Bischof S.,ETH Zurich |
Bischof S.,University of California at Los Angeles |
Umhang M.,ETH Zurich |
Eicke S.,ETH Zurich |
And 3 more authors.
Plant Cell | Year: 2013
The branched glucans glycogen and starch are the most widespread storage carbohydrates in living organisms. The production of semicrystalline starch granules in plants is more complex than that of small, soluble glycogen particles in microbes and animals. However, the factors determining whether glycogen or starch is formed are not fully understood. The tropical tree Cecropia peltata is a rare example of an organism able to make either polymer type. Electron micrographs and quantitative measurements show that glycogen accumulates to very high levels in specialized myrmecophytic structures (Müllerian bodies), whereas starch accumulates in leaves. Compared with polymers comprising leaf starch, glycogen is more highly branched and has shorter branches-factors that prevent crystallization and explain its solubility. RNA sequencing and quantitative shotgun proteomics reveal that isoforms of all three classes of glucan biosynthetic enzyme (starch/glycogen synthases, branching enzymes, and debranching enzymes) are differentially expressed in Müllerian bodies and leaves, providing a system-wide view of the quantitative programming of storage carbohydrate metabolism. This work will prompt targeted analysis in model organisms and cross-species comparisons. Finally, as starch is the major carbohydrate used for food and industrial applications worldwide, these data provide a basis for manipulating starch biosynthesis in crops to synthesize tailor-made polyglucans. © 2013 American Society of Plant Biologists. All rights reserved.
Kaufmann S.,ETH Zurich |
Sobek J.,Functional Genomics Center Zurich |
Textor M.,ETH Zurich |
Reimhult E.,University of Natural Resources and Life Sciences, Vienna
Lab on a Chip - Miniaturisation for Chemistry and Biology | Year: 2011
Arrays of supported lipid bilayers (SLBs) provide great potential for future drug development and multiplexed biological research, but are difficult to prepare due to the sensitivity of both the lipid and protein structural arrangement to air exposure. A novel way to produce arrays of SLBs is presented based on non-contact dispensing of vesicles to a substrate through a thin surface confined water film. The approach presents many degrees of freedom since it is not limited to a specific substrate, lipid composition, linker or controlled environment. The method allows adjustment of spot size (180-360 μm) by repeated dispensing as well as control over the composition of the spots and subsequent analytes. SLB formation by vesicle adsorption and rupture allows for incorporation of membrane proteins through pre-formed proteoliposomes. Dispensing through a dip-and-rinse water film avoids contamination, disruptive drying and the need for complex buffer compositions. Furthermore, no humidity control is necessary which simplifies the production step and prolongs the life-time of the spotting system. We characterize the method with respect to control over spot size, bilayer mobility and the formation process as well as demonstrate the possibility to fuse bilayer spots with subsequently added vesicles. Since complex lipid compositions and multiple spotting nozzles can be used, this novel technique is expected to be a promising platform for future applications, e.g. patterning to monitor peptide/protein-lipid interactions, for glycomics using glycolipids or lipopolysaccharides, and to study mixing of spatially confined lipid membranes. © 2011 The Royal Society of Chemistry.
Quaroni L.,University of Fribourg |
Quaroni L.,Functional Genomics Center Zurich |
Obst M.,University of Tubingen |
Nowak M.,University of Tubingen |
Zobi F.,University of Fribourg
Angewandte Chemie - International Edition | Year: 2015
Microscopy in the mid-infrared spectral range provides detailed chemical information on a sample at moderate spatial resolution and is being used increasingly in the characterization of biological entities as challenging as single cells. However, a conventional cellular 2D imaging measurement is limited in its ability to associate specific compositional information to subcellular structures because of the interference from the complex topography of the sample. Herein we provide a method and protocols that overcome this challenge in which tilt-series infrared tomography is used with a standard benchtop infrared microscope. This approach gives access to the quantitative 3D distribution of molecular components based on the intrinsic contrast provided by the sample. We demonstrate the method by quantifying the distribution of an exogenous metal carbonyl complex throughout the cell and by reporting changes in its coordination sphere in different locations in the cell. ©2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim chemical imaging • infrared tomography • single-cell studies.
Quaroni L.,Paul Scherrer Institute |
Quaroni L.,Functional Genomics Center Zurich |
Zlateva T.,Paul Scherrer Institute
Analytical Chemistry | Year: 2014
In recent years, major efforts have been devoted to the application of microscopy with mid-infrared light to the study of living cells and tissue. Despite this interest, infrared (IR) microscopy has not realized its full potential in the molecular characterization of living systems. This is partly due to the fact that current approaches for data mining and analysis of IR absorption spectra have not evolved comparably to measurement technology and are not up to the interpretation of the complex spectra of living systems such as cells and tissue. In this work we show that the use of two-dimensional correlation spectroscopy coupled to IR absorption spectro-microscopy allows us to extract the spectral components of individual metabolites from time-resolved IR spectra of living cells. We call this method correlated cellular spectro-microscopy, and we implement it in the study of the glycolytic metabolism of cancer cells. We show that the method can detect intermediates of the glycolytic pathway, quantify their rate of formation, and correlate this with variations in pH, all in a single measurement. We propose the method as a useful tool for the quantitative description of metabolic processes in living cells and for the validation of drug candidates aimed at suppressing glycolysis in cancer cells. © 2014 American Chemical Society.
Wollenick K.,University of Zurich |
Hu J.,University of Duisburg - Essen |
Kristiansen G.,University of Bonn |
Schraml P.,University of Zurich |
And 5 more authors.
Nucleic Acids Research | Year: 2012
The human prolyl-4-hydroxylase domain (PHD) proteins 1-3 are known as cellular oxygen sensors, acting via the degradation of hypoxia-inducible factor (HIF) α-subunits. PHD2 and PHD3 genes are inducible by HIFs themselves, suggesting a negative feedback loop that involves PHD abundance. To identify novel regulators of the PHD2 gene, an expression array of 704 transcription factors was screened by a method that allows distinguishing between HIF-dependent and HIF-independent promoter regulation. Among others, the E-twenty six transcription factor ETS translocation variant 4 (ETV4) was found to contribute to PHD2 gene expression particularly under hypoxic conditions. Mechanistically, complex formation between ETV4 and HIF-1/2α was observed by mammalian two-hybrid and fluorescence resonance energy transfer analysis. HIF-1α domain mapping, CITED2 overexpression and factor inhibiting HIF depletion experiments provided evidence for cooperation between HIF-1α and p300/CBP in ETV4 binding. Chromatin immunoprecipitation confirmed ETV4 and HIF-1α corecruitment to the PHD2 promoter. Of 608 hypoxically induced transcripts found by genome-wide expression profiling, 7.7 required ETV4 for efficient hypoxic induction, suggesting a broad role of ETV4 in hypoxic gene regulation. Endogenous ETV4 highly correlated with PHD2, HIF-1/2α and several established markers of tissue hypoxia in 282 human breast cancer tissue samples, corroborating a functional interplay between the ETV4 and HIF pathways. © 2011 The Author(s).