Plugs in nuclear pores: Transcripts in early oocyte development identified with nanotechniques

Abstract

Throughout oogenesis, huge amounts of RNA are produced that are needed for early development. Early stages of oocyte development are characterized by high transcriptional activity whereas translation of maternal RNA dominates late stages. Nuclear pore complexes (NPCs), located in the nuclear envelope (NE), mediate bidirectional macromolecule exchange between the nuclear and cytosolic compartments including RNA export. Here, we report on structural correlates of this transport pathway at single NPC level. Using atomic force microscopy (AFM), we imaged the nucleoplasmic ("inner") surface of the NE of Xenopus laevis oocytes in different stages of development. We found that NPC frequency per nucleus increases with maturation. However, individual NPCs are more active in immature stages. In early stages, known for high transcriptional activity, we found nearly 10% of NPC central channels plugged with a 400-800 kDa mass. In contrast, the incidence of plugged NPCs was below 1% in late oocyte stages. On-site RNA digestion led to a change in plug shape from prominent to flat while plug mass decreased by almost 20%. Quantitative AFM analysis revealed that RNase exposure reduced total nucleoplasmic NPC mass by about 58 and 25% in early and late stage oocytes, respectively. We conclude: (i) NPCs of immature oocytes are more active in RNA transport, (ii) Plugs identified at the nucleoplasmic entrance of NPC central channels represent ribonucleoproteins exiting the nucleus, (iii) RNA is a structural component of the NPC nanomachine.

Nuclear pore complex plasticity during developmental process as revealed by super-resolution microscopy

“…Stage II oocytes had a lower number of NPCs (1.25 × 10 7 ) than stage IV oocytes (2.3 × 10 7 ) and stage VI oocytes (3.01 × 10 7 ). These results corroborate recent measurements carried out on native nuclear membranes from late stage oocytes by atomic force microscopy 28 and super-resolution microscopy 17 where the density of nuclear pores and the total number of NPCs per nucleus are in agreement with our observations. In contrast, these figures differ from the measurements obtained previously using negative staining methods and EM 38 .…”

“…Despite the fact that X. Laevis oocyte is one of the favorite model for the study of NPCs (see 6 for a review), only two studies were carried out on early stage oocytes using electron microscopy 38 or Atomic Force Microscopy (AFM) 28 . The other investigations involve late stage of oogenesis, i.e.…”

“…The modifications of the nuclear pore structure, also called nuclear plasticity, have been assessed using electron microscopy 19 – 25 . Cryo-electron microscopy studies have shown the existence of natural variants and modifications of its structure 9 , 26 , 27 , while atomic force microscopy has demonstrated a reversible mechanical gating of the nuclear pore 28 – 31 .…”

Nup358, a nucleoporin, functions as a key determinant of the nuclear pore complex structure remodeling during skeletal myogenesis

“…3A, AFM images of the NPCs in myoblasts and myotubes were successfully captured. It was previously observed that the centers of some NPCs are plugged by complexes passing through the NPC [15–17]. Consistent with these studies, we observed that 29.3% of the NPCs in myoblasts were plugged, whereas 54.4% of NPCs in myotubes were plugged (Fig.…”

Towards monitoring transport of single cargos across individual nuclear pore complexes by time-lapse atomic force microscopy

“…In contrast, the AFM, albeit at typically 3-5 times lower resolution, has mainly been employed to depict at the single-event level structural changes of the NPC that correlate with different functional states (cf. Jaggi et al, 2003;Lim et al, 2006aLim et al, , 2007Lim et al, , 2006bMaco et al, 2006;Mooren et al, 2004;Sanner et al, 2005;Schlune et al, 2006;Stoffler et al, 1999c;Stolz et al, 2007;Wang and Clapham, 1999). Taken together, while the EM provides static views of NPC structure or, at best, snapshots of its structural dynamics or plasticity, time-lapse AFM can directly watch single NPCs at work.…”