O'Brien LL and Wiese C (2006), TPX2 is required for postmitotic nuclear assemb...

XB-ART-314

J Cell Biol 2006 Jun 05;1735:685-94. doi: 10.1083/jcb.200512107.

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TPX2 is required for postmitotic nuclear assembly in cell-free Xenopus laevis egg extracts.

O'Brien LL , Wiese C .

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Cell division in many metazoa is accompanied by the disassembly of the nuclear envelope and the assembly of the mitotic spindle. These dramatic structural rearrangements are reversed after mitosis, when the mitotic spindle is dismantled and the nuclear envelope reassembles. The targeting protein for XKlp2 (TPX2) plays important roles in mitotic spindle assembly. We report that TPX2 depletion from nuclear assembly extracts prepared from Xenopus laevis eggs results in the formation of nuclei that are only about one fifth the size of control nuclei. TPX2-depleted nuclei assemble nuclear envelopes, nuclear pore complexes, and a lamina, and they perform nuclear-specific functions, including DNA replication. We show that TPX2 interacts with lamina-associated polypeptide 2 (LAP2), a protein known to be required for nuclear assembly in interphase extracts and in vitro. LAP2 localization is disrupted in TPX2-depleted nuclei, suggesting that the interaction between TPX2 and LAP2 is required for postmitotic nuclear reformation.

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Species referenced: Xenopus laevis
Genes referenced: actl6a dnai1 hsp90aa1.1 igf2bp3 kif15 lmnb3 npm1 tmpo tpx2

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	Figure 1. Disrupting the levels of TPX2 in interphase extracts affects nuclear size. (A) TPX2 immunofluorescence in X. laevis tissue culture cells. A typical microscope field with several interphase cells stained for DNA (a) or TPX2 (b) is shown. The arrowhead denotes a cell in which TPX2 localizes to the nuclear rim. (B) Addition of exogenous TPX2 to interphase extracts disrupts nuclear size. Nuclear assembly reactions were supplemented with buffer (a), or 0.2 μM (b), 1.5 μM (c), 2.5 μM (d), 3.5 μM (e), or 6 μM (f) recombinant TPX2, incubated for 80 min at room temperature, fixed, spun onto coverslips, and stained for DNA with Hoechst dye. The concentration of TPX2 added ranged from twofold (0.2 μM) to 60-fold (6 μM) over endogenous TPX2 levels. (C) Interphase X. laevis egg extracts were depleted with either nonspecific IgG (a) or anti-TPX2 antibodies (b). The extracts were then induced to form nuclei by addition of demembranated X. laevis sperm chromatin, incubated at room temperature for 80 min, fixed, and spun onto coverslips. The coverslips were stained with Hoechst dye to visualize the DNA. (D) Samples were prepared as in C, but were supplemented with a small amount of rhodamine-labeled tubulin to visualize microtubule structures in mock-depleted (a) or TPX2-Δ (b) extracts. (E) TPX2 levels are reduced by >98% in depleted extracts. TPX2 Western blot of mock-depleted (lane 1) or increasing amounts of TPX2-Δ (lanes 2–4) extracts. The volume of extract loaded ranged from 1 to 10 μl for the depleted extract as indicated above the lanes. (F) Nuclear size can be rescued by readdition of bacterially expressed recombinant TPX2, but TPX2 must be present at the start of the assembly reaction. Nuclei are visualized by DNA staining. Typical nuclei assembled in mock-depleted extract (a), TPX2-Δ extract supplemented with buffer (b), TPX2-Δ extract supplemented with 100 nM TPX2 at the beginning of the assembly reaction (c), or TPX2-Δ extract supplemented with 100 nM TPX2 60 min after the beginning of the assembly reaction and incubated for an additional 60 min (d) are shown. Buffer controls were supplemented with an equivalent volume of buffer. (G) Quantitation of the area enclosed by the nuclei assembled in mock-depleted extracts, TPX2-Δ extracts, or extracts reconstituted with TPX2 at the beginning of the assembly reaction, as described for the experiment shown in F. At least 50 nuclei were measured per sample in five independent experiments. Error bars indicate standard deviation. Bars, 25 μm.
	Figure 2. TPX2-Δ nuclei have nuclear membranes, NPCs, and a lamina. (A) Nuclei assembled in mock (a–c) or TPX2-Δ (d–f) extracts were stained for DNA with Hoechst dye (a and d) or for membranes with DHCC (b and e) or were visualized by Nomarski (DIC) optics (c and f). (b', c', e', and f') Higher magnification views of the areas indicated by white rectangles in b, c, e, and f, respectively. (B) TPX2-Δ nuclei stain positively for the NPC probe, mAb414. Nuclei were assembled in mock-depleted (a and b) or TPX2-Δ (c and d) extracts, fixed, and spun onto coverslips. Nuclei were then processed for immunofluorescence with mAb414 (b and d) and for DNA staining with Hoechst dye (a and c). (C) TPX2-Δ nuclei assemble a nuclear lamina. Nuclei were prepared as in B and were stained for lamin B3 (b and d) or DNA (a and c). (D) Thin-section electron micrographs of untreated (a and d), TPX2-Δ (b and e), or mock-depleted (c and f) nuclei. Lower magnifications (a–c) reveal the overall appearance of the nuclei. Higher magnifications (d–f) reveal double nuclear membranes and NPCs (arrowheads). Bars: (A, B, and C) 25 μm; (D, a–c) 500 nm; (D, d–f) 200 nm.
	Figure 3. TPX2-Δ nuclei form fully functional NEs. (A) TPX2-Δ nuclei import a nuclear substrate. Mock-depleted (a–d) or TPX2-Δ (e–h) nuclear assembly reactions were supplemented with rhodamine-labeled nucleoplasmin to assay nuclear import. To distinguish import from nonspecific association of nucleoplasmin with the nuclei, some reactions (c, d, g, and h) were supplemented with the inhibitor of nuclear import, wheat germ agglutinin (WGA). Nuclei were visualized by DNA stain (a, c, e, e′, and g) or rhodamine fluorescence (b, d, f, f′, and h). (e′ and f′) Higher magnification views of e and f, respectively. (B) TPX2-Δ nuclei exclude a nonnuclear substance. Mock-depleted (a and b) or TPX2-Δ (c and d) nuclear assembly reactions were spiked with rhodamine-labeled 155-kD dextran, and a small amount of the reaction was spotted onto a coverslip. Nuclei were visualized by Hoechst staining (a and c) and the dextran by fluorescence (b and d). (C) TPX2-Δ nuclei replicate their DNA. Autoradiographs of two representative replication assays are shown. [32P]dCTP was added to control (C), mock-depleted (M), or TPX2-Δ (T) extracts supplemented with ∼100 sperm chromatin/microliter, and the reactions were incubated for 3 h at room temperature to allow DNA replication. Reactions were then stopped, separated on a 0.8% agarose gel, and visualized by autoradiography. Two types of control extracts were used, with similar results: (a) duplicate reactions were treated with the inhibitor of replication, aphidicolin (AC); (b) [32P]dCTP was added to mitotic extracts. (D) Quantitation of DNA replication in four independent experiments, normalized to the amount of label incorporated in the mock-depleted controls. Error bars indicate standard deviation. Bars: (A [a–f, g, and h] and B) 25 μm; (A, e' and f') 10 μm.
	Figure 4. TPX2 interacts with LAP2. (A) Coomassie-stained SDS-PAGE gel of proteins that copurify with GST-TPX2 from interphase or mitotic extracts. GST-TPX2, GST, or buffer were incubated in interphase or mitotic extracts and were retrieved using glutathione-agarose beads. Proteins were eluted from the beads by treatment with a protease that cuts between the GST and TPX2 (the GST remains bound to the beads), and eluted proteins were boiled in SDS sample buffer and separated on a 10% gel. Sizes of molecular mass markers are indicated on the left, and the position of TPX2, importin β, and actin are indicated on the right. (B, a) LAP2 associates with GST-TPX2 reisolated from egg extracts. LAP2 Western blots of GST pull downs from interphase and mitosis, prepared as in A. Buffer, GST, or GST-TPX2 was added to interphase or mitotic extract. (b) LAP2 coimmunoprecipitates with TPX2. LAP2 Western blot of proteins that coimmunoprecipitate with TPX2. Lane 1 shows IgG control, lane 2 shows TPX2 immunoprecipitation, and lane 3 shows extract control. (c) TPX2 (top) or LAP2 (bottom) Western blots of proteins that coimmunoprecipitate with LAP2. Lane 1 shows IgG control, and lane 2 shows LAP2 immunoprecipitation. The positions of TPX2 and LAP2 are indicated on the right. (d) LAP2 is not codepleted in extracts depleted of TPX2. LAP2 Western blot of extracts immunodepleted with control or TPX2 antibodies. (e) Western blot of X. laevis egg extracts probed with LAP2 antibodies. Two doublets (A and B) are recognized by two different antibodies raised against X. laevis LAP2. (C) LAP2 interacts with TPX2 in vitro. Bacterially expressed GST-XLAP2 was incubated in vitro with increasing amounts of untagged TPX2 (lanes 2–7). GST-LAP2 was then retrieved with glutathione-agarose beads, and the amount of TPX2 that copurified (top) was examined on Coomassie-stained SDS-PAGE gels and compared with the amount that remained in the supernatant (bottom). Lane 1 shows TPX2 incubated with beads in the absence of GST-XLAP2. The positions of TPX2 and LAP2 are indicated on the left of each panel.
	Figure 5. TPX2 and LAP2 colocalize at the nuclear rim during interphase in X. laevis tissue culture cells. X. laevis tissue culture cells triple stained for TPX2 (green), LAP2 (red), and DNA. (a–d) A field containing several interphase cells; (e–h) higher magnification of an individual interphase cell; (i–l) metaphase; (m–p) late anaphase; (q–t) telophase; (u–x) early G1. Bars: (d) 25 μm; (h, l, p, t, and x) 10 μm.
	Figure 6. TPX2 depletion disrupts LAP2 accumulation in nuclei. (A) LAP2 IIF in mock-depleted (a) or TPX2-Δ (b) nuclei fixed at various times after initiating nuclear assembly. (a, c, e, g, and i) Stained for DNA; (b, d, f, h, and j) stained for LAP2. Time (in minutes) is indicated in the top right corner of the DNA panels. (B) TPX2 IIF (b, d, and f) of nuclei assembled in reactions supplemented with buffer (a and b), 1.5 μM recombinant LAP2 (c and d), or 3.0 μM recombinant LAP2 (e and f). Nuclei were stained with Hoechst dye (a, c, and e) to visualize the DNA. Bars, 25 μm.

References [+] :

Alsheimer, Nuclear envelope remodelling during rat spermiogenesis: distribution and expression pattern of LAP2/thymopoietins. 1998, Pubmed