A systems biology analysis of feedback control in pheromone signalling of fission yeast

Cell signalling comprises the systems used by cells to detect changes in their environment and to transduce the information into appropriate adjustments enforced by regulatory proteins. Due to its central role in all life processes, the study of cell signalling is a major focus of current biomedical research. The fission yeast Schizosaccharomyces pombe (S. pombe) is a single-celled organism used as a model to simplify the study of eukaryotic cell signalling, as it shares many features of interest with human cells. In this thesis a systems biology approach was used to investigate the roles of feedback regulation to control the dynamics of pheromone signalling in S. pombe. To this end, a quantitative dynamical model was built describing the pheromone-induced activation of the master transcription factor Ste11, as well as the coupled positive and negative feedback loops that arise from Ste11 activity. To constrain the model, a collection of data sets were generated by performing absolute quantification measurements of pheromone-dependent changes in the concentration of the model species. Structural identifiability analyses were used to select the measured species, while confidence intervals of the estimated parameters were determined through profile likelihood estimation. Analysis of the resulting model revealed a role for the pheromone signalling feedback loops to aid in the discrimination of different pheromone input doses. Through their combined action, feedback control defines the concentration and time thresholds in Ste11 activity that must be satisfied for the cell to commit to a sexual development fate

Feedback activation of neurofibromin terminates growth factor-induced Ras activation.

BACKGROUND: Growth factors induce a characteristically short-lived Ras activation in cells emerging from quiescence. Extensive work has shown that transient as opposed to sustained Ras activation is critical for the induction of mitogenic programs. Mitogen-induced accumulation of active Ras-GTP results from increased nucleotide exchange driven by the nucleotide exchange factor Sos. In contrast, the mechanism accounting for signal termination and prompt restoration of basal Ras-GTP levels is unclear, but has been inferred to involve feedback inhibition of Sos. Remarkably, how GTP-hydrolase activating proteins (GAPs) participate in controlling the rise and fall of Ras-GTP levels is unknown. RESULTS: Monitoring nucleotide exchange of Ras in permeabilized cells we find, unexpectedly, that the decline of growth factor-induced Ras-GTP levels proceeds in the presence of unabated high nucleotide exchange, pointing to GAP activation as a major mechanism of signal termination. Experiments with non-hydrolysable GTP analogues and mathematical modeling confirmed and rationalized the presence of high GAP activity as Ras-GTP levels decline in a background of high nucleotide exchange. Using pharmacological and genetic approaches we document a raised activity of the neurofibromatosis type I tumor suppressor Ras-GAP neurofibromin and an involvement of Rsk1 and Rsk2 in the down-regulation of Ras-GTP levels. CONCLUSIONS: Our findings show that, in addition to feedback inhibition of Sos, feedback stimulation of the RasGAP neurofibromin enforces termination of the Ras signal in the context of growth-factor signaling. These findings ascribe a precise role to neurofibromin in growth factor-dependent control of Ras activity and illustrate how, by engaging Ras-GAP activity, mitogen-challenged cells play safe to ensure a timely termination of the Ras signal irrespectively of the reigning rate of nucleotide exchange.We acknowledge funding by the German research council (DFG), grant # RU 860/4-1 (AH), by the Federal Ministry of Education and Research (BMBF), Germany, FKZ: 01EO1002 (I.R., R.M.), by the BBSRC and through the BBSRC Midlands Interdisciplinary BioSciences Training Partnership (MAE-F) (GL - BB/G01227X/1 and BB/M00015X/1) and the National Council on Science and Technology of Mexico (CONACYT) (MAE-F).This is the final published version. It first appeared at http://biosignaling.biomedcentral.com/articles/10.1186/s12964-016-0128-z

The role of the RACK1 ortholog Cpc2p in modulating pheromone-induced cell cycle arrest in fission yeast

The detection and amplification of extracellular signals requires the involvement of multiple protein components. In mammalian cells the receptor of activated C kinase (RACK1) is an important scaffolding protein for signal transduction networks. Further, it also performs a critical function in regulating the cell cycle by modulating the G1/S transition. Many eukaryotic cells express RACK1 orthologs, with one example being Cpc2p in the fission yeast Schizosaccharomyces pombe. In contrast to RACK1, Cpc2p has been described to positively regulate, at the ribosomal level, cells entry into M phase. In addition, Cpc2p controls the stress response pathways through an interaction with Msa2p, and sexual development by modulating Ran1p/Pat1p. Here we describe investigations into the role, which Cpc2p performs in controlling the G protein-mediated mating response pathway. Despite structural similarity to Gβ-like subunits, Cpc2p appears not to function at the G protein level. However, upon pheromone stimulation, cells overexpressing Cpc2p display substantial cell morphology defects, disorientation of septum formation and a significantly protracted G1 arrest. Cpc2p has the potential to function at multiple positions within the pheromone response pathway. We provide a mechanistic interpretation of this novel data by linking Cpc2p function, during the mating response, with its previous described interactions with Ran1p/Pat1p. We suggest that overexpressing Cpc2p prolongs the stimulated state of pheromone-induced cells by increasing ste11 gene expression. These data indicate that Cpc2p regulates the pheromone-induced cell cycle arrest in fission yeast by delaying cells entry into S phase

Overexpression of cpc2 mediates its pheromone effects in a G1-dependent manner.

<p>(A) Cell morphology and size, at division (micrometers ± S.D.) for strains JY1520 (h⁻, cyr1⁻, sxa2>lacZ, rum1⁻), JY1637 (h⁻, cyr1⁻, sxa2>lacZ, rum1⁻+ oe-cpc2⁺), JY1714 (h⁻, sxa2⁻, rum1⁻) and JY1715 (h⁻, sxa2⁻, rum1⁻+oe-cpc2⁺) grown in minimal medium at 29°C and stained with calcofluor. Scale bars 10 µm. (B) Number of non-septated, septated and multiple septa containing cells for the strains JY448 (h⁻, sxa2⁻), JY1714 (h⁻, sxa2⁻, rum1⁻), JY1715 (h⁻, sxa2⁻, rum1⁻+oe-cpc2⁺), JY546 (h⁻, cyr1⁻, sxa2>lacZ), JY1520 (h⁻, cyr1⁻, sxa2>lacZ, rum1⁻ ) and JY1637 (h⁻, cyr1⁻, sxa2>lacZ, rum1⁻+oe-cpc2⁺) were determined from 400 individual cells. Values shown correspond to the percentage of the total population. Cells were stained with calcofluor white, to enable visualization of septum material. (C) Pheromone-dependent transcription for the strains JY546, JY1520, JY1637 and JY1710 (h⁻, cyr1⁻, sxa2>lacZ, rum1⁻, cpc2⁻) was determined using the sxa2>lacZ reporter. Cells were stimulated with pheromone for 16 h in minimal media and assayed for β-galactosidase production using ONPG. Activity is expressed as OD₄₂₀ units per 10⁶ cells. Values are means of triplicate determinations ± S.E.M. (D) The strains JY1520 and JY1637 were grown in minimal medium containing 10 µM of pheromone for the times indicated. Cells were harvested and fixed prior to staining with propidium iodide prior to analysis using flow cytometry (see methods). The proportion of cells exhibiting 1C or 2C DNA content was determined using FACSDiva v4.1 software for the assigned gates indicated by the blue and red shapes (E) The strains JY1520, JY1710 and JY1637 were grown to mid-exponential phase over 32 h in minimal media. Cells were then stained with calcofluor white to visualize septation (top panel) and imaged bright field microscopy (bottom panel) after 32 h exposure to pheromone. Scale bars 10 µm.</p

Cpc2p has profound morphological effects upon pheromone-stimulated cells.

<p>The strains (A) JY448 (h⁻, sxa2⁻) and JY546 (h⁻, cyr1⁻, sxa2>lacZ); (B) JY1712 (h⁻, sxa2⁻, cpc2⁻) and JY1628 (h⁻, cyr1⁻, sxa2>lacZ, cpc2⁻); (C) JY1711 (h⁻, sxa2⁻+oe-cpc2⁺), JY1578 (h⁻, cyr1⁻, sxa2>lacZ+oe-cpc2⁺), were grown to mid-exponential phase over 32 h in minimal media. Cells were then imaged using bright field microscopy on pads containing 10 µM of pheromone (see methods). Cells were also stained with calcofluor white (lower panels A-C) to visualize septation. Scale bars 10 µm. Prolonged exposure to pheromone for cells overexpressing Cpc2p (oe-cpc2⁺) results in multiple projection tips and a failure to undergo cytokinesis. Cells lacking Cpc2p fail to generate the classical shmoo formation as observed for control cells.</p

A physiologically required G protein-coupled receptor (GPCR)-regulator of G protein signaling (RGS) interaction that compartmentalizes RGS activity

G protein-coupled receptors (GPCRs) can interact with regulator of G protein signaling (RGS) proteins. However, the effects of such interactions on signal transduction and their physiological relevance have been largely undetermined. Ligand-bound GPCRs initiate by promoting exchange of GDP for GTP on the Gα subunit of heterotrimeric G proteins. Signaling is terminated by hydrolysis of GTP to GDP through intrinsic GTPase activity of the Gα subunit, a reaction catalyzed by RGS proteins. Using yeast as a tool to study GPCR signaling in isolation, we define an interaction between the cognate GPCR (Mam2) and RGS (Rgs1), mapping the interaction domains. This reaction tethers Rgs1 at the plasma membrane and is essential for physiological signaling response. In vivo quantitative data inform the development of a kinetic model of the GTPase cycle, which extends previous attempts by including GPCR-RGS interactions. In vivo and in silico data confirm that GPCR-RGS interactions can impose an additional layer of regulation through mediating RGS subcellular localization to compartmentalize RGS activity within a cell, thus highlighting their importance as potential targets to modulate GPCR signaling pathways

Cpc2p control the G1/S transition in pheromone-stimulated cells.

<p>(A) The strains JY546 (h⁻, cyr1⁻, sxa2>lacZ), JY1578 (h⁻, cyr1⁻, sxa2>lacZ, +oe-cpc2⁺) and JY1628 (h⁻, cyr1⁻, sxa2>lacZ, cpc2⁻) were grown in minimal medium and (B) minimal media containing 10 µM of pheromone for the times indicated. Cells were harvested and fixed prior to staining with propidium iodide prior to analysis using flow cytometry (see methods). The proportion of cells exhibiting 1C or 2C DNA content was determined using FACSDiva v4.1 software for the assigned gates indicated by the blue and red shapes. (C) The percentage of cells containing a 1C content (arrested in G₁) as determined from B. Cells containing an additional content of Cpc2 fail to desensitize following pheromone stimulation and remain arrested for the time frame analyzed. Cells lacking Cpc2p fail to generate a significant arrest in G₁ following exposure to 10 µM pheromone. (D) The percentage of cells from the strains JY448 (h⁻, sxa2⁻), JY1711 (h⁻, sxa2⁻+oe-cpc2⁺), JY1712 (h⁻, sxa2⁻, cpc2⁻) containing a 1C content (arrested in G₁) following nitrogen starvation and stimulation with 10 µM pheromone.</p

<i>S. pombe</i> strains used in this study.

<p><i>S. pombe</i> strains used in this study.</p

Overexpression of Cpc2 in pheromone stimulated cells mimics prolonged pheromone stimulation.

<p>(A) Cell morphology and size, at division (micrometers ± S.D.) for the strains JY1716 (h⁻, sxa2⁻, pmp1⁻) and JY948 (h⁻, cyr1⁻, sxa2>lacZ, pmp1⁻) grown in minimal medium at 29°C and stained with calcofluor white. (B) Strains from A strains were exposure to 10 µM of pheromone for 32 h and stained with calcofluor white. (C) The percentage of cells containing a 1C content (arrested in G₁) for the strains JY448, JY1716, JY546 and JY948 as determined using flow cytometry. Cells lacking Pmp1p show a failure to exit from a G₁ arrest analogous to strains where the cpc2 ORF has been deleted. (D) Cell morphology and size, at division (micrometers ± S.D.) for strains JY710 (h⁻, sxa2⁻ pyp2⁻) and JY1717 (h⁻, sxa2⁻, pyp2⁻+oe-cpc2⁺) grown in minimal medium at 29°C and stained with calcofluor white (top panel). Cell morphology and size at division (micrometers ± S.D.) for the strains JY709 (h⁻, cyr1⁻, sxa2>lacZ, pyp2⁻) and JY1661 (h⁻, cyr1⁻, sxa2>lacZ, pyp2⁻+oe-cpc2⁺) grown in minimal medium at 29°C and stained with calcofluor white (bottom panel). (E) Numbers of non-septated, septated and multiple septa containing cells for the strains JY448, JY1714, JY1715, JY546, JY987 and JY1661 were determined from 400 individual cells. Values shown correspond to the percentages of the total population. Cells were stained with calcofluor white, to enable visualization of septum material. (F) Pheromone-dependent transcription for the strains JY546, JY709, JY1661 and JY948 was determined using the sxa2>lacZ reporter. Cells were stimulated with pheromone for 16 h in minimal media and assayed for β-galactosidase production using ONPG. Activity is expressed as OD₄₂₀ units per 10⁶ cells. Values are means of triplicate determinations ± S.E.M. (G) The strains JY709 and JY1661 were grown in minimal medium containing 10 µM of pheromone for the times indicated. Cells were harvested and fixed prior to staining with propidium iodide prior to analysis using flow cytometry (see methods). The proportion of cells exhibiting 1C or 2C DNA content was determined using FACSDiva v4.1 software for the assigned gates indicated by the blue and red shapes. (H) The percentage of cells containing a 1C content (arrested in G₁) as determined for the strains JY448, JY546, JY710, JY1717, JY709 and JY1661.</p