Deciphering animal development through proteomics: requirements and prospects

In recent years proteomic techniques have started to become very useful tools in a variety of model systems of developmental biology. Applications cover many different aspects of development, including the characterization of changes in the proteome during early embryonic stages. During early animal development the embryo becomes patterned through the temporally and spatially controlled activation of distinct sets of genes. Patterning information is then translated, from gastrulation onwards, into regional specific morphogenetic cell and tissue movements that give the embryo its characteristic shape. On the molecular level, patterning is the outcome of intercellular communication via signaling molecules and the local activation or repression of transcription factors. Genetic approaches have been used very successfully to elucidate the processes behind these events. Morphogenetic movements, on the other hand, have to be orchestrated through regional changes in the mechanical properties of cells. The molecular mechanisms that govern these changes have remained much more elusive, at least in part due to the fact that they are more under translational/posttranslational control than patterning events. However, recent studies indicate that proteomic approaches can provide the means to finally unravel the mechanisms that link patterning to the generation of embryonic form. To intensify research in this direction will require close collaboration between proteome scientists and developmental researchers. It is with this aim in mind that we first give an outline of the classical questions of patterning and morphogenesis. We then summarize the proteomic approaches that have been applied in developmental model systems and describe the pioneering studies that have been done to study morphogenesis. Finally we discuss current and future strategies that will allow characterizing the changes in the embryonic proteome and ultimately lead to a deeper understanding of the cellular mechanisms that govern the generation of embryonic form

Contraction and polymerization cooperate to assemble and close actomyosin rings around Xenopus oocyte wounds

Xenopus oocytes assemble an array of F-actin and myosin 2 around plasma membrane wounds. We analyzed this process in living oocytes using confocal time-lapse (four-dimensional) microscopy. Closure of wounds requires assembly and contraction of a classic “contractile ring” composed of F-actin and myosin 2. However, this ring works in concert with a 5–10-μm wide “zone” of localized actin and myosin 2 assembly. The zone forms before the ring and can be uncoupled from the ring by inhibition of cortical flow and contractility. However, contractility and the contractile ring are required for the stability and forward movement of the zone, as revealed by changes in zone dynamics after disruption of contractility and flow, or experimentally induced breakage of the contractile ring. We conclude that wound-induced contractile arrays are provided with their characteristic flexibility, speed, and strength by the combined input of two distinct components: a highly dynamic zone in which myosin 2 and actin preferentially assemble, and a stable contractile actomyosin ring

How Plasma Membrane and Cytoskeletal Dynamics Influence Single-Cell Wound Healing: Mechanotransduction, Tension and Tensegrity

Organisms are able to recover from injuries by replacing damaged tissues, which recover by replacing damaged cells and extracellular structures. Similarly, a cell recovers from injuries by replacing damaged components of its structural integrity: its plasma membrane and cytoskeletal structures. Cells can be thought of as tensegral structures, their structural integrity relying on the interplay between tensile forces generated within and without the cell, and the compressive elements that counteracts them. As such, direct or indirect insults to the plasma membrane or cytoskeleton of a cell may not only result in the temporary loss of structural integrity, but also directly impact its ability to respond to its environment. This chapter will focus on the various aspects linking tensile forces and single-cell wound healing: where and how are they generated, how does the cell counteract them and how does the cell return to its previous tensegrity state? These questions will be explored using ubiquitous and cell-type specific examples of single-cell repair processes. Special attention will be given to changes in plasma membrane composition and area to cytoskeletal dynamics, and how these factor each other to influence and effect single-cell repair

Untangling the Roles of Anti-Apoptosis in Regulating Programmed Cell Death using Humanized Yeast Cells

Genetically programmed cell death (PCD) mechanisms, including apoptosis, are important for the survival of metazoans since it allows, among things, the removal of damaged cells that interfere with normal function. Cell death due to PCD is observed in normal processes such as aging and in a number of pathophysiologies including hypoxia (common causes of heart attacks and strokes) and subsequent tissue reperfusion. Conversely, the loss of normal apoptotic responses is associated with the development of tumors. So far, limited success in preventing unwanted PCD has been reported with current therapeutic approaches despite the fact that inhibitors of key apoptotic inducers such as caspases have been developed. Alternative approaches have focused on mimicking anti-apoptotic processes observed in cells displaying increased resistance to apoptotic stimuli. Hormesis and pre-conditioning are commonly observed cellular strategies where sub-lethal levels of pro-apoptotic stimuli lead to increased resistance to higher or lethal levels of stress. Increased expression of anti-apoptotic sequences is a common mechanism mediating these protective effects. The relevance of the latter observation is exemplified by the observation that transgenic mice overexpressing anti-apoptotic genes show significant reductions in tissue damage following ischemia. Thus strategies aimed at increasing the levels of anti-apoptotic proteins, using gene therapy or cell penetrating recombinant proteins are being evaluated as novel therapeutics to decrease cell death following acute periods of cell death inducing stress. In spite of its functional and therapeutic importance, more is known regarding the processes involved in apoptosis than anti-apoptosis. The genetically tractable yeast Saccharomyces cerevisiae has emerged as an exceptional model to study multiple aspects of PCD including the mitochondrial mediated apoptosis observed in metazoans. To increase our knowledge of the process of anti-apoptosis, we screened a human heart cDNA expression library in yeast cells undergoing PCD due to the conditional expression of a mammalian pro-apoptotic Bax cDNA. Analysis of the multiple Bax suppressors identified revealed several previously known as well as a large number of clones representing potential novel anti-apoptotic sequences. The focus of this review is to report on recent achievements in the use of humanized yeast in genetic screens to identify novel stress-induced PCD suppressors, supporting the use of yeast as a unicellular model organism to elucidate anti-apoptotic and cell survival mechanisms

Genetic interaction network of the Saccharomyces cerevisiae type 1 phosphatase Glc7

<p>Abstract</p> <p>Background</p> <p>Protein kinases and phosphatases regulate protein phosphorylation, a critical means of modulating protein function, stability and localization. The identification of functional networks for protein phosphatases has been slow due to their redundant nature and the lack of large-scale analyses. We hypothesized that a genome-scale analysis of genetic interactions using the Synthetic Genetic Array could reveal protein phosphatase functional networks. We apply this approach to the conserved type 1 protein phosphatase Glc7, which regulates numerous cellular processes in budding yeast.</p> <p>Results</p> <p>We created a novel <it>glc7 </it>catalytic mutant (<it>glc7-E101Q</it>). Phenotypic analysis indicates that this novel allele exhibits slow growth and defects in glucose metabolism but normal cell cycle progression and chromosome segregation. This suggests that <it>glc7-E101Q </it>is a hypomorphic <it>glc7 </it>mutant. Synthetic Genetic Array analysis of <it>glc7-E101Q </it>revealed a broad network of 245 synthetic sick/lethal interactions reflecting that many processes are required when Glc7 function is compromised such as histone modification, chromosome segregation and cytokinesis, nutrient sensing and DNA damage. In addition, mitochondrial activity and inheritance and lipid metabolism were identified as new processes involved in buffering Glc7 function. An interaction network among 95 genes genetically interacting with <it>GLC7 </it>was constructed by integration of genetic and physical interaction data. The obtained network has a modular architecture, and the interconnection among the modules reflects the cooperation of the processes buffering Glc7 function.</p> <p>Conclusion</p> <p>We found 245 genes required for the normal growth of the <it>glc7-E101Q </it>mutant. Functional grouping of these genes and analysis of their physical and genetic interaction patterns bring new information on Glc7-regulated processes.</p

Mitochondria Localize to the Cleavage Furrow in Mammalian Cytokinesis

<div><p>Mitochondria are dynamic organelles with multiple cellular functions, including ATP production, calcium buffering, and lipid biosynthesis. Several studies have shown that mitochondrial positioning is regulated by the cytoskeleton during cell division in several eukaryotic systems. However, the distribution of mitochondria during mammalian cytokinesis and whether the distribution is regulated by the cytoskeleton has not been examined. Using live spinning disk confocal microscopy and quantitative analysis of mitochondrial fluorescence intensity, we demonstrate that mitochondria are recruited to the cleavage furrow during cytokinesis in HeLa cells. After anaphase onset, the mitochondria are recruited towards the site of cleavage furrow formation, where they remain enriched as the furrow ingresses and until cytokinesis completion. Furthermore, we show that recruitment of mitochondria to the furrow occurs in multiple mammalian cells lines as well as in monopolar, bipolar, and multipolar divisions, suggesting that the mechanism of recruitment is conserved and robust. Using inhibitors of cytoskeleton dynamics, we show that the microtubule cytoskeleton, but not actin, is required to transport mitochondria to the cleavage furrow. Thus, mitochondria are specifically recruited to the cleavage furrow in a microtubule-dependent manner during mammalian cytokinesis. Two possible reasons for this could be to localize mitochondrial function to the furrow to facilitate cytokinesis and / or ensure accurate mitochondrial inheritance.</p></div

Mitochondria localize to the cytokinetic cleavage furrow in dividing HeLa cells.

<p>(A) Spinning disk confocal time-lapse images of HeLa cells stained with 40 nM MitoTracker Red to visualize mitochondria. The time points are representative of five stages of division from metaphase to late-cytokinesis. Shown are the following: a single focal plane from the center of the confocal stack (upper row), a maximum projection of the full Z-stack (middle row) and a merge of the DIC image with the mitochondrial single focal plane (bottom row). The full time-lapse can be seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072886#pone.0072886.s005" target="_blank">Movie S1</a>. Note the enrichment of mitochondria in the region of the cleavage furrow and reduction at the cell poles as division progresses. Yellow arrowheads indicate the position of the cleavage furrow. Time is given in minutes after anaphase onset. Bar, 10 µm. (B) Quantification of the distribution of mitochondria from cell pole to equator at each representative stage of division. An overlay of all five stages is also shown (last panel). The normalized distance from cell pole to equator is displayed on the x-axis and the average mitochondrial fluorescence intensity is displayed on the y-axis. Data are represented as mean +/– SEM (25 cells, N = 100) and lines fitted by non-linear regression.</p

Mitochondrial recruitment to the cleavage furrow is dependent on microtubules.

<p>(A) Live-imaging of HeLa cells treated with DMSO (top row), Early Nocodazole (second row), Late Nocodazole (third row) or Taxol (bottom row) and stained with MitoTracker Red to visualize mitochondria. Five representative frames are shown from metaphase to late cytokinesis. Treatments were added to live cells either 2 (DMSO, Noco Early, Tax) or 4 minutes (Noco Late) after anaphase onset. Note the mislocalized mitochondria at the cell poles (blue arrows) in drug-treated cells compared with DMSO-treated control cells. Red asterisks indicate the first time point following drug-addition and yellow arrowheads indicate the position of the cleavage furrow. The full time-lapse can be seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072886#pone.0072886.s008" target="_blank">Movie S4</a>. Bar, 10 µm. (B) Quantification of the distribution of mitochondria from cell pole to equator at each stage of division in cells treated with DMSO (top left; 8 cells, N = 32), Early Nocodazole (top right; 8 cells, N = 32), Late Nocodazole (bottom left; 10 cells, N = 40) and Taxol (bottom right; 6 cells, N = 24). The normalized distance from cell pole to equator is displayed on the x-axis and the average mitochondrial fluorescence intensity is displayed on the y-axis. Data are represented as the mean and lines fitted by non-linear regression (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072886#pone.0072886.s004" target="_blank">Figure S4</a>). (C) Quantification of mitochondrial inheritance by daughter cells in control (Untreated and DMSO) and drug-treated (Noco Late and Taxol) cells. Data are represented by a box and whisker plot showing maximum, minimum, upper and lower quartiles and sample median. n.s  =  not significant.</p

GSK3 regulates mitotic chromosomal alignment through CRMP4

Background: Glycogen Synthase Kinase 3 (GSK3) has been implicated in regulating chromosomal alignment and mitoticprogression but the physiological substrates mediating these GSK3-dependent effects have not been identified. CollapsinResponse Mediator Protein 4 (CRMP4) is a cytosolic phosphoprotein known to regulate cytoskeletal dynamics and is aknown physiological substrate of GSK3. In this study, we investigate the role of CRMP4 during mitosis.Methodology and Principal Findings: Here we demonstrate that during mitosis CRMP4 phosphorylation is regulated in aGSK3-dependent manner. We show that CRMP4 localizes to spindle microtubules during mitosis and loss of CRMP4 disruptschromosomal alignment and mitotic progression. The effect of CRMP4 on chromosomal alignment is dependent onphosphorylation by GSK3 identifying CRMP4 as a critical GSK3 substrate during mitotic progression. We also providemechanistic data demonstrating that CRMP4 regulates spindle microtubules consistent with its known role in the regulationof the microtubule cytoskeleton.Conclusion and Significance: Our findings identify CRMP4 as a key physiological substrate of GSK3 in regulatingchromosomal alignment and mitotic progression through its effect on spindle microtubules