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First published online 31 July 2007
doi: 10.1242/jcs.010181

Journal of Cell Science 120, 2895-2903 (2007)
Published by The Company of Biologists 2007
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NSF- and SNARE-mediated membrane fusion is required for nuclear envelope formation and completion of nuclear pore complex assembly in Xenopus laevis egg extracts

Tina Baur1, Kristijan Ramadan1, Andreas Schlundt1, Jürgen Kartenbeck2 and Hemmo H. Meyer1,*

1 Institute of Biochemistry, ETH Zurich, CH-8093 Zurich, Switzerland
2 Institute of Applied Physics, ETH Zurich, CH-8093 Zurich, Switzerland


Figure 1
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  		Fig. 1. NSF is required for NE formation. (A) NEs were formed by incubating Xenopus laevis egg cytosol and membranes with demembranated sperm chromatin. Reactions were performed in the presence of 1.2 µM (hexamer) bacterially expressed wild-type NSF or the dominant-negative E329Q variant, or NSF buffer as control. After 90 minutes, the samples were fixed. Chromatin and membranes were stained with 4',6-diamidino-2-phenylindole (DAPI) and 3,3'-dihexyloxacarbocyanine iodide (DiOC6), respectively, and imaged by confocal fluorescence microscopy. Bar, 5 µm. (B) The percentage of chromatin particles with closed NEs (CNE) was determined visually by light microscopy in reactions performed as in A. Shown are means from three independent experiments (n=3) with >50 particles counted in each (error bars indicate ± s.d.).

 

Figure 2
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  		Fig. 2. (A,B) Reactions were performed as in Fig. 1 after addition of either 7 µg/µl affinity-purified antibodies raised against Xenopus laevis NSF, purified preimmune IgG or antibody buffer as indicated. Samples were fixed and imaged as in Fig. 1. The percentages of CNE were determined as in Fig. 1B. Bar, 5 µm. (C) Equal fractions of total egg extract, cytosol and light membranes were analyzed by western blotting with either preimmune or anti-NSF serum. Migration of molecular mass markers in kDa is indicated. (D) Xenopus laevis egg cytosol was immunodepleted with anti-xNSF or preimmune (mock) antibodies. Depletion efficiency was analyzed by western blotting as indicated with Nup107 as control. (E,F) Membranes were either incubated without ATP for 5 minutes at 19°C to inactivate membrane-bound NSF (treated) or mock treated (untreated). Reactions were performed for 90 minutes with treated or untreated membranes in combinations with either mock- or NSF-depleted cytosol as indicated. Imaging and quantification was performed as in Fig. 1A and Fig. 1B, respectively. Note that only the combination of NSF inactivation on membranes and NSF depletion from cytosol inhibits envelope formation. Bar, 5 µm. (G,H) ER formation assays with egg extracts were performed in flow chambers in the presence of 7 µg/µl purified preimmune IgG or anti-NSF antibodies, or 1.2 µM NSFwt or NSFE329Q for 30 minutes. Samples were carefully washed, stained with DiOC6 and imaged by epifluorescence microscopy. ER formation was quantified by counting the number of three-way junctions per area. Results are presented as percentage of control. All results are presented as means (n=3, error bars indicate ± s.d.).

 

Figure 3
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  		Fig. 3. NSF and p97 are required throughout NE formation. (A) The dominant-negative mutant p97{Delta}D2 inhibits NE formation specifically. NE formation reactions were performed in egg extract in the presence of buffer, or 12.5 µM (hexamer) of p97{Delta}D2 or the double-mutant protein p97{Delta}D2-K251A. The percentage of closed NE was determined after 75 minutes (n=3, error bars indicate ± s.d.). (B-D) Staging experiments. Three sets of NE formation reactions were performed in parallel. From the first, samples were taken, fixed and stained at indicated time points. Representative intermediates were imaged by confocal microscopy and presented as maximum intensity projections (B), and the percentage of closed NEs was determined (C). The other two sets were supplemented with the dominant-negative mutants NSFE329Q (2.3 µM) or p97{Delta}D2 (12.5 µM), respectively, at the indicated times. The reactions were further incubated for a total of 75 minutes, then fixed and the percentage of CNE determined (D). Note that the reactions remained sensitive to addition of the mutants until completion of NE formation. Results are presented as means (n=3, error bars indicate ± s.d.).

 

Figure 4
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  		Fig. 4. NE formation is SNARE-dependent. (A) Nuclear formation reactions were performed in egg extracts. Reactions contained either buffer or 37.5 µM or 100 µM exogenous, bacterially expressed {alpha}-SNAPwt, {alpha}-SNAPL294A or the {alpha}-SNAP{Delta}N80 variant that lacks the SNARE-binding site. Samples were processed and imaged as in Fig. 1A. Note that only the full-length variants with SNARE-binding activity blocked NE formation. Bar, 5 µm. (B) Quantification of three experiments performed as in Fig. 1B. Results are presented as percentage of control. (C) Maximum intensity projections of confocal sections taken as in Fig. 1A. Note that excess {alpha}-SNAPL294A efficiently blocked the formation of tubules or cisternae on the chromatin surface. (D) Nuclear formation reactions were performed in the presence of 50 µM wild-type or L294A {alpha}-SNAP for 45 minutes. Samples were taken and fixed (0 minutes). The remaining reaction was split and either diluted 1:2 in fresh cytosol (+ dilution) or not (– dilution) and incubated for a further 120 minutes, followed by fixation. NE membranes were imaged as before. Note that the effect of both {alpha}-SNAP variants was equally well restored with fresh cytosol, which was confirmed by quantification (data not shown). (E,F) ER formation was performed for 60 minutes in the presence of 70 µM {alpha}-SNAPL294A or buffer and quantified as in Fig. 1E. Bar, 10 µm. All results are presented as means (n=3, error bars indicate ± s.e.m.).

 

Figure 5
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  		Fig. 5. Ultrastructural analysis. Nuclear formation was performed as before with cytosol and light membranes. Reactions were run with mock-treated membranes along with either buffer (control) or in the presence of 100 µM {alpha}-SNAP. Shown are results for {alpha}-SNAPL294A that were identical to {alpha}-SNAPwt (data not shown). In parallel, a reaction was performed with membranes pretreated with NEM, but without exogenous {alpha}-SNAP (NEM). Samples were fixed, processed and imaged by transmission electron microscopy. Note the flattened cisternae and nuclear pores (arrowheads) in control reactions and the tight attachment of vesicles (arrows) upon inhibition with {alpha}-SNAP, but not with NEM. Bars, 500 nm.

 

Figure 6
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  		Fig. 6. Block of membrane fusion arrests NPC assembly at a defined stage. (A) Nuclear formation assays were performed as before with extract and sperm chromatin. Reactions were run after addition of buffer or 100 µM {alpha}-SNAPL294A with or without sperm chromatin, as indicated. After 60 minutes, reactions were diluted, chromatin recovered by centrifugation through a sucrose cushion and analyzed by western blotting with antibodies specific to proteins indicated on the right. (B) The experiment as shown in A was repeated, but this time including a reaction lacking membranes. Nup107 was detected as indicated. LAP2 served as a membrane marker and the DNA-binding protein MCM3 as a control for equal chromatin recovery. (C) Samples from an identical experiment to that in A were fixed. Nup107- and FxFG-containing nucleoporins were detected by immunofluorescence microscopy using Nup107-specific or mAb414 antibodies, respectively, as indicated.

 

Figure 7
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  		Fig. 7. RanQ69L addition cannot overcome the arrest of NPC assembly caused by inhibition of membrane fusion. Sperm chromatin was incubated with the 200,000 g supernatant of egg extract. Reactions were performed after addition of buffer, 5 µM RanQ69L, RanQ69L plus 100 µM {alpha}-SNAPL294A or RanQ69L plus 0.1% Triton X-100, as indicated. Samples were fixed and chromatin, membranes and FxFG-containing nucleoporins were visualized with DAPI, DiOC6 or by fluorescently labeled mAb414 antibody, respectively. Note the increased recruitment of membrane and FxFG-nucleoporin in the presence of RanQ69L, but not with RanQ69L in combination with detergent or excess {alpha}-SNAP.

 

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© The Company of Biologists Ltd 2007