RNAi screening reveals a large signaling network controlling the Golgi apparatus in human cells

Molecular Systems Biology

Joanne Chia¹  Germaine Goh¹  Victor Racine¹  Susanne Ng¹  Pankaj Kumar¹ 

[1] Laboratory Regulation of Membrane Traffic, Institute of Molecular and Cell Biology, Singapore, Singapore

关键词: glycosylation;    Golgi;    imaging;    RNAi screening;    signaling;   

DOI  :  10.1038/msb.2012.59

来源: Wiley

【 摘 要 】

Abstract

The Golgi apparatus has many important physiological functions, including sorting of secretory cargo and biosynthesis of complex glycans. These functions depend on the intricate and compartmentalized organization of the Golgi apparatus. To investigate the mechanisms that regulate Golgi architecture, we developed a quantitative morphological assay using three different Golgi compartment markers and quantitative image analysis, and performed a kinome- and phosphatome-wide RNAi screen in HeLa cells. Depletion of 159 signaling genes, nearly 20% of genes assayed, induced strong and varied perturbations in Golgi morphology. Using bioinformatics data, a large regulatory network could be constructed. Specific subnetworks are involved in phosphoinositides regulation, acto-myosin dynamics and mitogen activated protein kinase signaling. Most gene depletion also affected Golgi functions, in particular glycan biosynthesis, suggesting that signaling cascades can control glycosylation directly at the Golgi level. Our results provide a genetic overview of the signaling pathways that control the Golgi apparatus in human cells.

Synopsis

RNAi screening and automated image analysis reveal 180 kinases and phosphatases regulating the organization of the Golgi apparatus. Most of these genes also control the expression of specific glycans, pointing to a web of interactions between signaling cascades and glycosylation at the Golgi.

• Golgi organization was probed with three markers of different Golgi compartments and quantitative morphological analysis.
• Knockdowns of ∼20% of all known kinases and phosphatases affected the Golgi globally or in a compartment-specific manner, and were comparable in degree to the depletion of known membrane traffic regulators such as SNAREs.
• Several cell surface receptors, their cognate ligands and downstream effectors regulate Golgi organization, suggesting a large regulatory network.
• Most signaling genes affected both Golgi morphology and the expression of specific glycans.

【 授权许可】

CC BY-NC-SA   
Copyright © 2012 EMBO and Macmillan Publishers Limited

Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation without specific permission.