An RNAi screening platform to identify secretion machinery in mammalian cells.
Integrative approaches to study protein function in a cellular context are a vital aspect of understanding human disease. Genome sequencing projects provide the basic catalogue of information with which to unravel gene function, but more systematic applications of this resource are now necessary. Here, we describe and test a platform with which it is possible to rapidly use RNA interference in cultured mammalian cells to probe for proteins involved in constitutive protein secretion. Synthetic small interfering RNA molecules are arrayed in chambered slides, then incubated with cells and an assay for secretion performed. Automated microscopy is used to acquire images from the experiments, and automated single-cell analysis rapidly provides reliable quantitative data. In test arrays of 92 siRNA spots targeting 37 prospective membrane traffic proteins, our approach identifies 7 of these as being important for the correct delivery of a secretion marker to the cell surface. Correlating these findings with other screens and bioinformatic information makes these candidates highly likely to be novel membrane traffic machinery components.
Simpson
Jeremy C
JC
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany. simpson@embl.de
Cetin
Cihan
C
Erfle
Holger
H
Joggerst
Brigitte
B
Liebel
Urban
U
Ellenberg
Jan
J
Pepperkok
Rainer
R
D000818:Animals; D002478:Cells, Cultured; D006367:HeLa Cells; D006801:Humans; D008322:Mammals; D010865:Pilot Projects; D021381:Protein Transport / Q000502:physiology*; D034622:RNA Interference; D034741:RNA, Small Interfering; D049490:Systems Biology / Q000379:methods*
D034741:RNA, Small Interfering