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First published online 17 July 2006
doi: 10.1242/jcs.03058

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Dynamic and reversible restructuring of the ER induced by PDMP in cultured cells

Teresa Sprocati¹, Paolo Ronchi¹, Andrea Raimondi^1,*, Maura Francolini¹ and Nica Borgese^1,2,

¹ Consiglio Nazionale delle Ricerche Institute of Neuroscience and Department of Medical Pharmacology, via Vanvitelli 32, University of Milano, 20129 Milano, Italy
² Department of Pharmacobiological Science, `Magna Graecia' University of Catanzaro, 88100 Catanzaro, Italy

Author for correspondence (e-mail: n.borgese{at}in.cnr.it)

Accepted 18 May 2006

In many cells, the endoplasmic reticulum (ER) contains segregated smooth and rough domains, but the mechanism of this segregation is unclear. Here, we used a HeLa cell line, inducibly expressing a GFP fusion protein [GFP-b(5)tail] anchored to the ER membrane, as a tool to investigate factors influencing ER organisation. Induction of GFP-b(5)tail expression caused proliferation of the ER, but its normal branching polygonal meshwork architecture was maintained. Experiments designed to test the effects of drugs that alter ceramide levels revealed that treatment of these cells with Phenyl-2-decanoyl-amino-3-morpholino-1-propanol-hydrocholride (PDMP) generated patches of segregated smooth ER, organised as a random tubular network, which rapidly dispersed after removal of the drug. The effect of PDMP was independent of its activity as sphingolipid synthesis inhibitor, but could be partially reversed by a membrane-permeant Ca²⁺ chelator. Although the smooth ER patches maintained connectivity with the remaining ER, they appeared to represent distinct domains differing in protein and lipid composition from the remaining ER. PDMP did not cause detachment of membrane-bound ribosomes, indicating that smooth ER patch generation was due to a reorganisation of pre-existing ribosome-free areas. Our results demonstrate a dynamic relationship between smooth and rough ER and have implications for the mechanisms regulating ER architecture.

Key words: HeLa TetOff cells, Membrane proliferation, Rough ER, Smooth ER, Tail-anchored GFP

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