Development of microplate screening system and construction of fluorescein-based library

Master'sMASTER OF SCIENC

Chemical Biology Tools to Study Lipids and their Metabolism with Increased Spatial and Temporal Resolution

Lipids are important cellular components providing many essential functions. To fulfill these various functions evolution has selected for a diverse set of lipids and this diversity is seen at the organismal, cellular and subcellular level. Understanding how cells maintain this complex lipid organization is a very challenging problem, which for lipids, is not easily addressed using biochemical and genetic techniques. Therefore, chemical tools have an important role to play in our quest to understand the complexities of lipid metabolism. Here we discuss new chemical tools to study lipids, their distribution and metabolism with increased spatial and temporal resolution

A single-cell model of PIP3 dynamics using chemical dimerization

AbstractMost cellular processes are driven by simple biochemical mechanisms such as protein and lipid phosphorylation, but the sum of all these conversions is exceedingly complex. Hence, intuition alone is not enough to discern the underlying mechanisms in the light of experimental data. Toward this end, mathematical models provide a conceptual and numerical framework to formally evaluate the plausibility of biochemical processes. To illustrate the use of these models, here we built a mechanistic computational model of PI3K (phosphatidylinositol 3-kinase) activity, to determine the kinetics of lipid metabolizing enzymes in single cells. The model is trained to data generated upon perturbation with a reversible small-molecule based chemical dimerization system that allows for the very rapid manipulation of the PIP3 (phosphatidylinositol 3,4,5-trisphosphate) signaling pathway, and monitored with live-cell microscopy. We find that the rapid relaxation system used in this work decreased the uncertainty of estimating kinetic parameters compared to methods based on in vitro assays. We also examined the use of Bayesian parameter inference and how the use of such a probabilistic method gives information on the kinetics of PI3K and PTEN activity

Structure and conserved function of iso-branched sphingoid bases from the nematode Caenorhabditis elegans

Sphingolipids are bio-active metabolites that show structural diversity among eukaryotes. They are essential for growth of all eukaryotic cells but when produced in an uncontrolled manner can lead to cell death and pathologies including auto-immune reactions, cancer, diabetes and neurodegeneration. Caenorhabditis elegans is an important genetic model organism both to find new drug-targets against parasitic nematodes and to study the conserved roles of sphingolipids in animals like their essential functions in very basic cellular processes ranging from maintenance of cell polarity and mitochondrial repair to growth and survival. C. elegans produces sphingoid bases which are structurally distinct from those of other animals as both iso- and anteiso-branched species have been reported. Using metabolic labeling we show that most worm sphingoid bases are iso-branched. We have synthesized the nematode- specific C17 iso-branched sphinganine and its 1-deoxy analogue and could show that both the iso-branch and the 1-hydroxyl group are essential to form functional nematode sphingolipids which are needed to maintain intestinal function. The organism specificity was examined by complementation experiments in Saccharomyces cerevisiae yeast cells lacking sphingoid base synthesis. We found that iso-branched sphingoid base did not support growth of mutant cells and was toxic to wild type yeast. 1-Deoxy sphingolipids have been linked to the hereditary disease HSAN1A and other metabolic disorders including diabetes. We found that in C. elegans the 1-deoxy analogue cannot rescue the intestinal phenotype caused by sphingoid base depletion. In fact, in wild-type animals with normal sphingoid base biosynthesis, exogenous 1- deoxy analogue had a disruptive effect on apical cytoskeletal organization of intestinal cells indicating that atypical bases can interfere with normal sphingolipid function

Recruitment of a splicing factor to the nuclear lamina for its inactivation

Precursor messenger RNA splicing is a highly regulated process, mediated by a complex RNA-protein machinery, the spliceosome, that encompasses several hundred proteins and five small nuclear RNAs in humans. Emerging evidence suggests that the spatial organization of splicing factors and their spatio-temporal dynamics participate in the regulation of splicing. So far, methods to manipulate the spatial distribution of splicing factors in a temporally defined manner in living cells are missing. Here, we describe such an approach that takes advantage of a reversible chemical dimerizer, and outline the requirements for efficient, reversible re-localization of splicing factors to selected sub-nuclear compartments. In a proof-of-principle study, the partial re-localization of the PRPF38A protein to the nuclear lamina in HEK293T cells induced a moderate increase in intron retention. Our approach allows fast and reversible re-localization of splicing factors, has few side effects and can be applied to many splicing factors by fusion of a protein tag through genome engineering. Apart from the systematic analysis of the spatio-temporal aspects of splicing regulation, the approach has a large potential for the fast induction and reversal of splicing switches and can reveal mechanisms of splicing regulation in native nuclear environments

Luciferase Controlled Protein Interactions

Protein trafficking and protein–protein interactions (PPIs) are central to regulatory processes in cells. Induced dimerization systems have been developed to control PPIs and regulate protein trafficking (localization) or interactions. Chemically induced dimerization (CID) has proven to be a robust approach to control protein interactions and localization. The most recent embodiment of this technology relies on CID conjugates that react with a self-labeling protein on one side and a photocaged ligand on the other side to provide spatiotemporal control of the interaction with the protein of interest. Advancing this technology further is limited by the light delivery problem and the phototoxicity of intense irradiation necessary to achieve photouncaging. Herein, we designed a novel chemically induced dimerization system that was triggered by bioluminescence, instead of external light. Protein dimerization showed fast kinetics and was validated by an induced change of localization of a target protein (to and from the nucleus or plasma membrane) upon trigger. The technology was used transiently to activate the phosphatidylinositol 3-kinase (PI3K)/mTOR pathway and measure the impact on lipid synthesis/metabolism, assessed by lipidomics

Luciferase-Induced Photouncaging: Bioluminolysis

Bioluminescence resonance energy transfer (BRET) has been widely used for studying dynamic processes in biological systems such as protein–protein interactions and other signaling events. Aside from acting as a reporter, BRET can also turn on functions in living systems. Herein, we report the application of BRET to performing a biorthogonal reaction in living cells; namely, releasing functional molecules through energy transfer to a coumarin molecule, a process termed bioluminolysis. An efficient BRET from Nanoluc‐Halotag chimera protein (H‐Luc) to a coumarin substrate yields the excited state of coumarin, which in turn triggers hydrolysis to uncage a target molecule. Compared to the conventional methods, this novel uncaging system requires no external light source and shows fast kinetics (t1/2<2 min). We applied this BRET uncaging system to release a potent kinase inhibitor, ibrutinib, in living cells, highlighting its broad utility in controlling the supply of bioactive small molecules in vivo

Lysosome-targeted photoactivation reveals local sphingosine metabolism signatures

Lipids are essential components of eukaryotic cell membranes and play crucial roles in cellular signaling and metabolism. While increasing evidence shows that the activities of lipids are dependent upon subcellular localization, tools to study local lipid metabolism and signaling are limited. Herein, we report an approach that enabled us to selectively deliver photo-caged lipids into lysosomes and thereafter to quickly release the lipid molecules by illumination. On combining this method with genetic techniques and lipidomics, we were able to investigate the localization-dependent metabolism of an important intermediate of sphingolipid metabolism, sphingosine. Our data reveal a distinct metabolic pattern of lysosomal sphingosine. In general, this method has the potential to serve as a platform to study lysosomal metabolism and signaling of various lipids and metabolites in living cells