Rainer Pepperkok

..This modular platform is scalable and makes the power of time-lapse microscopy available for genome-wide RNAi screens...

..Therefore, they are ideal tools for comparative genomic studies aimed at understanding how aspects of biological complexity such as cellular compartmentalization evolved...

..A report on the conference 'Systems Genomics 2008', Heidelberg, Germany, 2-3 May 2008...

..These data reveal the existence of an early COPI-dependent, pre-Golgi cargo sorting step in mammalian cells...

..Together, this suggests that a recycling pathway operates at the level of the trans-Golgi linking directly to the ER. This pathway would be the preferred route for both toxins and resident Golgi proteins...

..We conclude that secretory cargo retains the COPII complex on membranes, after Sar1p release has occurred, and prevents premature disassembly of COPII during cargo sorting and transport carrier formation...

..Two automated functional assays addressing protein secretion and the integrity of the Golgi complex were developed and tested. This shows the potential of the system in large-scale, cell-based functional proteomic projects...

..Our data strongly support a role for kinesin-2 in the KDEL-R-/COPI-dependent retrograde transport pathway from the Golgi complex to the ER...

..As part of the Mitocheck consortium, this study provides an in-depth analysis of cell division phenotypes and makes the entire high-content data set available as a resource to the community...

..We further describe the use of software macros and plugins in Image J that allow the quantification of the kinetics of membrane transport intermediates in fluorescence microscopy time-lapse sequences...

..This indicates that chromosome-induced microtubule production is a general requirement for the formation of functional spindles in animal cells...

..We extend the application of the method by demonstrating induced dynamic behavior of actin-stress fibers after severing. This new method enables the quantitative investigation of cytoskeleton dynamics in a local confinement...

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..Correlating these findings with other screens and bioinformatic information makes these candidates highly likely to be novel membrane traffic machinery components...

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..To enable these techniques to be applied to functional genomics experiments, fluorescence microscopy is making the transition to a quantitative and high-throughput technology...

..Here we discuss this new work in the context of other strategies for large-scale subcellular localization...

..Altogether, we demonstrate the potential of the systematic microscope-based screening approaches described here to tackle the complex biological process of neurite outgrowth regulation...

..Different human cell lines that have been successfully transfected using the protocol are presented here. The present protocol has been applied to two genome-wide siRNA screens addressing mitosis and constitutive protein secretion...

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..We demonstrate the use of these methods applying them on a genome wide RNAi screen by time-lapse microscopy...

..Consistent with this, membrane turnover of the COPII component Sec23p is delayed in sterol-depleted cells. Altogether, our results demonstrate the importance of sterol levels in COPII mediated ER-export...

..We demonstrate such an siRNA cell array in a microscope-based functional assay in living cells to determine the effect of various siRNA oligonucleotides against endogenous targets on cellular secretion...

..This method should facilitate drug target validation by small-interfering RNAs...

..It will also serve as an example for similar microscope-based screens addressing different biological questions...

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..Moreover, GFP2 has its excitation maximum at 396 nm at which the YFP acceptor is excited only below the detection level and thus this FRET pair is ideal for applications involving sensitized emission...

..Here we discuss recent progress on the technical implementation of the method, its limitations and applications to the imaging of biological samples...

..These advances, combined with an increasing number and diversity of gene perturbing systems, such as RNAi and ectopic gene expression, provide tools for expanding our understanding of biology at the systems level...

..A feedback forum allows users to comment on Harvester and to report database inconsistencies. AVAILABILITY: The service is freely available to the academic community at http://harvester.embl.de...

..Comparison with less comprehensive studies of mammalian cells provides insights into the localization of the mammalian proteome...

..The method is suitable for high content screening microscopy at a high spatial and temporal resolution allowing even time-lapse analysis of hundreds of samples in parallel...

..This work establishes membrane-permeant phosphoinositides as powerful tools to study PI3K signaling and directly demonstrates that 3-phosphorylated phosphoinositides are instrumental for neurite initiation...

..We propose a model of retinal morphogenesis whereby opo-mediated formation of focal contacts is required to transmit the mechanical tensions that drive the macroscopic folding of the vertebrate optic cup...

..The Harvester search engine works similar to Google, offering genome-wide ranked results at very high speed. Here we describe how to use this bioinformatic tool along with selected examples...

..Furthermore, the glass surface is laser-etched and upon fixation and flat embedding, the inverse ridge can be localized as well as the cell of interest, which is then processed for electron microscopy...

..These and previous results suggest that under physiological conditions, TPX2 is essential for microtubule nucleation around chromatin and functions in a network of other molecules, some of which also are regulated by RanGTP...

..Our results identify novel inhibitors of SV40 infection and show that SV40 requires COPI- and COPII-dependent transport steps for successful infection...

..Rab18-S22N also potentiated brefeldin-A-induced ER-Golgi fusion. This study is the first comprehensive application of large-scale proteomics to the cell biology of small GTPases of the secretory pathway...

..We conclude that solid-phase reverse transfection in multiwell plates is a cost-efficient and flexible tool for multiplexing cellular assays...

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..This suggests that the direct interaction of 2c18 and gamma1-adaptin is crucial for membrane association and thus the function of the AP-1 complex in living cells. We propose to name this protein gamma-BAR...

..A web interface enables the scientific community to explore and visualize features of the annotated cDNAs and ORFs combined with experimental results, and thus helps to unravel new features of proteins with as yet unknown functions...

..All information is presented in a Web database (http://www.dkfz.de/LIFEdb). It is exploited for the identification of disease-relevant genes and proteins for diagnosis and therapy...

..Together, our data suggest a mechanism by which membranes of the early secretory pathway can be linked to motors and microtubules for subsequent organization and movement to the Golgi apparatus...

..LIFEdb enables researchers to systematically select and characterize genes and proteins of interest, and presents data and information via its user-friendly web-based interface...

..These data show a differential requirement for efficient GTP hydrolysis by the Sar1p GTPase in export of cargo from the ER...

..By contrast, Golgi stacks associate intermittently and sometimes concurrently with several ERES as they move...

..These results suggest that the ERGIC is stationary and not simply a collection of mobile carriers that mediate protein traffic from endoplasmic reticulum to Golgi...

..Our classification method can be adapted to virtually any microscopic assay based on cell morphology, opening a wide range of applications including large-scale RNAi screening in human cells...

..Our results suggest that the subcellular distribution of cholesterol may be an important factor in how cholesterol alters Abeta production and the risk of Alzheimer's disease...

..Imaging of EGFR phosphorylation may thus serve as a valuable tool to investigate the role of receptor tyrosine kinase activity in malignant cell growth...

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..Our results support the concept that [Ca(2+)](i) is a key regulator of NF-(kappa)B activation in CF airway epithelial cells...

..We have established a novel functional profiling strategy that links genomics to cell biology and showed its potential for discerning cancer relevant modulators of the cell cycle in the candidate lists from microarray studies...

..The resulting information is integrated with functional genomics and proteomics data for further understanding of protein functions in the cellular context...

..We propose that the positioning of the Golgi complex and the centrosome depends on the geometrical constraints applied to the cell rather than on a precise migratory function in the leading region...

..Our data provide new details about the secretory route of FVIII, which may ultimately help to identify factors currently limiting the efficient and physiological expression of FVIII in gene therapy and manufacture...

..It has the potential to be extended to study interactions of proteins or multi-protein complexes with different biopolymers like actin microfilaments or organelle membranes...