Kiseleva E et al. (2007), Reticulon 4a/NogoA locates to regions of high m...

XB-ART-36539

J Struct Biol 2007 Nov 01;1602:224-35. doi: 10.1016/j.jsb.2007.08.005.

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Reticulon 4a/NogoA locates to regions of high membrane curvature and may have a role in nuclear envelope growth.

Kiseleva E , Morozova KN , Voeltz GK , Allen TD , Goldberg MW .

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Reticulon 4a (Rtn4a) is a membrane protein that shapes tubules of the endoplasmic reticulum (ER). The ER is attached to the nuclear envelope (NE) during interphase and has a role in post mitotic/meiotic NE reassembly. We speculated that Rtn4a has a role in NE dynamics. Using immuno-electron microscopy we found that Rtn4a is located at junctions between membranes in the cytoplasm, and between cytoplasmic membranes and the outer nuclear membrane in growing Xenopus oocyte nuclei. We found that during NE assembly in Xenopus egg extracts, Rtn4a localises to the edges of membranes that are flattening onto the chromatin. These results demonstrate that Rtn4a locates to regions of high membrane curvature in the ER and the assembling NE. Previously it was shown that incubation of egg extracts with antibodies against Rtn4a caused ER to form into large vesicles instead of tubules. To test whether Rtn4a contributes to NE assembly, we added the same Rtn4a antibody to nuclear assembly reactions. Chromatin was enclosed by membranes containing nuclear pore complexes, but nuclei did not grow. Instead large sacs of ER membranes attached to, but did not integrate into the NE. It is possible therefore that Rtn4a may have a role in NE assembly.

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Species referenced: Xenopus
Genes referenced: sacs

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References [+] :

Audhya, A role for Rab5 in structuring the endoplasmic reticulum. 2007, Pubmed, Xenbase

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	Fig. 1. Membrane structures associated with stage III oocyte NEs have Rtn4a at their membrane-membrane junction. (a and b) feiSEM of strings of vesicle-like structures at the NE showing 20 nm bridges (arrows) and ribosomes (arrowheads). (c) TEM of strings of vesicle-like structure at the NE showing bridges (arrows). (d and e) Rtn4a immuno-gold labelling of strings of vesicle-like structures at the connecting tubule. (f) Some vesicle-like structures have dense labelling of Rtn4a more evenly distributed over the surface. (g and h) Localisation of Rtn4a at the junction between larger membrane structures. (i) TEM sections of oocyte cytoplasmic membranes immuno-gold labelled for Rtn4a (arrows). (j) Our interpretation of these structures and the localisation of Rtn4a. Circles mark the position of immuno-gold particles determined by superimposing a simultaneously obtained backscatter electron image (see Section 4.1).
	Fig. 2. Immuno-gold labelling shows Rtn4a localises to NE associated membranes. (a) FeiSEM image of the surface of a stage III oocyte NE showing rough ribosome containing vesicles (RV), smooth vesicles (SV) and flattened membrane (FM). The position of anti-Rtn4a immuno-gold particles detected using a backscatter detector is indicated by circles. (b–d) Rtn4a localises to regions of contact between larger membrane structures and the ONM. (e) ER protein, ribophorin, has a more even distribution over flattened membranes, vesicles and the outer nuclear membrane.
	Fig. 3. Flattened membranes at the ONM of stage III oocyte. (a–c) Attached membranes with different inferred degrees of flattening: (a) is attached but not flattened; (b) is flattened with a few apparent points of fusion (arrows) and (c) is more integrated in the ONM and NPCs (arrowheads) are present around the edges (d–f) Immuno-gold labelling of Rtn4a in flattened membranes showing edge position of Rtn4a (circles). Scale bar in (c) refers to (a–c), scale bar in (f) refers to (d–f).
	Fig. 4. Thin section TEM of NE with cytoplasmic membranes attached. Membranes may be continuous with the ONM (black arrows) or attached but apparently not continuous (white arrows).
	Fig. 5. Rtn4a locates to the edges of flattened membranes. The average number of gold particles on each flattened membrane structure that are located less than 30 nm from the edge was compared to those located more than 30 nm from its edge. This was compared to the distribution of the ER protein, ribophorin. Bars represent standard error of the mean.
	Fig. 6. Membranes flattening onto chromatin during the early (a, b and d) stages of NE assembly in Xenopus egg extracts and labelled with antibodies to Rtn4a (a–c) or ribophorin (antibody CEL5C), marked by circles (d). (c) Fully assembled NE labelled for Rtn4a. (d) Quantification of gold labels for the two antibodies plotted as a distance from the edge. Scale bar in (d) refers to (b–d).
	Fig. 7. Rtn4a antibody perturbs NE growth in Xenopus egg extract. The antibody prevents the usual formation of ER tubules (a, arrows), instead forming large vesicles (b). In control nuclear assembly reactions (c) nuclei are large and roughly round, whereas in the presence of the antibody they remain sperm shaped with large membrane extensions (arrows) (d–g). (h) Nuclear growth is significantly retarded by the Rtn4a antibody as shown by quantification of the two-dimensional area of nuclei observed by feiSEM. (i) High magnification image of nucleus assembled in the presence of Rtn4a antibody, showing NPCs (black arrows) assembled on the sperm chromatin shaped region and the NPC-free ER-like extension with ribosomes (arrowheads). White arrows indicate the junction between the chromatin bound NE and the ER-like extension. Scale bar in (a) applies to (a and b). Scale bar in (f) applies to (c–g).