Dumas F et al. (2010), Spatial regulation of membrane fusion controlle...

ECB-ART-41747

PLoS One 2010 Aug 17;58:e12208. doi: 10.1371/journal.pone.0012208.

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Spatial regulation of membrane fusion controlled by modification of phosphoinositides.

Dumas F , Byrne RD , Vincent B , Hobday TM , Poccia DL , Larijani B .

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Membrane fusion plays a central role in many cell processes from vesicular transport to nuclear envelope reconstitution at mitosis but the mechanisms that underlie fusion of natural membranes are not well understood. Studies with synthetic membranes and theoretical considerations indicate that accumulation of lipids characterised by negative curvature such as diacylglycerol (DAG) facilitate fusion. However, the specific role of lipids in membrane fusion of natural membranes is not well established. Nuclear envelope (NE) assembly was used as a model for membrane fusion. A natural membrane population highly enriched in the enzyme and substrate needed to produce DAG has been isolated and is required for fusions leading to nuclear envelope formation, although it contributes only a small amount of the membrane eventually incorporated into the NE. It was postulated to initiate and regulate membrane fusion. Here we use a multidisciplinary approach including subcellular membrane purification, fluorescence spectroscopy and Förster resonance energy transfer (FRET)/two-photon fluorescence lifetime imaging microscopy (FLIM) to demonstrate that initiation of vesicle fusion arises from two unique sites where these vesicles bind to chromatin. Fusion is subsequently propagated to the endoplasmic reticulum-derived membranes that make up the bulk of the NE to ultimately enclose the chromatin. We show how initiation of multiple vesicle fusions can be controlled by localised production of DAG and propagated bidirectionally. Phospholipase C (PLCgamma), GTP hydrolysis and (phosphatidylinsositol-(4,5)-bisphosphate (PtdIns(4,5)P(2)) are required for the latter process. We discuss the general implications of membrane fusion regulation and spatial control utilising such a mechanism.

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Genes referenced: LOC100888919 LOC115919910

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	Figure 1. GTP-induced fusion is bi-polarised.(A) S10s containing total MVs (MV0) were independently labelled with either BODIPY-C12 (donor) or diIC12 (acceptor) and mixed together. Sperm nuclei and ATP-GS (ATP) were added. Nuclear envelope remnants of the nuclei were pre-labelled with hydroxycoumarin. Epifluorescence patterns of labelled nuclei with bound MVs were visualised by phase contrast and two-photon fluorescence microscopy using a 100X objective. MVs were bound around the entire periphery of the nucleus. The nuclear envelope remnants mark the former apex and base of the sperm nucleus (white arrowheads). Fluorescence lifetime of BODIPY was measured before (tâ€Š=â€Š0) and after (tâ€Š=â€Š5, 15, 30, 45 and 60 minutes) the induction of NE formation by photo activation of caged-GTP. (B) Quantification of FRET FLIM images. For analyses, nuclei were divided in four quadrants: p1 and p2 correspond to the poles of the nuclei that include NER while e1 and e2 correspond to the equatorial regions. The averaged mean lifetime was for each quadrant was plotted for each time point showing that MVs fusion is initiated in the polar quadrants and propagates toward the equator. Errors bars correspond to the standard deviation from 7 independent experiments.
	Figure 2. 3D view of nuclei visualised by FLIM.(A) Stack measurements of a nucleus: the first image corresponds to the fluorescence lifetime of the BODIPY measured at the top of a nucleus 15 minutes after photo-activation of caged GTP. Ten successive layers of the same nucleus were obtained. For each layer the focus of the objective was moved 0.25 Âµm along the Z-axis. Since the acquisition of one image lasts for 1 minute, the last image corresponding to the bottom of the nucleus was measured 25 minutes after the induction of NE formation. (B) 3D reconstructions from the Z-stacks of the same nucleus to form fluorescence lifetime 3D views. The indicated times correspond to the time elapsed after photo activation when the first image of each stack was measured.
	Figure 3. Inhibition of PLC prevents membrane fusion.The same experiment as in Fig. 2 was carried out in the presence of 30 ÂµM U73122, a specific PLC inhibitor. The images (A) and lifetime graph (B) show complete inhibition of MV fusion. Data representative of 3 independent experiments
	Figure 4. DAG is required for vectorial progression of fusion.(A) The same experiments as in Fig. 2 were performed using non-ER vesicles (MV1) labelled with BODIPY-C12 and ER vesicles (MV2) labelled with diIC12. Membrane fusion induces both a decrease of the lifetime and a spreading of BODIPY-C12 all around the nucleus. (B) Same experiment as in Fig. 4A carried out in the presence of 30 ÂµM of U73122, indicating that fusion of the non-ER with the ER membranes requires PLC activity. (C) Same experiment as Fig. 4A using SKIP pre-treated MV1 vesicles. Dephosphorylation of PtdIns(4,5)P2 to PtdIns(4)P inhibits fusion.

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