Acute depletion of diacylglycerol from the cis-Golgi affects localized nuclear envelope morphology during mitosis
2018; Elsevier BV; Volume: 59; Issue: 8 Linguagem: Inglês
10.1194/jlr.m083899
ISSN1539-7262
AutoresGary H. C. Chung, Marie‐Charlotte Domart, Christopher J. Peddie, Judith Mantell, Kieran Mclaverty, Angela Arabiotorre, Lorna Hodgson, Richard Byrne, Paul Verkade, Kenton P. Arkill, Lucy Collinson, Banafshé Larijani,
Tópico(s)Microtubule and mitosis dynamics
ResumoDysregulation of nuclear envelope (NE) assembly results in various cancers; for example, renal and some lung carcinomas ensue due to NE malformation. The NE is a dynamic membrane compartment and its completion during mitosis is a highly regulated process, but the detailed mechanism still remains incompletely understood. Previous studies have found that isolated diacylglycerol (DAG)-containing vesicles are essential for completing the fusion of the NE in nonsomatic cells. We investigated the impact of DAG depletion from the cis-Golgi in mammalian cells on NE reassembly. Using advanced electron microscopy, we observed an enriched DAG population of vesicles at the vicinity of the NE gaps of telophase mammalian cells. We applied a mini singlet oxygen generator-C1-domain tag that localized DAG-enriched vesicles at the perinuclear region, which suggested the existence of NE fusogenic vesicles. We quantified the impact of Golgi-DAG depletion by measuring the in situ NE rim curvature of the reforming NE. The rim curvature in these cells was significantly reduced compared with controls, which indicated a localized defect in NE morphology. Our novel results demonstrate the significance of the role of DAG from the cis-Golgi for the regulation of NE assembly. Dysregulation of nuclear envelope (NE) assembly results in various cancers; for example, renal and some lung carcinomas ensue due to NE malformation. The NE is a dynamic membrane compartment and its completion during mitosis is a highly regulated process, but the detailed mechanism still remains incompletely understood. Previous studies have found that isolated diacylglycerol (DAG)-containing vesicles are essential for completing the fusion of the NE in nonsomatic cells. We investigated the impact of DAG depletion from the cis-Golgi in mammalian cells on NE reassembly. Using advanced electron microscopy, we observed an enriched DAG population of vesicles at the vicinity of the NE gaps of telophase mammalian cells. We applied a mini singlet oxygen generator-C1-domain tag that localized DAG-enriched vesicles at the perinuclear region, which suggested the existence of NE fusogenic vesicles. We quantified the impact of Golgi-DAG depletion by measuring the in situ NE rim curvature of the reforming NE. The rim curvature in these cells was significantly reduced compared with controls, which indicated a localized defect in NE morphology. Our novel results demonstrate the significance of the role of DAG from the cis-Golgi for the regulation of NE assembly. The nuclear envelope (NE) is a subcompartment of the endoplasmic reticulum (ER) and it breaks down and reforms during an open mitosis. However, the detailed molecular mechanism regarding its reassembly remains to be determined. Although envelopment of the chromatin by the ER is one of the NE reassembly models (1.Anderson D.J. Hetzer M.W. Reshaping of the endoplasmic reticulum limits the rate for nuclear envelope formation.J. Cell Biol. 2008; 182: 911-924Crossref PubMed Scopus (154) Google Scholar) and contact between ER cisternae and chromosomes has been shown to initiate mammalian NE reassembly at anaphase (2.Lu L. Ladinsky M.S. Kirchhausen T. Formation of the postmitotic nuclear envelope from extended ER cisternae precedes nuclear pore assembly.J. Cell Biol. 2011; 194: 425-440Crossref PubMed Scopus (81) Google Scholar), these descriptions do not explain the necessity of membrane fusion or the possible requirement of correctly formed curved regions for the localization of the nuclear pore complexes. Proteins play an important role in membrane fusion and morphology (3.Martens S. McMahon H.T. Mechanisms of membrane fusion: disparate players and common principles.Nat. Rev. Mol. 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Domart M.C. Collinson L. Poccia D.L. Principle of duality in phospholipids: regulators of membrane morphology and dynamics.Biochem. Soc. Trans. 2014; 42: 1335-1342Crossref PubMed Scopus (5) Google Scholar). The understanding of how this lipid behaves during membrane fusion has mostly resulted from in vitro or reconstitution experiments (10.Das S. Rand R.P. Diacylglycerol causes major structural transitions in phospholipid bilayer membranes.Biochem. Biophys. Res. Commun. 1984; 124: 491-496Crossref PubMed Scopus (91) Google Scholar, 11.Das S. Rand R.P. Modification by diacylglycerol of the structure and interaction of various phospholipid bilayer membranes.Biochemistry. 1986; 25: 2882-2889Crossref PubMed Scopus (205) Google Scholar, 15.Huang W. Jiang D. Wang X. Wang K. Sims C.E. Allbritton N.L. Zhang Q. Kinetic analysis of PI3K reactions with fluorescent PIP2 derivatives.Anal. Bioanal. Chem. 2011; 401: 1881-1888Crossref PubMed Scopus (17) Google Scholar), and its complex behavior has not been directly examined in live cells or model organisms. Our group has isolated a population of membrane vesicles known as MV1, a distinctive membrane compartment in vivo with atypical polyphosphoinositide compositions (16.Byrne R.D. Veeriah S. Applebee C.J. Larijani B. Conservation of proteo-lipid nuclear membrane fusion machinery during early embryogenesis.Nucleus. 2014; 5: 441-448Crossref PubMed Scopus (5) Google Scholar), as one of the critical NE precursors of echinoderm male pronuclei. MV1 has elevated levels of phosphoinositides, a lipid-modifying enzyme, PLCγ, and its upstream regulator, src family kinase 1 (SFK1) (16.Byrne R.D. Veeriah S. Applebee C.J. Larijani B. Conservation of proteo-lipid nuclear membrane fusion machinery during early embryogenesis.Nucleus. 2014; 5: 441-448Crossref PubMed Scopus (5) Google Scholar, 17.Byrne R.D. Garnier-Lhomme M. Han K. Dowicki M. Michael N. Totty N. Zhendre V. Cho A. Pettitt T.R. Wakelam M.J. et al.PLCgamma is enriched on poly-phosphoinositide-rich vesicles to control nuclear envelope assembly.Cell. Signal. 2007; 19: 913-922Crossref PubMed Scopus (37) Google Scholar). These three proteo-lipid components are the fusion "tool-kit" responsible for localized production of DAG. In mammals, DAG localizes at the NE, ER, and Golgi (18.Domart M.C. Hobday T.M. Peddie C.J. Chung G.H. Wang A. Yeh K. Jethwa N. Zhang Q. Wakelam M.J. Woscholski R. et al.Acute manipulation of diacylglycerol reveals roles in nuclear envelope assembly & endoplasmic reticulum morphology.PLoS One. 2012; 7: e51150Crossref PubMed Scopus (50) Google Scholar). Using a rapamycin analog (rapalog)-based heterodimer system (19.Fili N. Calleja V. Woscholski R. Parker P.J. Larijani B. Compartmental signal modulation: Endosomal phosphatidylinositol 3-phosphate controls endosome morphology and selective cargo sorting.Proc. Natl. Acad. Sci. USA. 2006; 103: 15473-15478Crossref PubMed Scopus (81) Google Scholar, 20.Hammond G.R. Fischer M.J. Anderson K.E. Holdich J. Koteci A. Balla T. Irvine R.F. PI4P and PI(4,5)P2 are essential but independent lipid determinants of membrane identity.Science. 2012; 337: 727-730Crossref PubMed Scopus (305) Google Scholar), we have demonstrated the structural role of DAG in mammalian NE reassembly where localized depletion of DAG at the NE and ER results in failure of NE reassembly and abnormal ER morphology (18.Domart M.C. Hobday T.M. Peddie C.J. Chung G.H. Wang A. Yeh K. Jethwa N. Zhang Q. Wakelam M.J. Woscholski R. et al.Acute manipulation of diacylglycerol reveals roles in nuclear envelope assembly & endoplasmic reticulum morphology.PLoS One. 2012; 7: e51150Crossref PubMed Scopus (50) Google Scholar). The abnormal ER morphology and fragmented NE from these experiments resulted in taking our experiments forward to determine whether localized highly curved regions, such as the rim curvature of the NE, would be affected by the acute and inducible depletion of DAG. To examine this, we set out to investigate the impact of DAG depletion from the cis-Golgi, a reservoir of DAG in mammalian cells, on mammalian NE reassembly using the rapalog dimerization approach. Here, we show that locally depleted DAG from the cis-Golgi impacts the NE rim curvature of the reforming NE. The rim curvature in these cells is significantly reduced compared with the controls. We measured the in situ rim curvature and the pore diameters to illustrate the localized defect in NE morphology. These membranous defects may result in affecting the proper nuclear pore insertions. Our results demonstrate the significance of the role of DAG from the cis-Golgi for the regulation of NE assembly. The HeLa cell line was obtained from the ATCC (ATCC #CCL2). The cells were grown in DMEM containing 10% FBS (Gibco) supplemented with penicillin (100 U/ml) and streptomycin (100 μg/ml) and incubated at 37°C in 10% CO2. Golgi reassembly stacking protein 65 (GRASP65)-GFP and its non-Golgi-targeting mutation, GRASP65.G2A-GFP, were gifts from Professor Yan-Zhuang Wang (University of Michigan). GRASP65-GFP-2FKBP and GRASP65.G2A-GFP-2FKBP were created by adding a 2FKBP domain into the given constructs at its C terminus with flanking BsrGI sites. The GRASP65, GFP tag, and 2FKBP rapalog-binding domain were made into such a sequence so that the constructs could successfully localize to the Golgi and recruit DAG kinase εK (DGKεK). GFP-2FKBP-LBR, RFP-Flag-FRB-DGKεK, and RFP-Flag-FRB-DGKεK.D434N were created as described in (18.Domart M.C. Hobday T.M. Peddie C.J. Chung G.H. Wang A. Yeh K. Jethwa N. Zhang Q. Wakelam M.J. Woscholski R. et al.Acute manipulation of diacylglycerol reveals roles in nuclear envelope assembly & endoplasmic reticulum morphology.PLoS One. 2012; 7: e51150Crossref PubMed Scopus (50) Google Scholar). Cells were grown on gridded glass coverslips in 3.5 cm dishes (MatTek Corporation) and double transfected with the rapalog dimerization constructs described above. Cells were transfected with 0.5 μg of the DNA of each construct using Lipofectamine LTX and PLUS reagent (Invitrogen) in OPTIMEM medium (Gibco BRL) as recommended by the manufacturer. The cells were left overnight in the transfection mix in antibiotic-free medium before replacing it with fresh medium. Experiments were performed 36–48 h post transfection. The transfected cells were stained with ER tracker Blue-White DPX (Invitrogen) at a final concentration of 1 μM according to manufacturer's protocol before the rapalog experiment (18.Domart M.C. Hobday T.M. Peddie C.J. Chung G.H. Wang A. Yeh K. Jethwa N. Zhang Q. Wakelam M.J. Woscholski R. et al.Acute manipulation of diacylglycerol reveals roles in nuclear envelope assembly & endoplasmic reticulum morphology.PLoS One. 2012; 7: e51150Crossref PubMed Scopus (50) Google Scholar, 19.Fili N. Calleja V. Woscholski R. Parker P.J. Larijani B. Compartmental signal modulation: Endosomal phosphatidylinositol 3-phosphate controls endosome morphology and selective cargo sorting.Proc. Natl. Acad. Sci. USA. 2006; 103: 15473-15478Crossref PubMed Scopus (81) Google Scholar). Cells were incubated in a 5 l/h humidified chamber with 10% CO2 adapted to a Zeiss confocal microscope (Zeiss LSM 710). Images were acquired prior to and after the recruitment of DAG kinase to the targeted membrane compartment, ∼30 min to 1 h after addition of 500 nM rapalog heterodimerizer (Clontech). Cells were then imaged every 30–45 min at low pixel resolution (512 × 512 with a 0.6× zoom) from interphase to late anaphase to limit laser damage. A high pixel resolution (1,024 × 1,024 with a 2× zoom) series of images was also acquired from late anaphase to cytokinesis. Each experiment was performed at least three times. All images were treated in the same manner, i.e., only minor adjustments of brightness and contrast were applied to every pixel. Live cells were followed to the required stage of mitosis using confocal microscopy. Cells were then fixed in 4% electron microscopy (EM) grade formaldehyde (TAAB) in 0.1 M phosphate buffer (PB) (pH 7.4) to halt the cell cycle prior to reimaging for bright-field and high-magnification fluorescence signals. Cells were subsequently located using the grid number of the gridded glass coverslip (MatTek). Secondary fixation was performed in 1.5% glutaraldehyde/2% formaldehyde in 0.1 M PB for 30–60 min. After fixation, coverslips were carefully removed from the MatTek dishes and washed several times in 0.1 M PB. For transmission EM (TEM), the cells were postfixed in 1.5% potassium ferricyanide/1% osmium tetroxide for 1 h before rinsing in PB and incubating in 1% tannic acid in 0.05 M PB for 45 min to enhance membrane contrast. After a brief rinse in 1% sodium sulfate in 0.05 M PB, the coverslips were washed twice in distilled water, dehydrated through an ascending series of ethanol to 100% prior to infiltration with epoxy resin and polymerization overnight at 60°C. The coverslips were removed from the resin blocks by plunging briefly into liquid nitrogen. The cells of interest were identified by correlating the grid and cell pattern on the surface of the block with previously acquired confocal images. The area of interest was cut from the block and further trimmed by hand using a single edged razor blade to form a small trapezoid block face for serial ultrathin sectioning. Using a diamond knife, serial ultrathin sections of 70 nm thickness were cut through the entire extent of the cells of interest (80–140 sections) and collected on 1.5% Formvar-coated single slot grids. The sections were counterstained with lead citrate to further enhance contrast prior to viewing in the electron microscope (FEI Tecnai G2 Spirit BioTWIN with Gatan Orius CCD camera). Serial images were stacked and aligned, and the NE, ER, and centrioles and vesicles were manually segmented using Amira (FEI), based on their electron density and morphological features. Circular membrane structures with similar x, y, and z diameter were segmented as vesicles. Movies were created from 2D tiff stacks using Quicktime Player 7 Pro and compressed using Stomp (Shinywhitebox Ltd.). For serial block-face scanning EM (SBF SEM), the fixed cells were processed following the method of the National Centre for Microscopy and Imaging Research (21.Deerinck T.J. Bushong E.A. Thor A. Ellisman M.H. NCMIR methods for 3D EM: a new protocol for preparation of biological specimens for serial block face scanning electron microscopy.Microscopy. 2010; Google Scholar), which impregnates the sample with high concentrations of heavy metals to introduce maximal contrast and conductivity when viewed in the scanning electron microscope. The cell of interest was identified by correlation of grid reference with previously acquired confocal images; this area was trimmed to a small trapezoid, excised from the resin block, and attached to a SBF SEM specimen holder using conductive epoxy resin (Circuitworks; CW2400). Prior to commencement of a SBF SEM imaging run, the sample was coated with a 2 nm layer of platinum to further enhance conductivity. SBF SEM data were collected using a 3View2XP (Gatan, Pleasanton, CA) attached to a scanning electron microscope (Zeiss, Cambridge). To relocate the cell of interest in the scanning electron microscope, an overview was first acquired at 5 kV, sufficient to penetrate the platinum coating and generate an image of the underlying sample. Inverted backscattered electron images were then acquired through the entire extent of the cell of interest at a resolution of 8,192 × 8,192 pixels (horizontal frame width of 36.74 μm; pixel size of 4.5 nm) using a 2 μs dwell time and 50 nm slice thickness. The scanning electron microscope was operated in variable pressure mode at 5–10 Pa, with high current mode active, 20 μm aperture, an accelerating voltage of 2 kV, and an indicated magnification of 7,000. Typically, around 400 slices were necessary to image an entire cell, representing a total volume of approximately 27,000 μm3. As data were collected in variable pressure mode, only minor adjustments in image alignment were needed, particularly where the field of view was altered in order to track the cell of interest. For electron tomography (ET), samples were prepared as detailed above, but 200 nm-thick serial sections were collected through the entire extent of the cells of interest. Tomograms were acquired from the 200 nm sections at targeted regions in the reforming NE, either at specific gaps or where collections of vesicles were evident in close proximity to the reforming envelope. Images were collected at 1° intervals across a maximal tilt range of ±70°, with 0.79 nm width per pixel for 2,048 × 2,048 pixels, with a per pixel resolution of 0.79 nm. Tomograms were processed with IMOD (22.Kremer J.R. Mastronarde D.N. McIntosh J.R. Computer visualization of three-dimensional image data using IMOD.J. Struct. Biol. 1996; 116: 71-76Crossref PubMed Scopus (3580) Google Scholar), using patch tracking for alignment and simultaneous iterative reconstruction technique for volume reconstruction. The reconstructed volume was exported as a series of 2D tiff images, and the NE and adjacent membranous structures, including vesicles, were manually segmented and reconstructed using Amira (Visage Imaging, Berlin). Movies were created from the 2D tiff stacks using Quicktime Player 7 Pro, and compressed using Stomp (Shinywhitebox Ltd.). It is of note that one cannot negate embedding artifacts enough to have a definitive measurement of curvature and so cryo-microscopy is preferred; however, the cryo-tomography required is extremely technically challenging and cannot currently be performed for rare correlative events due to the complexity of sample preparation and difficulties in targeting specific cells through the cryo workflow. It should be noted that if this methodology were to be used for a larger study, or under varying imaging conditions, there would be a justification to verify these trends in a case-by-case manner. Serial micrographs were stacked and aligned using Amira (Visage Imaging, Berlin). The pixels of NE, ER, centrioles, and vesicles were manually traced based on their electron density and morphological features on the micrograph. Circular membrane structures with similar xy and z diameter were segmented as vesicles in the TEM analysis of the telophase cell. Discontinuities of the reforming NE around the chromatin were segmented as the NE gaps. Cells were transfected with GFP-PKCεC1aC1b-SOG and light microscopy was performed to identify cells of interest prior to initial fixation with 4% formaldehyde in 0.1 M PB, and secondary fixation with 2.5% glutaraldehyde in 0.1 M PB. The cells were washed in 0.1 M PB and incubated in blocking buffer (50 mM glycine, 10 mM KCN, and 20 mM aminotriazole in 0.1 M PB) for 30 min. The dishes were then loaded into a customized cooling chamber to maintain the samples at ∼4°C. The lid of the chamber was connected to an oxygen supply at a rate of 2.5 l/min. The cells were then incubated in diaminobenzidene solution (5.4 mg in 10 ml 0.1 M PB) and photo-oxidized for 30 min using a Hg lamp, after which they were further fixed on ice with 2% formaldehyde/1.5% glutaraldehyde in 0.1 M PB for 30 min, and postfixed with 0.5% osmium tetroxide in 0.1 M PB for 30 min before embedding as described above for correlative light microscopy and EM. The photo-oxidized cells were relocated using the MatTek grid coordinates and serially sectioned for examination using TEM. Blocks, stained as per the serial block-face method, were instead serial sectioned at 300 nm onto Pioloform-coated slot grids. Fifteen nanometer gold particles (Aurion) were drop cast for 5 min on both sides with the excess blotted off. Micrographs were acquired through the cell and two areas selected on each of the daughter cells. Single tilt tomograms were taken of the selected areas using a 200 KeV transmission electron microscope (T20; FEI) with a LaB6 filament and a 4k × 4k Eagle (FEI) bottom mounted camera. The tilt series was taken, using the FEI software, at 2° intervals between ±60° where possible, at a nominal magnification of 14,500×, giving pixel sizes of 0.74 × 0.74 nm2. The tilt series were reconstructed using IMOD (22.Kremer J.R. Mastronarde D.N. McIntosh J.R. Computer visualization of three-dimensional image data using IMOD.J. Struct. Biol. 1996; 116: 71-76Crossref PubMed Scopus (3580) Google Scholar), with subsequent image analysis using Fiji (23.Schindelin J. Arganda-Carreras I. Frise E. Kaynig V. Longair M. Pietzsch T. Preibisch S. Rueden C. Saalfeld S. Schmid B. et al.Fiji: an open-source platform for biological-image analysis.Nat. Methods. 2012; 9: 676-682Crossref PubMed Scopus (30105) Google Scholar) and statistical analysis using Prism (GraphPad). Tortuosity of the nuclear membranes could not be analyzed quantifiably from the 3D tilt series reconstructions due to the volumetric field of view. Instead, if the membrane curvature has been disrupted, a requirement for pathological tortuosity, it would be expected to be evident at the tight curvatures between the inner and outer membranes, defined here as rim diameter, to be consistent with modeling of the ER (24.Knorr R.L. Dimova R. Lipowsky R. Curvature of double-membrane organelles generated by changes in membrane size and composition.PLoS One. 2012; 7: e32753Crossref PubMed Scopus (45) Google Scholar). To achieve an estimate of any altered curvature, it was decided to quantify the nuclear pores. Two measures were taken (Fig. 4): the diameter of the pore and the distance between the inner and outer nuclear membrane, its rim diameter. Initially, to achieve good membrane contrast, the 3D reconstructed image stacks were reduced by binning by a factor of four (leaving 3 × 3 × 3 nm3 voxels in x-y and z directions), and dynamic range to 8 bit after suitable windowing. Three-dimensional image clips were taken from every nuclear pore completely enclosed in the reconstruction, as described by Fig. 4. Each clip was rotated such that the membranes are parallel to the x axis, then rotated around the x axis by 10° increments until the pore appeared (by eye) as near to a circle as possible. From this point, the clip was rotated back by 90°, making the pore perpendicular to the viewed plan. The width and rim diameter were then measured at a depth of the widest diameter of the pore by measuring the distance between centers of the dark bilayer membranes where they just become parallel. In our previous work on the echinoderm model, we showed that MV1, a vesicular compartment enriched in PLCγ and phosphoinositides, was recruited to each stage of membrane fusion required for NE and zygote formation (17.Byrne R.D. Garnier-Lhomme M. Han K. Dowicki M. Michael N. Totty N. Zhendre V. Cho A. Pettitt T.R. Wakelam M.J. et al.PLCgamma is enriched on poly-phosphoinositide-rich vesicles to control nuclear envelope assembly.Cell. Signal. 2007; 19: 913-922Crossref PubMed Scopus (37) Google Scholar). Based on these results, we predicted that vesicles enriched in phosphoinositides or their derivative, DAG, might also play a role in NE assembly in mammalian cells. To demonstrate the presence of vesicles that might participate in NE completion at late mitotic stages, we investigated the ultrastructure of telophase cells by TEM. Figure 1A and C show that multiple gaps existed at the NE of the examined telophase cells, and vesicles were detected in the proximity of these gaps. ET confirmed that these membrane structures occupied a spherical space in the virtual volume of the tomogram (Fig. 1C, supplemental Movie S1), implying the presence of vesicles as opposed to tubules. Some vesicles were very close to the edge of the reforming NE, as indicated by the reconstructed 3D model (Fig. 1D). However, there were numerous vesicles in a telophase cell. A comprehensive 3D reconstruction revealed at least 14,000 vesicles in one of the daughter cells at this mitotic stage (Fig. 1B). Therefore, we focused on studying the vesicles that were close (500 nm) to the reforming NE because physical contact is required for membrane fusion. To understand how vesicles distributed around the reforming NE of a telophase cell, we compared the concentration of vesicles at the regions with gaps and the regions without gaps (Fig. 2). A gap was defined by the shortest distance between the two converging edges of the reforming NE in the x-direction multiplied by its thickness in the z-direction, not exceeding 140 nm. The size range of the gaps varied within three orders of magnitude; therefore, we grouped the gaps into small (5,800–9,900 nm2), medium (11,000–82,000 nm2), and large (100,000–350,000 nm2), based on their vertical area (x-z) in the micrographs (Fig. 2A). A cylindrical volume was defined as the "vicinity" of different regions of interest and the volume has a radius of 1 μm so that it did not exceed the boundary of the cell. For a gap, one of its ends was selected as the center of the cylindrical volume (Fig. 2B). We compared the concentration of the vesicles in the vicinity with the same volume at different regions. The vesicle concentration at the large-gap regions was significantly higher than that in other regions; whereas at medium-gap-regions and small-gap-regions, the vesicle concentration reduced in relation to the gap size (Fig. 2C). This might imply that the vesicles in the large-gap regions were being utilized to induce membrane fusion of the NE fragments. The mean diameter of all the vesicles in the close field was 57 ± 0.5 nm (Fig. 2D). To identify DAG-enriched vesicles, we exploited the C1a-C1b domain of PKCε and its nonbinding mutant, C1aC1b-W264G. This domain specifically recognizes the subcellular localization of DAG. To illustrate the DAG specificity, the nonbinding mutant was utilized (supplemental Fig. S2). Moreover, to enable the direct recognition of these vesicles at the EM level at the vicinity of the NE, a mini singlet oxygen generator (miniSOG) approach was utilized. The conventional methods (e.g., immunogold) for TEM were inappropriate because the permeabilization step extracted the lipids of interest. The miniSOG tag (25.Shu X. Lev-Ram V. Deerinck T.J. Qi Y. Ramko E.B. Davidson M.W. Jin Y. Ellisman M.H. Tsien R.Y. A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms.PLoS Biol. 2011; 9: e1001041Crossref PubMed Scopus (607) Google Scholar) provided TEM contrast via photo-oxidation. This step released oxygen to polymerize 3,3′-diaminobenzidine (DAB) at the targeted structures, which attracted preferential osmium staining. Figure 3A shows that many vesicles were labeled with GFP-PKCεC1aC1b-SOG reaction products in an interphase cell at the perinuclear region. The mean size of the miniSOG-labeled vesicles was greater due to the formation of the DAB polymer (Fig. 3B). Hence, this demonstrated that the C1a-C1b domain recognized the DAG-enriched vesicles. If the C1 domain did not bind to DAG, the size of the vesicles would remain unchanged. We suggest that these DAG-enriched vesicles are present at interphase for the maintenance of the NE morphology. To investigate the localized and inducible impact of Golgi-DAG depletion on NE reassembly during mitosis, we used the rapalog dimerization system and the Golgi-targeting domain of GRASP65, a cis-Golgi marker expected to be corecruited with
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