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Vol. 19, Issue 1, 237-247, January 2008

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Expansion of the Nucleoplasmic Reticulum Requires the Coordinated Activity of Lamins and CTP:Phosphocholine Cytidylyltransferase

Karsten Gehrig^*, Rosemary B. Cornell, and Neale D. Ridgway^*

*Departments of Pediatrics, and Biochemistry and Molecular Biology, Atlantic Research Centre, Dalhousie University, Halifax, NS, Canada B3H 4H7; and Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada V5A 1S6

Submitted February 27, 2007; Revised October 1, 2007; Accepted October 17, 2007
Monitoring Editor: Jennifer Lippincott-Schwartz

The nucleoplasmic reticulum (NR), a nuclear membrane network implicated in signaling and transport, is formed by the biosynthetic and membrane curvature-inducing properties of the rate-limiting enzyme in phosphatidylcholine synthesis, CTP:phosphocholine cytidylyltransferase (CCT) . The NR is formed by invagination of the nuclear envelope and has an underlying lamina that may contribute to membrane tubule formation or stability. In this study we investigated the role of lamins A and B in NR formation in response to expression and activation of endogenous and fluorescent protein-tagged CCT. Similarly to endogenous CCT, CCT-green fluorescent protein (GFP) reversibly translocated to nuclear tubules projecting from the NE in response to oleate, a lipid promoter of CCT membrane binding. Coexpression and RNA interference experiments revealed that both CCT and lamin A and B were necessary for NR proliferation. Expression of CCT-GFP mutants with compromised membrane-binding affinity produced fewer nuclear tubules, indicating that the membrane-binding function of CCT promotes the expansion of the NR. Proliferation of atypical bundles of nuclear membrane tubules by a CCT mutant that constitutively associated with membranes revealed that expansion of the double-bilayer NR requires the coordinated assembly of an underlying lamin scaffold and induction of membrane curvature by CCT.

Address correspondence to: Neale D. Ridgway (nridgway{at}dal.ca)

Abbreviations used: CCT, CTP:phosphocholine cytidylyltransferase; CPT, choline phosphotransferase; ConA, concanavalin A; DAG, diacylglycerol; DsRed, discosoma red fluorescent protein; DiOC6, 3,3'-dihexyloxacarbocyanine iodide; GFP, green fluorescent protein; NE, nuclear envelope; NPC, nuclear pore complex; NR, nucleoplasmic reticulum; PtdCho, phosphatidylcholine; RNAi, RNA interference; siRNA, short interfering RNA.