xNup43 and xNup37 are components of the Xenopus Nup107-160 complex. (A) An antibody raised to amino acids 1–375 of Xenopus Nup43 recognizes two bands in Xenopus interphase extract. The lower band is Nup43, and the higher band labeled with the asterisk is a cross-reacting protein. (B) An antibody raised to amino acids 1–267 of Xenopus Nup37 recognizes two bands in Xenopus interphase extract. The higher band is Nup37, and the lower band labeled with the asterisk is a cross-reacting protein. (C) Immunofluorescence on interphase XL177 Xenopus cells was performed with anti-xNup37 antibody (left), which localizes in a punctate pattern on the nuclear rim, characteristic of a nuclear pore stain. Right, DNA stained with 4,6-diamidino-2-phenylindole (DAPI). Bar, 10 μm. (D) Schematic of the nine-member Xenopus Nup107-160 complex is shown. Because the nearest neighbors of Nup43 and Nup37 are unknown, they are placed arbitrarily in the subcomplex. (E) Nup37 and Nup43 are members of the Xenopus Nup107-160 nuclear pore subcomplex. Anti-Nup43 antibody immunoprecipitates Nup43 as well as the other members of the Nup107-160 complex, including Nup160, Nup133, and Nup37, from interphase extract (lane 3). Note that the cross-reacting band indicated by the asterisk does not coimmunoprecipitate with the Nup107-160 complex. (F) Anti-Nup133 antibody reciprocally immunoprecipitates Nup43 as well as other complex members, such as Nup160 and Nup37.

The Nup107-160 complex localizes to kinetochores, spindle poles, and microtubules during prometaphase. (A and B) HeLa cells were prepared for immunofluorescence as described in Materials and Methods and stained with affinity-purified anti-hNup133 (red, A), affinity-purified anti-mNup85 (red, B), or an anti-hCENP-B monoclonal antibody (mAb) (green, A and B). A merge is shown in the right panels (A and B). Whereas CENP-B and the Nup107-160 complex were present throughout mitosis on centromeres and kinetochores, respectively, only the Nup107-160 complex was found at the spindle poles and proximal microtubules during prometaphase (A, prometaphase). Nup107-160 complex spindle pole localization was greatly diminished or absent after cells entered metaphase (A, metaphase and anaphase). Bar, 4 μm. (C) Immunofluorescence with anti-hNup160 and anti-hNup96 antibodies on HeLa cells stained the spindle poles during prometaphase (red, far left), identical to the localization seen for Nup133 and Nup85 in A and B. Immunofluorescence with affinity-purified anti-hNup93 polyclonal antibody or an anti-hNup62 mAb does not give a spindle stain. Affinity-purified antisera to Mad2, an outer kinetochore checkpoint protein found at the spindle poles and proximal microtubules during prometaphase (Howell et al., 2004), shows localization identical to that of the Nup107-160 complex at that stage of the cell cycle (far right). Bar, 5 μm. (D) Nup133 colocalizes with NuMA in prometaphase HeLa cells. In cells prepared for immunofluorescence with affinity-purified anti-hNup133 and affinity-purified anti-xNuMA antisera, the localization of Nup133 and NuMA overlap in the spindle pole area in prometaphase cells. Nup133 is also found at the kinetochores and free in the cytoplasm. Bar, 5 μm.

The Nup107-160 complex localizes to in vitro-assembled mitotic spindles. (A) Demembranated sperm chromatin and rhodamine-labeled tubulin were added to Xenopus CSF extracts. The reactions were induced to enter interphase through addition of 0.4 mM calcium chloride. After 1 h, the reactions were returned to mitosis by the addition of an equal volume of fresh CSF extract, and allowed to form spindles for 60 min. Reactions were fixed with formaldehyde, then the spindles were centrifuged onto coverslips, postfixed in methanol, and processed for immunofluorescence. The presence or absence of Nup133 (green, first row), Nup43 (green, second row), Nup62 (green, third row), or IgG (green, bottom row) on spindles was detected with the same affinity-purified antisera used in Figure 1. Presence of rhodamine-labeled tubulin (red, all rows), and DAPI-stained chromosomes (blue, all rows) is also shown. The right-most panels contain the merged images. Bar, 10 μm. (B) The staining of the in vitro assembled spindles by anti-Nup133 and Nup43 shown in A most often obscures concurrent kinetochore staining. However, occasional views reveal bright foci of kinetochore staining (indicated by arrowheads). An anti-Nup133 stain is shown. Bar, 10 μm.

The Nup107-160 complex shows increased accumulation on unattached kinetochores. (A) Mitotic (CSF) extract was incubated with demembranated sperm chromatin for 40 min at 23°C with (bottom) or without (top) nocodazole. Mitotic chromosomes were collected and spread by centrifugation onto coverslips and then analyzed by indirect immunofluorescence with antibodies to Xenopus Nup37 (green) and Bub1 (red). The chromosomal DNA was stained with DAPI. Note that these conditions do not maintain spindle structure. Both Nup37 and BubR1 staining of kinetochores increases greatly. Bar, 10 μm. (B) HeLa cells were treated with nocodazole for 2 h before performing immunofluorescence by using affinity-purified anti-hNup133 antisera. The amount of the Nup107-160 complex, visualized with affinity-purified anti-hNup133 antisera, increased greatly giving large crescents, and ring structures typical of those outer kinetochore proteins that are affected by microtubule disassembly. Bar, 5 μm.

Extracts lacking the Nup107-160 complex have an intact spindle checkpoint. (A) Nup107-160 complex depleted (Δ107-160) or mock depleted (mock) CSF extracts plus sperm chromatin and nocodazole were incubated for 40 min at 23°C. Mitotic chromosomes were analyzed by indirect immunofluorescence by using antibodies to the checkpoint proteins Bub1, BubR1, and Mad2, which accumulated at kinetochores even in the absence of the Nup107-160 complex. (B) The checkpoint was checked for responsiveness to Ran by the addition of the RanGEF RCC1 (lanes 2 and 4). The checkpoint protein Bub1 and Mad2 were released normally in both extracts. (C) CSF extracts with nuclei were treated with DMSO (top) or nocodazole (bottom). After incubation for 40 min, extracts were stimulated with 0.6 mM Ca²⁺ to destroy CSF. At 45 min after calcium addition, Hoechst 33258 dye was added for the examination of DNA morphology. (D) At the indicated times after calcium addition aliquots were removed for Western blot analysis with anti-cyclin B2 antibodies. (E) Anti-Nup133 immunodepletion of mitotic CSF extract removes all visible Nup133 (lanes 2 and 4; 0.2 and 0.4 μl of extract was loaded, respectively). Mock depletion of extract with control IgG antibodies did not (lanes 1 and 3; 0.2 and 0.4 μl). Levels of noncomplex nucleoporins, such as Nup62, were not altered. (F) An immunoblot of mock- and Nup107-160 complex-depleted extracts is shown probed with antibodies to Nup160, Nup133, Importin-α, the AAA ATPase p97, dynein, and Nup43. Lanes 1 and 2 contain 0.2 μl of extract; lanes 3 and 4 contain 0.4 μl of extract. Nup160, Nup133, and Nup43, all members of the complex, were depleted. Levels of Importins and noncomplex kinetochore components tested were not altered by Nup107-160 deletion.

Disruption of the Nup107-160 complex severely compromises spindle assembly. (A) Spindles were assembled for 60 min in vitro by adding sperm chromatin and rhodamine-labeled tubulin to mitotic CSF extracts that were either mock-depleted (top) or Nup107-160 complex-depleted (bottom). Aliquots were examined for spindle assembly by fixation, centrifugation, and a second fixation, before examination with a Zeiss Axioskop fluorescence microscope. In both A and B, DNA was stained with DAPI DNA dye (blue, all rows). Absence of the Nup107-160 complex produced a much reduced number of bipolar spindles and those that were seen were small and contained many fewer microtubules. (B) Nuclei were assembled in interphase Xenopus extract for 60 min. At that time, an equal volume of CSF extract was added to convert the interphase extract to mitosis, via the cyclin B present in the CSF extract. Five minutes later rhodamine-labeled tubulin and either control IgG (left), affinity-purified anti-Nup133 antibody (middle), or affinity-purified anti-Nup43 antibody (right) were added. Spindle assembly was assessed 60 min later, as in A. Both antibodies to the Nup107-160 complex significantly interfered with correct spindle assembly, mirroring the depletion defect of A. Bar, 10 μm.

Absence of the Nup107-160 complex causes an increasing regression in spindle assembly and stability. (A) Mock-depleted and Nup107-160 complex-depleted Xenopus mitotic (CSF) extracts were supplemented with rhodamine-labeled tubulin and sperm chromatin at t = 0′. Aliquots were withdrawn at 15, 25, and 60 min, prefixed with formaldehyde, spun down onto coverslips, postfixed with methanol, and examined with a Zeiss Axioskop 2 microscope. Bar, 10 μm. (B) Quantitation of in vitro spindle defects observed upon depletion of the Nup107-160 complex. The data from the 60-min time point in Figure 7A is shown graphically.