The Yeast Three-Hybrid System as an Experimental Platform to Identify Proteins Interacting with Small Signaling Molecules in Plant Cells: Potential and Limitations

Chemical genetics is a powerful scientific strategy that utilizes small bioactive molecules as experimental tools to unravel biological processes. Bioactive compounds occurring in nature represent an enormous diversity of structures that can be used to dissect functions of biological systems. Once the bioactivity of a natural or synthetic compound has been critically evaluated the challenge remains to identify its molecular target and mode of action, which usually is a time-consuming and labor-intensive process. To facilitate this task, we decided to implement the yeast three-hybrid (Y3H) technology as a general experimental platform to scan the whole Arabidopsis proteome for targets of small signaling molecules. The Y3H technology is based on the yeast two-hybrid system and allows direct cloning of proteins that interact in vivo with a synthetic hybrid ligand, which comprises the biologically active molecule of interest covalently linked to methotrexate (Mtx). In yeast nucleus the hybrid ligand connects two fusion proteins: the Mtx part binding to dihydrofolate reductase fused to a DNA-binding domain (encoded in the yeast strain), and the bioactive molecule part binding to its potential protein target fused to a DNA-activating domain (encoded on a cDNA expression vector). During cDNA library screening, the formation of this ternary, transcriptional activator complex leads to reporter gene activation in yeast cells, and thereby allows selection of the putative targets of small bioactive molecules of interest. Here we present the strategy and experimental details for construction and application of a Y3H platform, including chemical synthesis of different hybrid ligands, construction of suitable cDNA libraries, the choice of yeast strains, and appropriate screening conditions. Based on the results obtained and the current literature we discuss the perspectives and limitations of the Y3H approach for identifying targets of small bioactive molecules

Radial distribution function of semiflexible polymers

We calculate the distribution function of the end--to--end distance of a semiflexible polymer with large bending rigidity. This quantity is directly observable in experiments on single semiflexible polymers (e.g., DNA, actin) and relevant to their interpretation. It is also an important starting point for analyzing the behavior of more complex systems such as networks and solutions of semiflexible polymers. To estimate the validity of the obtained analytical expressions, we also determine the distribution function numerically using Monte Carlo simulation and find good quantitative agreement.Comment: RevTeX, 4 pages, 1 figure. Also available at http://www.cip.physik.tu-muenchen.de/tumphy/d/T34/Mitarbeiter/frey.htm

Stepwise bending of DNA by a single TATA-box Binding Protein

The TATA-box Binding Protein (TBP) is required by all three eukaryotic RNA polymerases for the initiation of transcription from most promoters. TBP recognizes, binds to, and bends promoter sequences called ``TATA-boxes'' in the DNA. We present results from the study of individual Saccharomyces cerevisia TBPs interacting with single DNA molecules containing a TATA-box. Using video microscopy, we observed the Brownian motion of beads tethered by short surface-bound DNA. When TBP binds to and bends the DNA, the conformation of the DNA changes and the amplitude of Brownian motion of the tethered bead is reduced compared to that of unbent DNA. We detected individual binding and dissociation events and derived kinetic parameters for the process. Dissociation was induced by increasing the salt concentration or by directly pulling on the tethered bead using optical tweezers. In addition to the well-defined free and bound classes of Brownian motion, we observed another two classes of motion. These extra classes were identified with intermediate states on a three-step, linear binding pathway. Biological implications of the intermediate states are discussed.Comment: Accepted for publication in: Biophysical Journa

In Vivo Determination of Fluctuating Forces during Endosome Trafficking Using a Combination of Active and Passive Microrheology

BACKGROUND: Regulation of intracellular trafficking is a central issue in cell biology. The forces acting on intracellular vesicles (endosomes) can be assessed in living cells by using a combination of active and passive microrheology. METHODOLOGY/PRINCIPAL FINDINGS: This dual approach is based on endosome labeling with magnetic nanoparticles. The resulting magnetic endosomes act both as probes that can be manipulated with external magnetic fields to infer the viscoelastic modulus of their surrounding microenvironment, and as biological vehicles that are trafficked along the microtubule network by means of forces generated by molecular motors. The intracellular viscoelastic modulus exhibits power law dependence with frequency, which is microtubule and actin-dependent. The mean square displacements of endosomes do not follow the predictions of the fluctuation-dissipation theorem, which offers evidence for active force generation. Microtubule disruption brings the intracellular medium closer to thermal equilibrium: active forces acting on the endosomes depend on microtubule-associated motors. The power spectra of these active forces, deduced through the use of a generalized Langevin equation, show a power law decrease with frequency and reveal an actin-dependent persistence of the force with time. Experimental spectra have been reproduced by a simple model consisting in a series of force steps power-law distributed in time. This model enlightens the role of the cytoskeleton dependent force exerted on endosomes to perform intracellular trafficking. CONCLUSIONS/SIGNIFICANCE: In this work, the influence of cytoskeleton components and molecular motors on intracellular viscoelasticity and transport is addressed. The use of an original probe, the magnetic endosome, allows retrieving the power spectrum of active forces on these organelles thanks to interrelated active and passive measures. Finally a computational model gives estimates of the force itself and hence of the number of the motors pulling on endosomes

LIBER Open Science Roadmap

Embracing Open Science is critical if we are to make science more collaborative, reproducible, transparent and impactful. Open Science undoubtedly has the power to positively influence society, but its implementation is not yet universal. A revolution is required: one which opens up research processes and changes mindsets in favour of a world where policies, tools and infrastructures universally support the growth and sharing of knowledge. Research libraries are well placed to make that revolution happen, and LIBER\u27s Open Science Roadmap outlines the specific actions libraries can take to champion Open Science, both within and beyond their own institutions. As we explain in detail throughout this document, libraries need to advocate for Open Science locally and internationally, to support Open Science through tools and services and to expand the impact of their work through collaboration and partnerships. LIBER has shaped its 2018-2022 Strategy to support and enable Open Science and it is our hope that this Roadmap will help Europe’s research libraries to do the same. This document was written during spring 2018, when the Open Science Policy Platform (OSPP) produced integrated advice for the EC and key stakeholders. People from across the LIBER community translated the OSPP recommendations for libraries and combined them with suggestions drawn from their own expertise and experiences

LIBER Open Science Roadmap

Embracing Open Science is critical if we are to make science more collaborative, reproducible, transparent and impactful. Open Science undoubtedly has the power to positively influence society, but its implementation is not yet universal. A revolution is required: one which opens up research processes and changes mindsets in favour of a world where policies, tools and infrastructures universally support the growth and sharing of knowledge. Research libraries are well placed to make that revolution happen, and LIBER\u27s Open Science Roadmap outlines the specific actions libraries can take to champion Open Science, both within and beyond their own institutions. As we explain in detail throughout this document, libraries need to advocate for Open Science locally and internationally, to support Open Science through tools and services and to expand the impact of their work through collaboration and partnerships. LIBER has shaped its 2018-2022 Strategy to support and enable Open Science and it is our hope that this Roadmap will help Europe’s research libraries to do the same. This document was written during spring 2018, when the Open Science Policy Platform (OSPP) produced integrated advice for the EC and key stakeholders. People from across the LIBER community translated the OSPP recommendations for libraries and combined them with suggestions drawn from their own expertise and experiences