates physiological targets are unclear. We have previously demonstrated that Drosophila FMRP (dFMRP) is required in early embryos for cleavage furrow formation. In an effort to identify new targets of dFMRP-dependent regulation and new effectors of cleavage furrow formation, we used two-dimensional difference gel electrophoresis and mass spectrometry to identify proteins that are misexpressed in dfmr1 mutant embryos. Of the 28 proteins identified, we have identified three subunits of the Chaperonin containing TCP-1 (CCT) complex as new direct targets of dFMRP-dependent regulation. Furthermore, we found that the septin Peanut, a known effector of cleavage, is a likely conserved substrate of fly CCT and is mislocalized in both cct and in dfmr1 mutant embryos. Based on these results we propose that dFMRP-dependent regulation of CCT subunits is required for cleavage furrow formation and that at least one of its substrates is affected in dfmr1- embryos suggesting that dFMRP-dependent regulation of CCT contributes to the cleavage furrow formation phenotype.

End‐on imaging: A new perspective on dorsoventral development in Drosophila embryos

Drosophila ventral furrow formation has frequently been used as a model to study developmentally‐regulated cell‐shape changes. However, a technique to follow all cellular changes during this process within a single living embryo has been lacking. We describe a novel technique, called “end‐on imaging”, to collect time‐lapse images of transversely mounted living embryos. End‐on imaging revealed several new features of dorsoventral development. First, we observed a wave of syncytial nuclear divisions predicting the location of the ventral furrow. Second, we determined that there is a 5‐min gap between the end of cellularization and the start of ventral furrow formation, suggesting that the two processes may share the same pool of cytoskeletal components. Lastly, we show that apical‐membrane flattening, the first step in ventral furrow formation, is due to the ventral cells pushing against the vitelline membrane, rather than flattening the dome‐shaped, apical surfaces of these cells by a pulling or constriction motion. Developmental Dynamics 237:3252–3259, 2008.