title: PLCgamma is enriched on poly-phosphoinositide-rich vesicles to control nuclear envelope assembly.
abstract: Nuclear envelope assembly is an essential event in each cell cycle but the proteins and lipids involved in its regulation remain mostly unknown. Assembly involves membrane fusions but neither specific SNAREs nor Rab GTPases have been identified in its control. We report that a precursor membrane population (MV1) required for NE assembly has a unique lipid composition consisting prominently of poly-phosphatidylinositides. The lipid composition was determined by adapting HPLC electrospray ionisation tandem mass spectrometry to phosphoinositide analysis, revealing the capacity of this technique to document dynamic lipid transitions of functional importance in natural membrane populations. MV1 is >100-fold enriched in endogenous PLCgamma and >25-fold enriched in the PLC substrate phosphatidylinositol bisphosphate (PtdInsP2) compared to the second membrane population, derived largely from endoplasmic reticulum (ER), that contributes most of the NE. During NE formation PLCgamma becomes transiently phosphorylated at the tyrosine 783 site indicative of its activation. In addition specific inhibition of PLCgamma blocks nuclear envelope formation. In vivo, PLCgamma is concentrated on vesicles of similar size to purified MV1. These associate with nuclei during the period of NE formation and are distinct from ER membranes. The unprecedented concentration of PLCgamma and its substrate PtdInsP2 in a subset of membranes that binds to only two regions of the nucleus, and activation of PLCgamma by GTP during initial stages of NE formation provide a mechanism for temporal control of NE assembly and offer an explanation for how such a process of membrane fusion can be spatially regulated.
authors.affiliation: Cell Biophysics Laboratory, Cancer Research UK (CRUK), London Research Institute, 44 Lincoln's Inn Fields, London, WC2A 3PX, UK.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Byrne
authors.forename: Marie
authors.initials: M
authors.lastname: Garnier-Lhomme
authors.forename: Kevin
authors.initials: K
authors.lastname: Han
authors.forename: Michael
authors.initials: M
authors.lastname: Dowicki
authors.forename: Nick
authors.initials: N
authors.lastname: Michael
authors.forename: Nick
authors.initials: N
authors.lastname: Totty
authors.forename: Vanessa
authors.initials: V
authors.lastname: Zhendre
authors.forename: Aeri
authors.initials: A
authors.lastname: Cho
authors.forename: Trevor R
authors.initials: TR
authors.lastname: Pettitt
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Wakelam
authors.forename: Dominic L
authors.initials: DL
authors.lastname: Poccia
authors.forename: Banafshé
authors.initials: B
authors.lastname: Larijani
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D047331:Lytechinus; D008297:Male; D008969:Molecular Sequence Data; D009685:Nuclear Envelope / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010716:Phosphatidylinositols / Q000737:chemistry / Q000378:metabolism*; D051966:Phospholipase C gamma / Q000378:metabolism*; D010766:Phosphorylation; D013094:Spermatozoa / Q000166:cytology* / Q000378:metabolism; D047270:Strongylocentrotus purpuratus; D053719:Tandem Mass Spectrometry
chemical_list: D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D051966:Phospholipase C gamma