Chemical-Biology-derived in vivo Sensors: Past, Present, and Future.

To 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

La dinámica de los estados de fosforilación de las vías HOG y de las feromonas en levaduras

Comunicaciones a congreso

Active-Site Inhibitors of mTOR Target Rapamycin-Resistant Outputs of mTORC1 and mTORC2

The mammalian target of rapamycin (mTOR) regulates cell growth and survival by integrating nutrient and hormonal signals. These signaling functions are distributed between at least two distinct mTOR protein complexes: mTORC1 and mTORC2. mTORC1 is sensitive to the selective inhibitor rapamycin and activated by growth factor stimulation via the canonical phosphoinositide 3-kinase (PI3K)→Akt→mTOR pathway. Activated mTORC1 kinase up-regulates protein synthesis by phosphorylating key regulators of mRNA translation. By contrast, mTORC2 is resistant to rapamycin. Genetic studies have suggested that mTORC2 may phosphorylate Akt at S473, one of two phosphorylation sites required for Akt activation; this has been controversial, in part because RNA interference and gene knockouts produce distinct Akt phospho-isoforms. The central role of mTOR in controlling key cellular growth and survival pathways has sparked interest in discovering mTOR inhibitors that bind to the ATP site and therefore target both mTORC2 and mTORC1. We investigated mTOR signaling in cells and animals with two novel and specific mTOR kinase domain inhibitors (TORKinibs). Unlike rapamycin, these TORKinibs (PP242 and PP30) inhibit mTORC2, and we use them to show that pharmacological inhibition of mTOR blocks the phosphorylation of Akt at S473 and prevents its full activation. Furthermore, we show that TORKinibs inhibit proliferation of primary cells more completely than rapamycin. Surprisingly, we find that mTORC2 is not the basis for this enhanced activity, and we show that the TORKinib PP242 is a more effective mTORC1 inhibitor than rapamycin. Importantly, at the molecular level, PP242 inhibits cap-dependent translation under conditions in which rapamycin has no effect. Our findings identify new functional features of mTORC1 that are resistant to rapamycin but are effectively targeted by TORKinibs. These potent new pharmacological agents complement rapamycin in the study of mTOR and its role in normal physiology and human disease

The Vam6 GEF controls TORC1 by activating the EGO complex

The target of rapamycin complex 1 (TORC1) is a central regulator of eukaryotic cell growth that is activated by a variety of hormones (e.g., insulin) and nutrients (e.g., amino acids) and is deregulated in various cancers. Here, we report that the yeast Rag GTPase homolog Gtr1, a component of the vacuolar-membrane-associated EGO complex (EGOC), interacts with and activates TORC1 in an amino-acid-sensitive manner. Expression of a constitutively active (GTP-bound) Gtr1GTP, which interacted strongly with TORC1, rendered TORC1 partially resistant to leucine deprivation, whereas expression of a growth inhibitory, GDP-bound Gtr1GDP, caused constitutively low TORC1 activity. We also show that the nucleotide-binding status of Gtr1 is regulated by the conserved guanine nucleotide exchange factor (GEF) Vam6. Thus, in addition to its regulatory role in homotypic vacuolar fusion and vacuole protein sorting within the HOPS complex, Vam6 also controls TORC1 function by activating the Gtr1 subunit of the EGO complex

Identification of a small molecule yeast TORC1 inhibitor with a flow cytometry-based multiplex screen

TOR (target of rapamycin) is a serine/threonine kinase, evolutionarily conserved from yeast to human, which functions as a fundamental controller of cell growth. The moderate clinical benefit of rapamycin in mTOR-based therapy of many cancers favors the development of new TOR inhibitors. Here we report a high throughput flow cytometry multiplexed screen using five GFPtagged yeast clones that represent the readouts of four branches of the TORC1 signaling pathway in budding yeast. Each GFP-tagged clone was differentially color-coded and the GFP signal of each clone was measured simultaneously by flow cytometry, which allows rapid prioritization of compounds that likely act through direct modulation of TORC1 or proximal signaling components. A total of 255 compounds were confirmed in dose-response analysis to alter GFP expression in one or more clones. To validate the concept of the high throughput screen, we have characterized CID 3528206, a small molecule most likely to act on TORC1 as it alters GFP expression in all five GFP clones in an analogous manner to rapamycin. We have shown that CID 3528206 inhibited yeast cell growth, and that CID 3528206 inhibited TORC1 activity both in vitro and in vivo with EC50s of 150 nM and 3.9 μM, respectively. The results of microarray analysis and yeast GFP collection screen further support the notion that CID 3528206 and rapamycin modulate similar cellular pathways. Together, these results indicate that the HTS has identified a potentially useful small molecule for further development of TOR inhibitors

A brief history of TOR

Abstract The TOR (target of rapamycin) serine/threonine kinases are fascinating in that they influence many different aspects of eukaryote physiology including processes often dysregulated in disease. Beginning with the initial characterization of rapamycin as an antifungal agent, studies with yeast have contributed greatly to our understanding of the molecular pathways in which TORs operate. Recently, building on advances in quantitative MS, the rapamycin-dependent phosphoproteome in the budding yeast Saccharomyces cerevisiae was elucidated. These studies emphasize the central importance of TOR and highlight its many previously unrecognized functions. One of these, the regulation of intermediary metabolism, is discussed

Growth control: function follows form

Cell division, intuitively, is often dependent upon increases in cellular mass and volume. Less obvious is the reciprocal regulation of growth by the cell division cycle. In budding yeast, this link is mediated by the cell-cycle-dependent polarization of actin