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See detailA microtubule-independent role of p150glued in secretory cargo concentration at endoplasmic reticulum exit sites
Verissimo, Fatima; Halavatyi, Aliaksandr UL; Pepperkok, Rainer et al

in Journal of Cell Science (2015), 128(22), 4160-4170

Newly synthesized proteins are sorted into COPII-coated transport carriers at the endoplasmic reticulum (ER). Assembly of the COPII coat complex, which occurs at ER exit sites (ERES), is initiated by ... [more ▼]

Newly synthesized proteins are sorted into COPII-coated transport carriers at the endoplasmic reticulum (ER). Assembly of the COPII coat complex, which occurs at ER exit sites (ERES), is initiated by membrane association and GTP loading of SAR1, followed by the recruitment of the SEC23-SEC24 and SEC13-SEC31 subcomplexes. Both of these two subcomplexes stimulate GTP hydrolysis and coat disassembly. This inherent disassembly capacity of COPII complexes needs to be regulated to allow sufficient time for cargo sorting and transport carrier formation. By performing fluorescence recovery after photobleaching (FRAP) and mathematical modeling, we show that p150glued (also known as DCTN1), a component of the dynactin complex, stabilizes the COPII pre-budding complex on ER membranes in a microtubule-independent manner. Concentration of the secretory marker ts-O45-G at ERES is reduced in the presence of a C-terminal p150glued fragment that prevents binding of endogenous p150glued to SEC23. A similar cargo reduction is observed upon p150glued knockdown. Taken together, our data suggest that cargo concentration at ERES is regulated by p150glued to coordinate protein sorting and transport carrier formation with the subsequent long-range transport towards the Golgi complex along microtubules. © 2015. Published by The Company of Biologists Ltd. [less ▲]

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See detailPhenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes
Neumann, Beate; Walter, Thomas; Heriche, Jean-Karim et al

in Nature (2010), 464(7289), 721-727

Despite our rapidly growing knowledge about the human genome, we do not know all of the genes required for some of the most basic functions of life. To start to fill this gap we developed a high ... [more ▼]

Despite our rapidly growing knowledge about the human genome, we do not know all of the genes required for some of the most basic functions of life. To start to fill this gap we developed a high-throughput phenotypic screening platform combining potent gene silencing by RNA interference, time-lapse microscopy and computational image processing. We carried out a genome-wide phenotypic profiling of each of the similar to 21,000 human protein-coding genes by two-day live imaging of fluorescently labelled chromosomes. Phenotypes were scored quantitatively by computational image processing, which allowed us to identify hundreds of human genes involved in diverse biological functions including cell division, migration and survival. 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. [less ▲]

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See detailFrom experimental setup to bioinformatics: An RNAi screening platform to identify host factors involved in HIV-1 replication
Boerner, Kathleen; Hermle, Johannes; Sommer, Christoph et al

in Biotechnology Journal (2010), 5(1), 39-49

RNA interference (RNAi) has emerged as a powerful technique for studying loss-of-function phenotypes by specific down-regulation of gene expression, allowing the investigation of virus-host interactions ... [more ▼]

RNA interference (RNAi) has emerged as a powerful technique for studying loss-of-function phenotypes by specific down-regulation of gene expression, allowing the investigation of virus-host interactions by large-scale high-throughput RNAi screens. Here we present a robust and sensitive small interfering RNA screening platform consisting of an experimental setup, single-cell image and statistical analysis as well as bioinformatics. The workflow has been established to elucidate host gene functions exploited by viruses, monitoring both suppression and enhancement of viral replication simultaneously by fluorescence microscopy. The platform comprises a two-stage procedure in which potential host factors are first identified in a primary screen and afterwards re-tested in a validation screen to confirm true positive hits. Subsequent bioinformatics allows the identification of cellular genes participating in metabolic pathways and cellular networks utilised by viruses for efficient infection. Our workflow has been used to investigate host factor usage by the human immunodeficiency virus-1 (HIV-1), but can also be adapted to other viruses. Importantly, we expect that the description of the platform will guide further screening approaches for virus-host interactions. The ViroQuant-Cell Networks RNAi Screening core facility is an integral part of the recently founded BioQuant centre for systems biology at the University of Heidelberg and will provide service to external users in the near future. [less ▲]

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