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Steps of nuclear pore complex disassembly and reassembly during mitosis in early Drosophila embryos

¹ CRC Department of Structural Cell Biology, Paterson Institute for Cancer Research, Christie Hospital, Wilmslow Road, Manchester, M20 9BX, UK
² Institute of Cytology and Genetics, Russian Academy of Sciences, Novosibirsk, 630090, Russia

View larger version (214K): [in a new window]   Fig. 1. Interphase nuclear morphology. (a) Nuclei vary in diameter from 4 µm in early interphase up to 9 µm at prophase, (b) chromatin is generally dispersed and (c) there are membrane protrusions (dark blue arrows) and numerous NPCs (d,e), which have a morphology that is similar to other higher eukaryotes such as Xenopus (g), containing a cytoplasmic ring (yellow arrows), cytoplasmic particles (light blue arrows), internal filaments (red arrows) and a central structure (pink arrows). (h) Cross sections of interphase NPCs showing typical NE profiles.

View larger version (145K): [in a new window]   Fig. 2. Prophase nuclear morphology. (a) Nuclei are spherical with 9.5 µm diameter. (b) NPCs appear to be partially dismantled and (c) the central channel is less electron dense in TEM thin sections (arrows). (d-f) FESEM also shows that they are empty in the middle, suggesting that the transporter has been removed leaving the star ring. (g-i) Other intermediates observed in prophase.

View larger version (113K): [in a new window]   Fig. 3. Prophase/metaphase nuclear morphology. (a) FESEM image of prophase nucleus where vesicles are found at defined regions on the nuclear surface (arrows), presumably the nuclear poles, which are about to disrupt. (b) TEM thin section of more advanced stage of polar disruption in late prophase, showing membranes accumulating on the surface of the NE (arrowheads). (c) Low magnification TEM image showing disruption of poles (arrows) and penetration of the spindle microtubules in metaphase. (d,e) Mitotic NEs showing accumulated spindle membranes near or around the NE (arrowheads).

View larger version (98K): [in a new window]   Fig. 4. Early telophase nuclear morphology. (a) Low magnification FESEM image showing bilobal morphology of a nucleus dividing into two 4 µm ‘daughters’. (b) Surface of such a nucleus showing a few early assembly intermediates (arrows) and bound membrane vesicles (stars), which could be spindle membranes. (c) TEM thin section showing reassembling NPCs. (d,e) Network of spindle envelope overlying the NE (stars) and early NPC intermediates (arrows) assembling on the NE between the spindle envelope tubules. (f-h) Thin section TEM showing fusion of spindle membrane with NE (arrows).

View larger version (117K): [in a new window]   Fig. 5. Late telophase nuclear morphology. (c) FESEM of almost separated ‘daughter’ nuclei, (d) also shown in thin section. (a,b) Spindle membrane (stars) has fused with NE and appears to be flattening into it. NPCs form at the junction between the spindle envelope and NE (arrows), and we also observe a novel ‘rosette’ structure (circled). (e-m) Montage of selected intermediates at this stage. (e-g) Stabilized pores with structures, probably the beginnings of the spoke ring complex, assembled into central channel. (h) Rosette structure. (i-m) Star rings.

View larger version (82K): [in a new window]   Fig. 6. Telophase/early interphase nucleus. (a) NPCs assemble at the base of bound vesicles. (b) After dispersal of the spindle membrane into the nuclear envelope, the nascent NPCs appear to be left in a semicircular arrangement. (c) NPC clusters are also observed. (d) The assembly of NPCs at the fusion junction between NE and spindle envelope or vesicles: (1) apposition of spindle vesicle to NE; (2) vesicle-NE fusion, initiating NPC assembly at the site of fusion; (3) further incorporation of vesicle membrane into NE.

View larger version (111K): [in a new window]   Fig. 7. Early interphase nucleus. (a) Nuclei are relatively small, (b) have mature NPCs (large arrow) but also many intermediates (small arrows), including (c) rosettes (arrows).

View larger version (44K): [in a new window]   Fig. 8. Quantification of each intermediate at each stage of mitosis. Data are presented as a percentage of the total NPC-like structures at each stage. The stage of mitosis is defined on the right-hand side with an example of a nucleus used in the quantification; the intermediate structures quantified are shown along the bottom. No data is given for metaphase (c) as these could not be visualized by FESEM. This shows that mature NPCs predominate in interphase (a) but disappear rapidly in prophase (b), in which star rings predominate. In early telophase (d), early assembly intermediates (particularly dimples and pores) predominate, whereas, in late telophase (e), assembly appears to progress to star rings and then thin rings in early interphase (f).

View larger version (112K): [in a new window]   Fig. 9. Montage and corresponding illustrations of the NPC assembly intermediates (as shown in Fig. 8) observed in early Drosophila embryos arranged into a proposed order of assembly. Disassembly is roughly the reverse of this order