Quenched substrates for live-cell labeling of SNAP-tagged fusion proteins with improved fluorescent background.

Recent developments in fluorescence microscopy raise the demands for bright and photostable fluorescent tags for specific and background free labeling in living cells. Aside from fluorescent proteins and other tagging methods, labeling of SNAP-tagged proteins has become available thereby increasing the pool of potentially applicable fluorescent dyes for specific labeling of proteins. Here, we report on novel conjugates of benzylguanine (BG) which are quenched in their fluorescence and become highly fluorescent upon labeling of the SNAP-tag, the commercial variant of the human O(6)-alkylguanosyltransferase (hAGT).

Model-based extension of high-throughput to high-content data.

Background: High-quality quantitative data is a major limitation in systems biology. The experimental data used in systems biology can be assigned to one of the following categories: assays yielding average data of a cell population, high-content single cell measurements and high-throughput techniques generating single cell data for large cell populations. For modeling purposes, a combination of data from different categories is highly desirable in order to increase the number of observable species and processes and thereby maximize the identifiability of parameters.

Systems biology of JAK/STAT signalling.

Signalling in multicellular organisms is mediated by complex networks that integrate extracellular and intracellular signals to generate appropriate responses regulating cell proliferation, differentiation and survival. Downstream of many cytokine and growth hormone receptors, receptor-associated JAKs (Janus kinases) activate transcription factors of the STAT (signal transducer and activator of transcription) protein family and thereby mediate signal transduction from the plasma membrane to the nucleus. The JAK/STAT pathway has been shown to be constitutively activated in a wide array of human malignancies.

Standardizing experimental protocols.

Systems biology aims at understanding the behavior of biological networks by mathematical modeling based on experimental data. However, frequently experimental data is derived from poorly defined cellular systems, the procedures of data generation are insufficiently documented and data processing is arbitrary. For the advancement of systems biology, standardization at multiple levels is essential.

A systems biology approach to analyse amplification in the JAK2-STAT5 signalling pathway.

Background: The amplification of signals, defined as an increase in the intensity of a signal through networks of intracellular reactions, is considered one of the essential properties in many cell signalling pathways. Despite of the apparent importance of signal amplification, there have been few attempts to formalise this concept.

Results: In this work we investigate the amplification and responsiveness of the JAK2-STAT5 pathway using a kinetic model.

Dynamic pathway modeling: feasibility analysis and optimal experimental design.

A major challenge in systems biology is to evaluate the feasibility of a biological research project prior to its realization. Since experiments are animals-, cost- and time-consuming, approaches allowing researchers to discriminate alternative hypotheses with a minimal set of experiments are highly desirable. Given a null hypothesis and alternative model, as well as laboratory constraints like observable players, sample size, noise level, and stimulation options, we suggest a method to obtain a list of required experiments in order to significantly reject the null hypothesis model M0 if a specified alternative model MA is realized.

Dynamics of GBF1, a Brefeldin A-sensitive Arf1 exchange factor at the Golgi.

Trafficking through the Golgi apparatus requires members of the Arf family of GTPases, whose activation is regulated by guanine nucleotide exchange factors (GEFs). Once activated, Arf-GTP recruits effectors such as coat complexes and lipid-modifying enzymes to specific membrane sites, creating a domain competent for cargo concentration and transport. GBF1 is a peripherally associated Arf GEF involved in both endoplasmic reticulum-Golgi and intra-Golgi transport.

CASP, the alternatively spliced product of the gene encoding the CCAAT-displacement protein transcription factor, is a Golgi membrane protein related to giantin.

Large coiled-coil proteins are being found in increasing numbers on the membranes of the Golgi apparatus and have been proposed to function in tethering of transport vesicles and in the organization of the Golgi stack. Members of one class of Golgi coiled-coil protein, comprising giantin and golgin-84, are anchored to the bilayer by a single C-terminal transmembrane domain (TMD). In this article, we report the characterization of another mammalian coiled-coil protein, CASP, that was originally identified as an alternatively spliced product of the CUTL1 gene that encodes CCAAT-displacement protein (CDP), the human homologue of the Drosophila homeodomain protein Cut.

Dissection of COPI and Arf1 dynamics in vivo and role in Golgi membrane transport.

Cytosolic coat proteins that bind reversibly to membranes have a central function in membrane transport within the secretory pathway. One well-studied example is COPI or coatomer, a heptameric protein complex that is recruited to membranes by the GTP-binding protein Arf1. Assembly into an electron-dense coat then helps in budding off membrane to be transported between the endoplasmic reticulum (ER) and Golgi apparatus.