PLCgamma is enriched on poly-phosphoinositide-rich vesicles to control nuclear envelope assembly.
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.
Byrne
Richard D
RD
Cell Biophysics Laboratory, Cancer Research UK (CRUK), London Research Institute, 44 Lincoln's Inn Fields, London, WC2A 3PX, UK.
Garnier-Lhomme
Marie
M
Han
Kevin
K
Dowicki
Michael
M
Michael
Nick
N
Totty
Nick
N
Zhendre
Vanessa
V
Cho
Aeri
A
Pettitt
Trevor R
TR
Wakelam
Michael J
MJ
Poccia
Dominic L
DL
Larijani
Banafshé
B
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
D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D051966:Phospholipase C gamma