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1.  Modification of Akt by SUMO conjugation regulates alternative splicing and cell cycle 
Cell Cycle  2013;12(19):3165-3174.
Akt/PKB is a key signaling molecule in higher eukaryotes and a crucial protein kinase in human health and disease. Phosphorylation, acetylation, and ubiquitylation have been reported as important regulatory post-translational modifications of this kinase. We describe here that Akt is modified by SUMO conjugation, and show that lysine residues 276 and 301 are the major SUMO attachment sites within this protein. We found that phosphorylation and SUMOylation of Akt appear as independent events. However, decreasing Akt SUMOylation levels severely affects the role of this kinase as a regulator of fibronectin and Bcl-x alternative splicing. Moreover, we observed that the Akt mutant (Akt E17K) found in several human tumors displays increased levels of SUMOylation and also an enhanced capacity to regulate fibronectin splicing patterns. This splicing regulatory activity is completely abolished by decreasing Akt E17K SUMO conjugation levels. Additionally, we found that SUMOylation controls Akt regulatory function at G₁/S transition during cell cycle progression. These findings reveal SUMO conjugation as a novel level of regulation for Akt activity, opening new areas of exploration related to the molecular mechanisms involved in the diverse cellular functions of this kinase.
doi:10.4161/cc.26183
PMCID: PMC3865012  PMID: 24013425
signal transduction; post-translational modification; SUMO; Akt/PKB; alternative splicing; cell cycle
2.  Messages Do Diffuse Faster than Messengers: Reconciling Disparate Estimates of the Morphogen Bicoid Diffusion Coefficient 
PLoS Computational Biology  2014;10(6):e1003629.
The gradient of Bicoid (Bcd) is key for the establishment of the anterior-posterior axis in Drosophila embryos. The gradient properties are compatible with the SDD model in which Bcd is synthesized at the anterior pole and then diffuses into the embryo and is degraded with a characteristic time. Within this model, the Bcd diffusion coefficient is critical to set the timescale of gradient formation. This coefficient has been measured using two optical techniques, Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Correlation Spectroscopy (FCS), obtaining estimates in which the FCS value is an order of magnitude larger than the FRAP one. This discrepancy raises the following questions: which estimate is "correct''; what is the reason for the disparity; and can the SDD model explain Bcd gradient formation within the experimentally observed times? In this paper, we use a simple biophysical model in which Bcd diffuses and interacts with binding sites to show that both the FRAP and the FCS estimates may be correct and compatible with the observed timescale of gradient formation. The discrepancy arises from the fact that FCS and FRAP report on different effective (concentration dependent) diffusion coefficients, one of which describes the spreading rate of the individual Bcd molecules (the messengers) and the other one that of their concentration (the message). The latter is the one that is more relevant for the gradient establishment and is compatible with its formation within the experimentally observed times.
Author Summary
Understanding the mechanisms by which equivalent cells develop into different body parts is a fundamental question in biology. One well-studied example is the patterning along the anterior-posterior axis of Drosophila melanogaster embryos for which the spatial gradient of the protein Bicoid is determinant. The localized production of Bicoid is implicated in its inhomogeneous distribution. Diffusion then determines the time and spatial scales of the gradient as it is formed. Estimates of Bicoid diffusion coefficients made with the optical techniques, FRAP and FCS resulted in largely different values, one of which was too slow to account for the observed time of gradient formation. In this paper, we present a model in which Bicoid diffuses and interacts with binding sites so that its transport is described by a "single molecule'' and a "collective'' diffusion coefficient. The latter can be arbitrarily larger than the former coefficient and sets the rate for bulk processes such as the formation of the gradient. In this way we obtain a self-consistent picture in which the FRAP and FCS estimates are accurate and where the gradient can be established within the experimentally observed times.
doi:10.1371/journal.pcbi.1003629
PMCID: PMC4046929  PMID: 24901638
3.  Pheromone-Induced Morphogenesis Improves Osmoadaptation Capacity by Activating the HOG MAPK Pathway** 
Science signaling  2013;6(272):ra26.
Environmental and internal conditions expose cells to a multiplicity of stimuli whose consequences are difficult to predict. Here, we investigate the response to mating pheromone of yeast cells adapted to high osmolarity. Events downstream of pheromone binding involve two mitogen-activated protein kinase (MAPK) cascades: the pheromone response (PR) and the cell-wall integrity response (CWI). Although these MAPK pathways share components with each and a third MAPK pathway, the high osmolarity response (HOG), they are normally only activated by distinct stimuli, a phenomenon called insulation. We found that in cells adapted to high osmolarity, PR activated the HOG pathway in a pheromone- and osmolarity- dependent manner. Activation of HOG by the PR was not due to loss of insulation, but rather a response to a reduction in internal osmolarity, which resulted from an increase in glycerol release caused by the PR. By analyzing single-cell time courses, we found that stimulation of HOG occurred in discrete bursts that coincided with the “shmooing” morphogenetic process. Activation required the polarisome, the cell wall integrity MAPK Slt2, and the aquaglyceroporin Fps1. HOG activation resulted in high glycerol turnover that improved adaptability to rapid changes in osmolarity. Our work shows how a differentiation signal can recruit a second, unrelated sensory pathway to enable responses to yeast to multiple stimuli.
doi:10.1126/scisignal.2003312
PMCID: PMC3701258  PMID: 23612707
4.  Using Cell-ID 1.4 with R for Microscope-Based Cytometry 
This unit describes a method for quantifying various cellular features (e.g., volume, total and subcellular fluorescence localization) from sets of microscope images of individual cells. It includes procedures for tracking cells over time. One purposefully defocused transmission image (sometimes referred to as bright-field or BF) is acquired to segment the image and locate each cell. Fluorescent images (one for each of the color channels to be analyzed) are then acquired by conventional wide-field epifluorescence or confocal microscopy. This method uses the image processing capabilities of Cell-ID (Gordon et al., 2007, as updated here) and data analysis by the statistical programming framework R (R-Development-Team, 2008), which we have supplemented with a package of routines for analyzing Cell-ID output. Both Cell-ID and the analysis package are open-source.
doi:10.1002/0471142727.mb1418s100
PMCID: PMC3485637  PMID: 23026908
image processing; fluorescence microscopy; Cell-ID; R
5.  Modulation of the Akt Pathway Reveals a Novel Link with PERK/eIF2α, which Is Relevant during Hypoxia 
PLoS ONE  2013;8(7):e69668.
The unfolded protein response (UPR) and the Akt signaling pathway share several regulatory functions and have the capacity to determine cell outcome under specific conditions. However, both pathways have largely been studied independently. Here, we asked whether the Akt pathway regulates the UPR. To this end, we used a series of chemical compounds that modulate PI3K/Akt pathway and monitored the activity of the three UPR branches: PERK, IRE1 and ATF6. The antiproliferative and antiviral drug Akt-IV strongly and persistently activated all three branches of the UPR. We present evidence that activation of PERK/eIF2α requires Akt and that PERK is a direct Akt target. Chemical activation of this novel Akt/PERK pathway by Akt-IV leads to cell death, which was largely dependent on the presence of PERK and IRE1. Finally, we show that hypoxia-induced activation of eIF2α requires Akt, providing a physiologically relevant condition for the interaction between Akt and the PERK branch of the UPR. These data suggest the UPR and the Akt pathway signal to one another as a means of controlling cell fate.
doi:10.1371/journal.pone.0069668
PMCID: PMC3726764  PMID: 23922774
6.  Modelling reveals novel roles of two parallel signalling pathways and homeostatic feedbacks in yeast 
Ensemble modelling is used to study the yeast high osmolarity glycerol (HOG) pathway, a prototype for eukaryotic mitogen-activated kinase signalling systems. The best fit model provides new insights into the function of this system, some of which are then experimentally validated.
The main mechanism for osmo-adaptation is a fast and transient non-transcriptional Hog1-mediated activation of glycerol production.The transcriptional response rather serves to maintain an increased steady-state glycerol production with low steady-state Hog1 activity after adaptation.A fast negative feedback of activated Hog1 on the upstream signalling branches serves to stabilise the adaptation response by preventing oscillatory behaviour.Two parallel redundant signalling branches elicit a more robust and swifter adaptation than a single branch alone, at least for low osmotic shock. This notion could be corroborated by dedicated measurements of single-cell volume recovery for the wild-type and single-branch mutants.
The high osmolarity glycerol (HOG) pathway in yeast serves as a prototype signalling system for eukaryotes. We used an unprecedented amount of data to parameterise 192 models capturing different hypotheses about molecular mechanisms underlying osmo-adaptation and selected a best approximating model. This model implied novel mechanisms regulating osmo-adaptation in yeast. The model suggested that (i) the main mechanism for osmo-adaptation is a fast and transient non-transcriptional Hog1-mediated activation of glycerol production, (ii) the transcriptional response serves to maintain an increased steady-state glycerol production with low steady-state Hog1 activity, and (iii) fast negative feedbacks of activated Hog1 on upstream signalling branches serves to stabilise adaptation response. The best approximating model also indicated that homoeostatic adaptive systems with two parallel redundant signalling branches show a more robust and faster response than single-branch systems. We corroborated this notion to a large extent by dedicated measurements of volume recovery in single cells. Our study also demonstrates that systematically testing a model ensemble against data has the potential to achieve a better and unbiased understanding of molecular mechanisms.
doi:10.1038/msb.2012.53
PMCID: PMC3531907  PMID: 23149687
adaptation; ensemble modeling; Hopf bifurcation; model discrimination; osmotic stress
7.  Phosphoproteomic Analysis Reveals Interconnected System-Wide Responses to Perturbations of Kinases and Phosphatases in Yeast 
Science signaling  2010;3(153):rs4.
The phosphorylation and dephosphorylation of proteins by kinases and phosphatases constitute an essential regulatory network in eukaryotic cells. This network supports the flow of information from sensors through signaling systems to effector molecules, and ultimately drives the phenotype and function of cells, tissues, and organisms. Dysregulation of this process has severe consequences and is one of the main factors in the emergence and progression of diseases, including cancer. Thus, major efforts have been invested in developing specific inhibitors that modulate the activity of individual kinases or phosphatases; however, it has been difficult to assess how such pharmacological interventions would affect the cellular signaling network as a whole. Here, we used label-free, quantitative phosphoproteomics in a systematically perturbed model organism (Saccharomyces cerevisiae) to determine the relationships between 97 kinases, 27 phosphatases, and more than 1000 phosphoproteins. We identified 8814 regulated phosphorylation events, describing the first system-wide protein phosphorylation network in vivo. Our results show that, at steady state, inactivation of most kinases and phosphatases affected large parts of the phosphorylation-modulated signal transduction machinery, and not only the immediate downstream targets. The observed cellular growth phenotype was often well maintained despite the perturbations, arguing for considerable robustness in the system. Our results serve to constrain future models of cellular signaling and reinforce the idea that simple linear representations of signaling pathways might be insufficient for drug development and for describing organismal homeostasis.
doi:10.1126/scisignal.2001182
PMCID: PMC3072779  PMID: 21177495
8.  The Alpha Project, a model system for systems biology research 
IET systems biology  2008;2(5):222-233.
One goal of systems biology is to understand how genome-encoded parts interact to produce quantitative phenotypes. The Alpha Project is a medium-scale, interdisciplinary systems biology effort that aims to achieve this goal by understanding fundamental quantitative behaviors of a prototypic signal transduction pathway, the yeast pheromone response system from Saccharomyces cerevisiae. The Alpha Project distinguishes itself from many other systems biology projects by studying a tightly-bounded and well-characterized system that is easily modified by genetic means, and by focusing on deep understanding of a discrete number of important and accessible quantitative behaviors. During the project, we have developed tools to measure the appropriate data and develop models at appropriate levels of detail for studying a number of these quantitative behaviors. We also have developed transportable experimental tools and conceptual frameworks for understanding other signaling systems. In particular, we have begun to interpret system behaviors and their underlying molecular mechanisms through the lens of information transmission, a principal function of signaling systems. The Alpha Project demonstrates that interdisciplinary studies that identify key quantitative behaviors and measure important quantities, in the context of well-articulated abstractions of system function and appropriate analytical frameworks, can lead to deeper biological understanding. Our experience may provide a productive template for system biology investigations of other cellular systems.
doi:10.1049/iet-syb:20080127
PMCID: PMC2806158  PMID: 19045818
9.  Cell-ID Software for Microscope-Based Cytometry 
This unit describes a method to quantify, from sets of microscope images, various cellular parameters from individual cells, and includes procedures to track cells over time. For example, the user can measure cell volume, total and subcellular localization (nuclear, plasma membrane) of fluorescence for multiple fluorescence channels. This method uses the image processing capabilities of Cell-ID (Gordon et al., 2007) and data analysis by the statistical programming framework R, both open source software packages. The first step for successful cytometry entails acquiring at least one set of images for each field of cells. Each set is composed of one purposefully defocused transmission image (sometimes referred to as brightfield, or BF) that will be used to locate each cell, and one fluorescence image for each of the color channels to be analyzed. Images may be conventional wide-field epifluorescence or confocal microscopy images. Cell-ID processes the images and outputs a tab-delimited file with information extracted from each cell, for each time point and each fluorescence channel. Finally, the user analyzes the data using R (R-Development-Team, 2008), which we have supplemented with a package tailored to analyze Cell-ID output.
doi:10.1002/0471142727.mb1418s84
PMCID: PMC2784696  PMID: 18972382
image processing; fluorescence microscopy; Cell-ID; R
10.  Fus3 generates negative feedback that improves information transmission in yeast pheromone response 
Nature  2008;456(7223):755-761.
Haploid Saccharomyces cerevisiae yeast cells use a prototypic cell signaling system to transmit information about the extracellular concentration of mating pheromone secreted by potential mating partners. The ability for cells to respond distinguishably to different pheromone concentrations depends on how much information about pheromone concentration the system can transmit. Here we show that the MAPK Fus3 mediates fast-acting negative feedback that adjusts the dose-response of downstream system response to match that of receptor-ligand binding. This “dose-response alignment”, defined by a linear relationship between receptor occupancy and downstream response, can improve the fidelity of information transmission by making downstream responses corresponding to different receptor occupancies more distinguishable and reducing amplification of stochastic noise during signal transmission. We also show that one target of the feedback is a novel signal-promoting function of the RGS protein Sst2. Our work suggests that negative feedback is a general mechanism used in signaling systems to align dose-responses and thereby increase the fidelity of information transmission.
doi:10.1038/nature07513
PMCID: PMC2716709  PMID: 19079053

Results 1-10 (10)