spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 12 February 2008
doi: 10.1242/jcs.019968

Journal of Cell Science 121, 706-716 (2008)
Published by The Company of Biologists 2008
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Salpingidou, G.
Right arrow Articles by Hutchison, C. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Salpingidou, G.
Right arrow Articles by Hutchison, C. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

NEP-A and NEP-B both contribute to nuclear pore formation in Xenopus eggs and oocytes

Georgia Salpingidou1, Ryszard Rzepecki1,2, Elena Kiseleva3, Carol Lyon4, Birgit Lane4, Kasia Fusiek1,2, Anja Golebiewska1,2, Shoena Drummond5, Terry Allen5, Juliet A. Ellis6, Carl Smythe7, Martin W. Goldberg1 and Christopher J. Hutchison1,*

1 Integrative Cell Biology Laboratories, School of Biological and Biomedical Sciences, The University of Durham, South Road, Durham DH1 3LE, UK
2 Laboratory of Nuclear Proteins, University of Wroclaw, Przybyszewskiego 63/77, 51–148 Wroclaw, Poland
3 Department of Morphology and Function of Cell Structure, Institute of Cytology and Genetics, Russian Academy of Sciences, Novosibirk-90, 630090, Russia
4 School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 4HN, UK
5 CRUK, Department of Structural Cell Biology, Paterson Institute for Cancer Research, Christie Hospital, Wilmslow Road, Manchester M20 9BX, UK
6 The Randall Division of Cell and Molecular Biophysics, Kings College, New Hunts House, Guy's Campus, London SE1 1UL, UK
7 Centre for Developmental Genetics, Department of Biomedical Sciences, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK


Figure 1
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 1. Characterisation of novel markers of NEP-A and NEP-B. (a) Affinity-purified antisera against XLAP2 were used to immunoblot Xenopus egg extracts (LSS) or cell extracts from Xenopus tissue culture cells (XTCs). Molecular mass standards are shown to the right of each blot. (b) Alternatively, XTC cells were prepared for immunofluorescence and stained with antibody against XLAP2 followed by TRITC-labelled goat anti-rabbit antibody and counter stained with DAPI. (c,d) NEP-A, NEP-B and membrane-free cytosol were resolved by 10% SDS-PAGE and either stained with Coomassie Blue (panel c) or immunoblotted (panel d) for the presence of CEL5C, CEL13A, LAP12, CEL1FF or XLAP2. M, molecular weight markers; MP1, NEP-A vesicles; MP2, NEP-B vesicles; CYT, membrane-free cytosol.

 

Figure 2
View larger version (196K):
[in this window]
[in a new window]

  		Fig. 2. Association of membrane vesicles with manually isolated stage III-IV GV envelopes. Whole GVs were manually isolated from stage III-IV Xenopus oocytes and prepared for either feSEM microscopy (a,c) or thin-section TEM (b). Arrowheads show large vesicles and arrows show small smooth vesicles in panel a. In thin-section TEM, vesicles that were close to (arrow), attached to (arrowheads) or fused with the ONM (*) are shown in panel b. NPC intermediates (arrowheads) that appear at high density in the vicinity of areas of membrane fusion are illustrated in panel c. Samples were prepared for immunogold labelling with either CEL13A (NEP-A, panels d,e) or antibody against XLAP2 (NEP-B, panels f,g). Panels d and f show the ONM, whereas panels e and g show the INM. In the micrographs, individual gold particles (appearing black) have been artificially highlighted with white rings to improve their visibility. CEL13A decorated a range of vesicles varying in diameter from 100–500 nm, as well as the ONM. Antibodies against XLAP2 decorated the INM and to some extent the ONM but did not decorate any of the vesicles. Bars, 1000 nm (a); 200 nm (b); 100 nm (c-g).

 

Figure 3
View larger version (108K):
[in this window]
[in a new window]

  		Fig. 3. NPCs form at boundaries between rough and smooth membranes. Sperm chromatin was preincubated with NEP-B and cytosol before addition of NEP-A. Assembling sperm pronuclei were isolated and prepared for feSEM microscopy. (a-c) Each micrograph shows examples of the morphology of fusing vesicles observed five minutes after addition of NEP-A. At very high frequency, membranes with dense concentrations of ribosomes (labelled `dense') can be seen to be joined to membranes with sparse concentrations of ribosomes (`sparse'), and these are interpreted as initial fusion boundaries between single NEP-A and NEP-B vesicles. NPCs (arrows) and NPC assembly intermediates (arrowheads) were observed at the boundaries between dense and sparse areas of membranes. Bars, 100 nm. (d) Quantification of the position of NPC assembly was performed by measuring the distance from the nearest fusion seam of >300 randomly selected NPC intermediates in three independent experiments. The graph shows the percentage of NPC intermediates that were either at a fusion seam, within 200 µm of a fusion seam or >200 µm from a fusion seam. Bars, ±s.e.m.

 

Figure 4
View larger version (36K):
[in this window]
[in a new window]

  		Fig. 4. Inhibition of NE assembly by human emerin constructs. (a) NEP-A (labelled `-A'), NEP-B (labelled `-B') and cytosol were immunoblotted with antibodies against gp210, POM121. Molecular mass markers are indicated on the left-hand side. (b,c) Human emerin peptides 1-70 or 73-180 were added to Xenopus egg extracts at concentrations ranging from 0.5 µM to 8.0 µM. The ability of extracts to assemble sperm pronuclei in the presence of each peptide was assessed by either immunoblotting assays (b) or immunofluorescence assays (c). Here, mAb 4G12 was used to detect NEP-B and mAb CEL13A was used to detect NEP-A in immunofluorescence assays, whereas CEL1FF was used to detect NEP-B, and LAP12 was used to detect NEP-A in immunoblotting assays. CEL5C was used to detect total membranes. Bar, 10 µm.

 

Figure 5
View larger version (38K):
[in this window]
[in a new window]

  		Fig. 5. Inhibition of NPC assembly by human emerin constructs. Human emerin peptides 1-70 (a,c) and 73-180 (b) were added to Xenopus egg extracts at concentrations between 0.5 and 8.0 µM. Alternatively, no peptides were added to extracts in order to measure normal assembly (labelled `control' in b,c). The effects of the peptides on NPC assembly were investigated by immunofluorescence using antibodies against FG-repeat nucleoporins (a,b) or Nup107 (c). Bars, 10 µm. (d) Alternatively, nuclei were assembled in extracts containing 4.0 µM peptide 73-180 (lane 1) or peptide 1-70 (lane 3). Nuclei were isolated and prepared for immunoblotting using antibodies against FG-repeat nucleoporins. Lane 2 contains material recovered from extracts containing no nuclei.

 

Figure 6
View larger version (28K):
[in this window]
[in a new window]

  		Fig. 6. Preferential depletion of Nup153 in nuclei assembled in the presence of peptide 1-70. Nuclei were assembled in egg extracts containing 4.0 µM peptides 1-70 or 73-180 and prepared for immunofluorescence. Nuclei were stained with antibodies against Nup153 (a) or Nup214 (b) followed by FITC-labelled goat anti-mouse antibody and counter stained with DAPI. Images are presented as mono images or two-colour merged images. Bar, 10 µm.

 

Figure 7
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 7. Depletion of NEP-A leads to assembly of nuclei missing components of the nucleoplasmic ring of NPCs. Fractionated extracts of Xenopus eggs were reconstituted with normal amounts of NEP-A and NEP-B (labelled `10') or with an amount such that NAP-A was depleted by a factor of ten relative to NEP-B (labelled `1'). Nuclei were prepared for either immunofluorescence with antibodies against FG-repeat nucleoporins 414 (a), Nup153 (b) and Nup214 (c) or immunoblotting (d) with antibodies against FG-repeat nucleoporins. Micrographs are presented as individual mono images in which the first image in each pair shows DAPI staining or as two-colour merged images. Bar, 10 µm.

 

Figure 8
View larger version (128K):
[in this window]
[in a new window]

  		Fig. 8. Analysis by feSEM of NEs and NPCs assembled in the presence of emerin peptides. Sperm pronuclei were assembled in either the absence (labelled `control' in a,b) or presence of 4.0 µM emerin peptide 73-180 (c,d) or emerin peptide 1-70 (e,f). Samples were prepared for feSEM microscopy and examined at either low (a,c,e) or high (b,d,f) magnification. Ribosomes are indicated by arrows. Apparently intact fully formed NPCs are indicated by white circles, but these are rare in panel f, where more-abnormal structures predominate (black circles). Bars, 2000 nm (a,c,e); 100 nm (b,d,f).

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2008