We describe a modification of two-dimensional (2-D) polyacrylamide gel electrophoresis that requires only a single gel to reproducibly detect differences between two protein samples. This was accomplished by fluorescently tagging the two samples with two different dyes, running them on the same 2-D gel, post-run fluorescence imaging of the gel into two images, and superimposing the images. The amine reactive dyes were designed to insure that proteins common to both samples have the same relative mobility regardless of the dye used to tag them. Thus, this technique, called difference gel electrophoresis (DIGE), circumvents the need to compare several 2-D gels. DIGE is reproducible, sensitive, and can detect an exogenous difference between two Drosophila embryo extracts at nanogram levels. Moreover, an inducible protein from E. coli was detected after 15 min of induction and identified using DIGE preparatively.
Two-dimensional difference gel electrophoresis (2D DIGE) is a modified form of 2D electrophoresis (2DE) that allows one to compare two or three protein samples simultaneously on the same gel. The proteins in each sample are covalently tagged with different color fluorescent dyes that are designed to have no effect on the relative migration of proteins during electrophoresis. Proteins that are common to the samples appear as 'spots' with a fixed ratio of fluorescent signals, whereas proteins that differ between the samples have different fluorescence ratios. With the appropriate imaging system, DIGE is capable of reliably detecting as little as 0.5 fmol of protein, and protein differences down to +/- 15%, over a >10,000-fold protein concentration range. DIGE combined with digital image analysis therefore greatly improves the statistical assessment of proteome variation. Here we describe a protocol for conducting DIGE experiments, which takes 2-3 d to complete.
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 x 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.
Mutant huntingtin (mHTT), the causative protein in Huntington’s disease (HD), associates with the translocase of mitochondrial inner membrane 23 (TIM23) complex, resulting in inhibition of synaptic mitochondrial protein import first detected in presymptomatic HD mice. The early timing of this event suggests that it is a relevant and direct pathophysiologic consequence of mHTT expression. We show that, of the 4 TIM23 complex proteins, mHTT specifically binds to the TIM23 subunit and that full-length wild-type huntingtin (wtHTT) and mHTT reside in the mitochondrial intermembrane space. We investigated differences in mitochondrial proteome between wtHTT and mHTT cells and found numerous proteomic disparities between mHTT and wtHTT mitochondria. We validated these data by quantitative immunoblotting in striatal cell lines and human HD brain tissue. The level of soluble matrix mitochondrial proteins imported through the TIM23 complex is lower in mHTT-expressing cell lines and brain tissues of HD patients compared with controls. In mHTT-expressing cell lines, membrane-bound TIM23-imported proteins have lower intramitochondrial levels, whereas inner membrane multispan proteins that are imported via the TIM22 pathway and proteins integrated into the outer membrane generally remain unchanged. In summary, we show that, in mitochondria, huntingtin is located in the intermembrane space, that mHTT binds with high-affinity to TIM23, and that mitochondria from mHTT-expressing cells and brain tissues of HD patients have reduced levels of nuclearly encoded proteins imported through TIM23. These data demonstrate the mechanism and biological significance of mHTT-mediated inhibition of mitochondrial protein import, a mechanism likely broadly relevant to other neurodegenerative diseases.
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.
MicroRNAs (miRNAs) are a class of small RNAs that silence gene expression. In animal cells, miRNAs bind to the 3 untranslated regions of specific mRNAs and inhibit their translation. Although some targets of a handful of miRNAs are known, the number and identities of mRNA targets in the genome are uncertain, as are the developmental functions of miRNA regulation. To identify the global range of miRNA-regulated genes during oocyte maturation of Drosophila, we compared the proteome from wild-type oocytes with the proteome from oocytes lacking the dicer-1 gene, which is essential for biogenesis of miRNAs. Most identified proteins appeared to be subject to translation inhibition. Their transcripts contained putative binding sites in the 3 untranslated region for a subset of miRNAs, based on computer modeling. The fraction of genes subject to direct and indirect repression by miRNAs during oocyte maturation appears to be small (4%), and the genes tend to share a common functional relationship in protein biogenesis and turnover. The preponderance of genes that control global protein abundance suggests this process is under tight control by miRNAs at the onset of fertilization.translation control S mall RNAs, including microRNAs (miRNAs) and short interfering RNAs, are components of a RNA-based mechanism of gene silencing (1, 2). The miRNA branch of RNA-based gene regulation is found in plants and animals (3, 4). miRNAs have a specific size of Ϸ22 nt, and they are processed from hairpin-loop RNA precursors by endoribonucleases of the Dicer class. These precursors are transcribed from genes within plant and animal genomes, with the number of miRNA genes found in different species roughly corresponding to 0.5-1% of the total number of genes in their genomes (5). Most miRNA genes are conserved between related species, and Ϸ30% of miRNA genes are highly conserved, with orthologs found in vertebrate and invertebrate genomes. This suggests that a significant fraction of miRNAs have evolutionarily conserved biological functions.miRNAs specifically repress gene expression by negatively regulating complementary mRNAs. Plant miRNAs generally cause degradation of complementary mRNAs by near-perfect basepairing (5). Conversely, most characterized miRNAs from animals repress gene expression by blocking the translation of complementary mRNAs into protein. They interact with their targets by imperfectly basepairing to mRNA sequences within the 3Ј UTR. The exact mechanism of translation inhibition is unknown, although miRNAs have been found not to interfere with translation initiation (5).A question of great interest concerns the functions of miRNAs in animals. Although the functions of only a few miRNAs are known, available evidence suggests that miRNAs play diverse and important roles in development. This conclusion is based on the mutant phenotypes of individual miRNA genes and on the identification of genes that are direct targets of miRNA regulation. In Caenorhabditis elegans, the genes lin-14, lin-28, lin-41, and hbl-1 are trans...
Abstract. One of the first signs of cell differentiation in the Drosophila melanogaster embryo occurs 3 h after fertilization, when discrete groups of cells enter their fourteenth mitosis in a spatially and temporally patterned manner creating mitotic domains (Foe, V. E., and G. M. Odell. 1989. Am. Zool. 29:617-652). To determine whether cell residency in a mitotic domain is determined solely by cell position in this early embryo, or whether cell lineage also has a role, we have developed a technique for directly analyzing the behavior of nuclei in living emb~os. By microinjecting fluorescently labeled histones into the syncytial embryo, the movements and divisions of each nucleus were recorded without perturbing development by using a microscope equipped with a high resolution, charge-coupled device. Two types of developmental maps were generated from three-dimensional time-lapse recordings: one traced the lineage history of each nucleus from nuclear cycle 11 through nuclear cycle 14 in a small region of the embryo; the other recorded nuclear fate according to the timing and pattern of the 14th nuclear division. By comparing these lineage and fate maps for two embryos, we conclude that, at least for the examined area, the pattern of mitotic domain formation in Drosophila is determined by the position of each cell, with no effect of cell lineage.
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 stressinduced 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. Cell Death and Differentiation (2008) 15, 589-599; doi:10.1038/sj.cdd.4402296; published online 11 January 2008 The endoplasmic reticulum (ER) serves as the primary processing site for membrane and secreted proteins. The ER recruits translating ribosomes, translocates newly synthesized polypeptides into its lumen, and promotes a variety of post-translational modifications and chaperone-facilitated protein folding. 1,2 Proper ER function is critical for numerous aspects of cell physiology, including vesicle trafficking, lipid and membrane biogenesis, and protein targeting and secretion. Accordingly, cells react rapidly to various forms of ER dysfunction -including the accumulation of unfolded, misfolded or excessive protein, ER lipid or glycolipid imbalances, or changes in the redox or ionic conditions of the ER lumenthrough a set of adaptive pathways known collectively as the ER stress response (ESR). [2][3][4][5] The ESR promotes cell survival both by increasing the capacity of the ER to fold and process client proteins and by reducing the amount of protein inside the ER. These effects are achieved through three major pathways: (1) the unfolded protein response, a transcription-dependent induction of ER lumenal chaperone proteins and many other components of the secretory apparatus, which augments the polypeptide processing capacity of the ER; 5,6 (2) the activation of proteasome-dependent ER-associated degradation to remove proteins from the ER; 7,8 and (3) the control of protein translation to modulate the polypeptide traffic into the ER. 9,10 Normally, this suite of responses succeeds in restoring ER homeostasis. However, in metazoans, persistent or intense ER stress can also trigger apoptosis. [11][12][13][14] ER stress and the apoptotic program coupled to it have been implicated in many important pathologies, including diabetes, obesity, neurodegenerativ...
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