1,721,068 research outputs found
CryoEM architecture of a near-native stretch-sensitive membrane microdomain.
No full textLipid diffusion, lipid sorting and yeast cell biology dat
Manuscript_DBPR-NSMB-A45852 RAW DATA
No full textRaw data of all Figures and Extended Figures of the manuscript DBPR-NSMB-A45852
Chemical-Biology-derived in vivo Sensors: Past, Present, and Future.
Full text linkTo understand the complex biochemistry and biophysics of biological systems, one needs to be able to monitor local concentrations of molecules, physical properties of macromolecular assemblies and activation status of signaling pathways, in real time, within single cells, and at high spatio-temporal resolution. Here we look at the tools that have been / are being / need to be provided by chemical biology to address these challenges. In particular, we highlight the utility of molecular probes that help to better measure mechanical forces and flux through key signalling pathways. Chemical biology can be used to both build biosensors to visualize, but also actuators to perturb biological processes. An emergent theme is the possibility to multiplex measurements of multiple cellular processes. Advances in microscopy automation now allow us to acquire datasets for 1000's of cells. This produces high dimensional datasets that require computer vision approaches that automate image analysis. The high dimensionality of these datasets are often not immediately accessible to human intuition, and, similarly to 'omics technologies, require statistical approaches for their exploitation. The field of biosensor imaging is therefore experiencing a multidisciplinary transition that will enable it to realize its full potential as a tool to provide a deeper appreciation of cell physiology
Structural and functional characterization of rapamycin-resistant TORC2
No full textTORC2 is nucleated by Target of Rapamycin (TOR), a protein kinase of the phosphatidylinositol 3-kinase-related kinase (PIKK) family. TOR kinases are validated drug targets which can be inhibited by rapamycin, facilitating elucidation of TORC1 signaling. TORC2 cannot be inhibited by rapamycin, and the lack of specific inhibitors has impeded progress in understanding the functions of this essential complex. The EM reconstruction revealed a rhomboid shape with C2 pseudo-symmetry and a prominent central cavity. We found that the TORC2-specific subunit Avo3 is proximal to the rapamycin binding domain of Tor2. Building on this observation, we successfully engineered a yeast strain in which TORC2, but not TORC1, is inhibited by rapamycin. We leveraged this unique tool to study TORC2 function and regulation, demonstrating that acute TORC2 inhibition abolishes actin polarization and leads to cell-cycle arrest
Control of ribosome biogenesis by the TORC1-Sch9 pathway in Saccharomyces cerevisiae
No full textYeast TORC1 is a evolutionarily conserved multiprotein complex whose kinase activity is ensured by the TOR catalytic subunit. In response to environmental cues, TORC1 directly phosphorylates and activates the AGC kinase Sch9 to regulate anabolic processes such as translation. We show here that Sch9 mediates TORC1 signals to all three nuclear RNA polymerases (RNAP I-III) via transcriptional repressors. Maf1 is directly phosphorylated an inhibited by Sch9 to prevent it from repressing RNAP III. Stb3, Dot6 and Tod6 are also directly phosphorylated by Sch9 to keep them from recruiting RPD3L upstream of ribosome biogenesis and ribosomal protein genes and thereby from repressing their transcription by RNAP II. In turn, expression of those genes serves to promote RNAP I activity
Chemical‐genetic approach to identify new TOR effectors
No full textThe TOR kinases are central regulators of cell growth and extremely well conserved throughout the eukaryotic kingdom. The model organism Saccharomyces cerevisiae possesses two paralogous genes encoding TOR kinases: TOR1 and TOR2. They form the catalytic core of two distinct multi-protein complexes TORC1 and TORC2. The projects of this thesis pursued two goals: identification of new TOR-inhibitors and characterization of phosphorylation events downstream of TORC1 and TORC2. A novel label-free mass-spectrometry approach was applied to quantify changes in abundance of phosphopeptides after TORC1 inhibition by rapamycin. More than 100 TORC1-dependent phosphorylation events were identified. To get a better understanding of TORC2-signalling, a high-throughput chemical screen for new small molecule TOR-inhibitors was performed. Finally, a compound that putatively targeted TORC1 and TORC2 was characterized: BHS345. This molecule was confirmed to be a potent and yeast-permeable TOR-inhibitor in vivo and in vitro
Identification of TORC1 and TORC2 upstream regulators
No full textAll eukaryote cells, and especially singled celled species, monitor their environment and use this information to appropriately adjust their intracellular physiology. In Saccharomyces cerevisiae AGC kinase family members appear to be a major link between environmental cues and the cell growth machinery. However, how environmental cues impinge upon Pkh1/2, TORC1 and TORC2 remains a mystery. We found that TORC1, TORC2 and Pkh1/2 are positively regulated by nutrient quantity and quality. Moreover, in a complementary, hypothesis-based approach we found that regulation of TORC2 downstream of plasma membrane stress is mediated by the Slm1/2 proteins
Small molecules as tool compounds to probe Target of Rapamycin (TOR) signaling
No full textThe development of tool compounds to interrogate TOR signaling greatly facilitates our understanding of the complex and multilayered pathways governing TOR. In this study, we present two new molecules that can be used to decipher both upstream and direct TOR regulation. We have shown that the use of palmitoylcarnitine can guide insights into new models for TORC2 inhibition, an area that is fundamentally challenging to interrogate with the current TOR toolbox repetoire. CMB5806 shows great promise as a novel class of TOR inhibitors and its use as an activity based probe will allow us to determine the active state of the TOR kinase in variable conditions. In conclusion, two valuable tool compounds are developed in this study and will assist future understanding of TOR signaling
Unravelling the target specificity of the major AGC kinases in Saccharomyces cerevisiae
No full textTo achieve a coordinated growth yeast cells have intimately linked signaling pathways that sense and transduce nutrient and stress signals to adjust different growth-determining processes in the cell. Of central importance to these signaling pathways are AGC family kinases, which play essential roles in many signaling pathways. Out of twenty AGC kinases in Saccharomyces cerevisiae, six are of particular importance since they are integrated in the major growth controlling network. Sch9, Ypk1, Ypk2 and PKA AGC kinases contribute to the control of cell growth, they must communicate with each other and through common targets. There is no consensus on how this is achieved. The main goal of this research is to study the target specificity and possible interactions of Tpk1, Tpk2, Tpk3, Sch9, and Ypk1 AGC kinases using a label free mass spectrometry approach. Analysis of the acquired phosphoproteomics data revealed new and known targets of these kinases and, in particular, we were able to identify the first common target between Ypk1 and PKA kinases
Upstream Regulation of TORC2 Signalling
No full textThe Target Of Rapamycin (TOR) kinase functions in two unique protein complexes, named TORC1 and TORC2. TORC1 and TORC2 differ in terms of their structure and localization, but they also present some similarities including the upstream stimuli they sense; i.e. both complexes appear to respond to changes in glucose levels. We found that TORC2 senses glucose in a manner that is at least partially distinct from the glucose signalling that is upstream of TORC1 which involves the SEA and EGO complexes. In contrast, our results demonstrate that the first step in glucose signalling to TORC2 requires any of the three yeast hexokinases bound to their substrate. We genetically uncoupled the catalytic and signalling function of hexokinases, further supporting the role of these glycolytic enzymes in glucose signalling per se. Interestingly, we found that this signalling pathway acts in parallel to inversely regulate the yeast AMPK - Snf1
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