Live-Cell Assays to Identify Regulators of ER-to-Golgi Trafficking
| Autor: | Petr Matula, Lars Lehmann, Christoph Claas, Tautvydas Lisauskas, Karl Rohr, Vytaute Starkuviene, Holger Erfle, Roland Eils, Brian Storrie, Peter Fischer, Susanne Reusing, Stefan Wiemann |
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| Rok vydání: | 2012 |
| Předmět: |
0303 health sciences
Effector Endoplasmic reticulum 030302 biochemistry & molecular biology USE1 Cell Biology Golgi apparatus Biology Brefeldin A Biochemistry Transport protein Cell biology 03 medical and health sciences symbols.namesake chemistry.chemical_compound chemistry Structural Biology RNA interference Genetics symbols Fluorescence microscope Molecular Biology 030304 developmental biology |
| Zdroj: | Traffic. 13:416-432 |
| ISSN: | 1398-9219 |
| DOI: | 10.1111/j.1600-0854.2011.01318.x |
| Popis: | We applied fluorescence microscopy-based quantitative assays to living cells to identify regulators of endoplasmic reticulum (ER)-to-Golgi trafficking and/or Golgi complex maintenance. We first validated an automated procedure to identify factors which influence Golgi-to-ER relocalization of GalT-CFP (β1,4-galactosyltransferase I-cyan fluorescent protein) after brefeldin A (BFA) addition and/or wash-out. We then tested 14 proteins that localize to the ER and/or Golgi complex when overexpressed for a role in ER-to-Golgi trafficking. Nine of them interfered with the rate of BFA-induced redistribution of GalT-CFP from the Golgi complex to the ER, six of them interfered with GalT-CFP redistribution from the ER to a juxtanuclear region (i.e. the Golgi complex) after BFA wash-out and six of them were positive effectors in both assays. Notably, our live-cell approach captures regulator function in ER-to-Golgi trafficking, which was missed in previous fixed cell assays, as well as assigns putative roles for other less characterized proteins. Moreover, we show that our assays can be extended to RNAi and chemical screens. |
| Databáze: | OpenAIRE |
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