Partial Nuclear Pore Complex Disassembly during Closed Mitosis in Aspergillus nidulans

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1 Current Biology, Vol. 14, , November 23, 2004, 2004 Elsevier Ltd. All rights reserved. DOI /j.cub Partial Nuclear Pore Complex Disassembly during Closed Mitosis in Aspergillus nidulans Colin P.C. De Souza, Aysha H. Osmani, Shahr B. Hashmi, and Stephen A. Osmani* Department of Molecular Genetics Ohio State University Columbus, Ohio Summary and yeast NPCs share many structural similarities and two-thirds of the nucleoporins are conserved between these organisms, although overall similarity of individual proteins is often low [4]. Simplistically, the NPC consists of a central channel through the nuclear envelope with cytoplasmic fibrils and a nuclear basket [2, 5, 6]. Phenylalanine-Glycine (FG)-repeat nucleoporins are thought to form a selective molecular sieve and also facilitate active transport [6 9]. These FG-repeat nucleoporins bind transport factors carrying their cargo [5, 10] for transport through the NPC in a Ran-dependent manner [3, 11]. Critical for karyopherin-mediated transport is the presence of a steep gradient of RanGTP across the nuclear envelope [6, 12]. This is achieved by localizing the Ran guanine nucleotide exchange factor (RanGEF) to the nucleus and the Ran GTPase-activating protein (Ran- GAP) to the cytoplasm [6, 12]. The compartmentalization of these Ran regulators generates a high nuclear con- centration of RanGTP [6, 12]. The closed mitosis of Aspergillus nidulans requires the kinase activities of both NIMA and the NIMX cdc2 / NIME cyclin B complex (Cdk1) [13, 14]. Regulation of the localization of both these kinases is important for mitotic entry [15, 16]. For example, even though Cdk1 is fully active in a G2 arrest caused by NIMA inactivation [13, 17], cells do not enter mitosis because both NIMA and Cdk1 are excluded from nuclei [15, 16]. If cells are ar- rested in G2 in the absence of Cdk1 activity, NIMA is only partially activated [14] and localized to the cyto- plasm [16]. Full activation of NIMA requires Cdk1-depen- dent phosphorylation [14], allowing nuclear accumula- Background: Many organisms undergo closed mitosis and locate tubulin and mitotic kinases to nuclei only during mitosis. How this is regulated is unknown. Inter- estingly, the NIMA kinase of Aspergillus nidulans inter- acts with two nuclear pore complex (NPC) proteins and NIMA is required for mitotic localization of the Cdk1 kinase to nuclei. Therefore, we wished to define the mechanism by which the NPC is regulated during A. nidulans closed mitosis. Results: The structural makeup of the NPC is dramatically changed during A. nidulans mitosis. At least five NPC proteins disperse throughout the cell during mitosis while at least three structural components remain at the NPC. These modifications correlate with marked changes in the function of the NPC. Notably, during mitosis, An-RanGAP is not excluded from nuclei, and five other nuclear or cytoplasmic proteins investigated fail to locate as they do during interphase. Mitotic modification of the NPC requires NIMA and Cdk1 kinase activation. NIMA appears to be particularly important. Most strikingly, ectopic induction of NIMA promotes mitoticlike changes in NPC structure and function during S phase. Furthermore, NIMA locates to the NPC during tion of NIMA and Cdk1 to promote mitotic entry [16]. entry into mitosis, and a dominant-negative version of Therefore, these two kinases require the activity of each NIMA that causes G2 delay dwells at the NPC. other to trigger mitosis. Conclusions: We conclude that partial NPC disassem- Genetic evidence suggests that the NIMA kinase plays bly under control of NIMA and Cdk1 in A. nidulans may a role in NPC function at the G2/M transition in A. nidurepresent a new mechanism for regulating closed mito- lans [15, 18]. We isolated mutations in genes encoding ses. We hypothesize that proteins locate by their relative the SONA Gle2 and SONBn Nup98 nucleoporins, which allow binding affinities within the cell during A. nidulans cells arrested at the nima1 G2 arrest point to enter closed mitosis, analogous to what occurs during open mitosis [15, 18]. SONBn Nup98 is an FG-repeat nucleoporin mitosis. related to human Nup98, and both are generated by autoproteolytic cleavage of precursor proteins [18 20]. Introduction SONA Gle2 is a homolog of the Gle2/Rae1WD-repeat nucleo- porins [15] and physically binds to SONBn Nup98 [18]. Nota- Regulation of the G2/M transition differs between verte- bly, mutants in the fission yeast SONA Gle2 homolog, brates, which undergo nuclear envelope breakdown Rae1, arrest in G2 with interphase microtubules and (NEBD), and many filamentous fungi and many yeasts, high Cdk1 kinase activity [21], suggesting a role at G2/M which carry out mitosis within an intact nuclear envelope for this nucleoporin in another species. During closed [1]. During such closed mitoses, the transport of tubulin mitosis in budding yeast, the NPC remains intact and and mitotic regulators into the nucleus must occur protein localization is thought to be regulated by mitotic through the nuclear pore complex (NPC). How nuclear specific modification of regulated transport, as has reimport of tubulin and mitotic regulators is controlled cently been demonstrated [22]. during closed mitoses is currently unknown. Here, we have investigated the status of the NPC The NPC is embedded in the nuclear envelope and during A. nidulans closed mitosis. Our data indicate provides the gateway for transport of proteins and nu- that the NPC is partially disassembled during mitosis cleic acids to and from the nucleus [2, 3]. Vertebrate and that NIMA kinase function is required for these modifications. The data provide a new paradigm for regulation *Correspondence: osmani.2@osu.edu of closed mitosis.

2 Current Biology 1974 Figure 1. Mitotic-Specific Dissociation of SONBn Nup98 from the NPC (A) Time-lapse imaging of GFP-SONBn Nup98 in an asynchronous culture of strain CDS165. Germlings in G1 and G2 at 33 min are indicated. GFP-SONBn Nup98 is present at the NPC during G2 but disperses during nuclear division before reaccumulating at the NPC as nuclei divide. The scale bar represents 8 m. (See also Movie 1.) (B) Graph showing the average fluorescence intensity of GFP-SONBn Nup98 in the nucleus and cytoplasm for the germling undergoing mitosis in (A). Nuclear fluorescence is not indicated during nuclear division because nuclei could not be precisely defined. The area defined as cytoplasmic in G2 was also considered cytoplasmic during nuclear division. During nuclear division, the average cytoplasmic fluorescence was 3-fold higher than in G2. (C) A germling in which the four nuclei are at the G2/M transition. The onset of spindle formation indicates three nuclei are in prophase (p) while the other nucleus is in very late G2 (i). GFP-SONBn Nup98 localizes at the nuclear periphery in late G2 (i) but begins to disperse during prophase (p). (D) A germling containing nuclei in metaphase (m) and telophase (t) as indicated by DNA and spindle morphology. GFP-SONBn Nup98 is absent from the nuclear periphery of the metaphase nucleus but has begun to reaccumulate at the nuclear periphery during telophase. The scale bar represents 6 m. (E) Quantification of GFP-SONBn Nup98 localization during different cell cycle stages as defined by DAPI staining and microtubule morphology, as in (C and D). At least 100 nuclei were counted for each of the data sets.

3 Partial NPC Disassembly during A. nidulans Mitosis 1975 Results The SONBn Nup98 Nucleoporin Disperses from the NPC during Mitosis We previously isolated the SONBn Nup98 nucleoporin due to its genetic interaction with the NIMA mitotic kinase [18]. As this suggests a potential role for SONBn Nup98 in mitotic regulation we examined the localization of SONBn Nup98 during the cell cycle by time lapse confocal microscopy. As expected, GFP-SONBn Nup98 generated by gene replacement displayed a characteristic NPC pattern in interphase nuclei (Figure 1A) [18]. However, as cells progressed from G2 into mitosis, GFP-SONBn Nup98 strikingly dispersed from the nuclear periphery and did not reappear at this locale again until after nuclear division (Figure 1A; min; see also Movie 1 in the Supplemental Data available with this article online). Immunoblotting of mitotic and interphase extracts demonstrated that SONBn Nup98 is not degraded during mitosis (see Figure 4I). Indeed we consistently measured an increase in cellular fluorescence throughout cells when GFP-SONBn Nup98 was not at the nuclear periphery (Figures 1B and 2C). GFP-SONBn Nup98 localized to the NPC of all cells displaying interphase microtubules and uncondensed DNA (Figure 1E). Mitosis in A. nidulans occurs in a synchronous manner allowing nuclei staggered in slightly different stages of the G2/M transition to be examined within one cellular compartment. Careful examination indicated that GFP-SONBn Nup98 began to dissipate from the nuclear periphery of prophase cells which had just begun spindle formation and DNA condensation (Figures 1C p and 1E). During metaphase cells displayed no nuclear periphery localization for GFP-SONBn Nup98 (Figure 1D m). GFP-SONBn Nup98 began to re-associate around DNA during anaphase and telophase, remarkably while the spindle was still present and before DNA decondensation was complete (Figure 1D t and E). We conclude that SONBn Nup98 disperses from the NPC during mitosis and returns to the NPC as cells exit mitosis and enter G1. The NPC Partially Disassembles during Mitosis in A. nidulans We next examined the localization of the SONBc Nup96 nucleoporin which is the C-terminal cleavage product of the SONB Nup98/Nup96 precursor [18]. SONBc Nup96 -GFP localized to the nuclear periphery during both interphase and mitosis (Figure 2A, see also Movie 2). Examination of fixed cells counterstained for DNA and microtubules Figure 2. SONBc Nup96 Remains at the NPC during Mitosis (A) Time-lapse imaging of SONBc Nup96 -GFP in strain CDS232 underconfirmed that SONBc Nup96 -GFP remained at the nuclear going mitosis as indicated by nuclear division at 5 6 min. The scale periphery throughout mitosis (data not shown). Therebar represents 5 m. (See also Movie 2.) fore, the SONBn Nup98 and SONBc Nup96 nucleoporins local- (B) Localization of GFP-SONBn Nup98 and SONBc Nup96 -mrfp in a fixed ize differently during mitosis as highlighted in Figure 2B. germling (strain CDS246) containing one mitotic (M) and one interphase The germling shown contains one cellular compartment (I) compartment. DAPI staining shows DNA morphology. in mitosis (M) and one in interphase (I) (Figure 2B, DAPI). An overlay of SONBc Nup96 -mrfp and GFP-SONBn Nup98 is also shown SONBc Nup96 -mrfp localizes to the nuclear periphery of (merge). Note that during mitosis, SONBc Nup96 -mrfp is present at both mitotic and interphase nuclei (Figures 2B and 2C). the nuclear periphery while GFP-SONBn Nup98 is dispersed through- out the cell. The scale bar represents 5 m. SONBc Nup96 localized In contrast, GFP-SONBn Nup98 is only found at the NPC to the nuclear periphery of all cells examined (n 1000). of interphase nuclei and is dispersed throughout the (C) Quantification of relative levels of GFP-SONBn Nup98 and mitotic compartment of the cell (Figures 2B and 2C). SONBc Nup96 -mrfp in the cytoplasm and nucleus during interphase The SONA Gle2 nucleoporin is known to bind SONBn Nup98 and mitosis for the cell shown in (B). [18]. Examination of SONA Gle2 -GFP localization during

4 Current Biology 1976 localized to the nuclear periphery during interphase but dispersed throughout the cell during mitosis (Figures 3A and 3B; see also Movies 3, 5, and 6). Cytoplasmic levels of An-Nsp1-GFP increased 2.9-fold during nuclear division when it dispersed from the NPC before relocating back to the nuclear periphery as cells exit mitosis (Figure 3A). We have confirmed the timing of An- Nsp1-GFP dispersal and re-accumulation at the nuclear periphery during mitosis by real time imaging of cells containing both An-Nsp1-GFP and GFP-tubulin (data not shown). To identify additional nucleoporins which may remain at the nuclear periphery during mitosis, we examined the localization of the A. nidulans homologs of budding yeast Pom152 [26] and vertebrate Nup133 [27]. Similar to budding yeast Pom152, An-Pom152 contains a transmembrane domain and likely performs a structural role at the NPC [26]. An-Nup133 is expected to behave similarly to SONBc Nup96 as homologs of these two nucleoporins are components of the Nup NPC sub-complex in other systems [27, 28]. Both An-Pom152-GFP and An-Nup133-GFP localized to the nuclear periphery throughout mitosis (Figure 3C; Movies 7 and 8). Together, these data establish that the composition of the NPC is changed dramatically during mitosis in A. nidulans. Figure 3. Changes in the Composition of the NPC during Mitosis in A. nidulans Time-lapse imaging of the indicated nucleoporins as cells transit through mitosis. Images are shown for cells following nuclear division (G1), during mitosis (M), and in G2. Full-time courses are shown in the indicated supplemental movies. (A) Localization of the FG-repeat nucleoporin An-Nsp1-GFP (strain SO440) during G2, M, and the subsequent G1. Relative grayscale levels of An-Nsp1-GFP along the indicated cross section are shown. See also Movie 3. (B) SONA Gle2 -GFP (strain CDS171), the FG-repeat nucleoporins An- Nup159-GFP (strain SO438), and An-Nup42-GFP (strain SO435) also disperse from the NPC during mitosis. See also Movies 4, 5, and 6, respectively. (C) The An-Pom152-GFP (strain FN19) and An-Nup133-GFP (strain CDS234) nucleoporins remain associated with the nuclear periphery during mitosis. An-Pom152-GFP and An-Nup133-GFP localized to the nuclear periphery in all cells examined (n 1000). See also Movies 7 and 8, respectively. The scale bar represents 5 m. mitosis indicated that it dispersed from the NPC in a manner similar to SONBn Nup98 (Figure 3B; see also Movie 4). Several other predicted FG-repeats nucleoporins were next examined to determine if they behaved in a similar manner. Homologs of budding yeast Nsp1 [23], Nup159, [24] and Nup42 [25] were identified and C-terminally tagged with GFP. All three of these nucleoporins Mitotic Dispersal of SONBn Nup98 and SONA Gle2 Is Dependent upon NIMA and Cdk1 Activation The isolation of mutations in SONBn Nup98 and SONA Gle2 as genetic suppressors of a nima1 G2 arrest [15, 18] suggests that their mitotic dispersal from the NPC may be regulated by the NIMA kinase. We therefore examined GFP-SONBn Nup98 in a nima5 strain which reversibly arrests in G2 with inactive NIMA5 at the restrictive temperature [29]. At nima5 arrest, GFP-SONBn Nup98 localized to the nuclear periphery of all cells (Figure 4A), but dispersed throughout the cell following release into mitosis (Figure 4B). Mitotic entry also requires activation of A. nidulans Cdk1 (NIMX cdc2 ) by its activating phosphatase NIMT Cdc25 [13]. Cells arrested with inactive Cdk1 at the nimt23 cdc25 G2 arrest point all displayed GFP-SONBn Nup98 localized to the nuclear periphery (Figure 4C). Following release into mitosis GFP-SONBn Nup98 dispersed throughout the cell (Figure 4D). Mitotic dispersal of SONA Gle2 -GFP from the NPC was similarly dependent upon both NIMA and Cdk1 activation (Figures 4E 4H). We next examined whether SONBn Nup98 may be phos- phorylated during mitosis using a nima5 strain containing a HA-tagged version of SONBn Nup98. Cells were first arrested in G2 at the nima5 arrest point and then released to permissive temperature for 20 min in the presence of nocodazole to cause a pseudo-metaphase arrest. Analysis of HA-SONBn Nup98 immunoprecipitates indicated that SONBn Nup98 underwent dramatic NIMAdependent mobility shifts following release into mitosis (Figure 4I). These mobility shifts were sensitive to phosphatase treatment (Figure 4J) indicating that SONBn NUP98 is phosphorylated during mitosis. No mobility shifts were observed in similar experiments using a nima5 strain containing a HA-tagged version of SONA Gle2 (Figure 4I). Levels of HA-SONBn Nup98 and SONA Gle2 -HA varied little

5 Partial NPC Disassembly during A. nidulans Mitosis 1977 Figure 4. SONBn Nup98 and SONA Gle2 Dispersal from the NPC Requires NIMA and Cdk1 Activation, and SONBn Nup98 Is Phosphorylated during Mitosis (A and C) GFP-SONBn Nup98 localizes to the NPC if (A) NIMA (nima5) or (C) Cdk1 (nimt23 cdc25 )is inactivated, causing G2 arrest. (B and D) GFP- SONBn Nup98 disperses throughout the cell during mitosis after (B) release from nima5 G2 arrest or (D) release from nimt23 cdc25 G2 arrest. (E and G) SONA Gle2 -GFP localizes to the NPC if (A) NIMA (nima1) or (C) Cdk1 is inactivated, causing G2 arrest. (F and H) SONA Gle2 - GFP disperses throughout the cell during mitosis following (F) release from nima1 G2 arrest or (H) release from nimt23 cdc25 G2 arrest. DAPI staining shows uncondensed DNA at arrest and condensed DNA during mitosis. The scale bar represents 5 m. (I) Immunoblotting for HA-SONBn NUP98 (strain CDS177) and SONA Gle2 -HA (strain CDS127) in anti-ha immunoprecipitates prepared from nima5 block release experiments. Electrophoretically retarded forms of HA-SONBn NUP98 are apparent following release into mitosis in the presence of 5 ug/ml nocodazole to maintain cells in a pseudometaphase state. (J) Electrophoretically retarded forms of HA-SONBn NUP98 are reduced by Lambda protein phosphatase activity. between G2 and mitotic samples indicating that neither tently dwelled at the nuclear periphery (e.g., 10 min is degraded during mitosis (Figure 4I). for the cell in Figure 5B), perhaps by preventing normal phosphorylation of NIMA substrates. After delaying at Cell Cycle-Dependent Localization of NIMA the nuclear periphery, N-NIMA entered the nuclei, preto the Nuclear Periphery sumably once NIMA was able to eventually phosphorylate NIMA is cytoplasmic in G2 but accumulates in the nucleus its substrates. Therefore, these data further impli- during mitosis [16], necessitating transit through cate NIMA in regulating NPC function. the NPC. Because NIMA is required for mitotic modification of the NPC, we determined if NIMA is detectable NIMA Induces Changes in NPC Composition at the NPC during the G2/M transition. Live-cell confocal and Function microscopy indicated that NIMA-GFP was excluded The above data implicate a role for NIMA in regulating from the nucleus at the nimt23 cdc25 G2 arrest point as NPC composition during mitosis. To examine this, the expected (see n in Figure 5A). Upon release into mito- effect of NIMA expression on the localization of sis, NIMA-GFP was conspicuous at the nuclear periphery SONBn Nup98 was examined. NIMA was expressed under before entering the nucleus (see p in Figure 5A the control of the alca promotor (alca::nima) in a strain and Movie 9). containing GFP-SONBn Nup98. For these experiments, The noncatalytic domain of NIMA ( N-NIMA) has dom- cells were first arrested in S phase by addition of hydroxyurea inant-negative effects over the G2/M transition [30, 31]. (HU) for 2 hr. During S phase arrest, GFPinant-negative It is likely that N-NIMA competes with full-length NIMA SONBn Nup98 localized to the NPC (e.g., Figure 6A c ). NIMA and hinders phosphorylation of NIMA substrates. We expression was then induced while maintaining the S expressed N-NIMA C-terminally tagged with mrfp phase arrest. By 90 min after NIMA induction, the number ( N-NIMA-mRFP) in a strain also containing SONBc Nup96 - of cells displaying a NPC localization of GFP- GFP to allow visualization of the NPC throughout the cell SONBn Nup98 had decreased by 56% (e.g., Figure 6A a ), cycle. In Figure 5B, N-NIMA-mRFP is initially excluded even though these cells were arrested in S phase. from nuclei (n) before accumulating at the nuclear pe- A. nidulans StuA is maintained in the nucleus throughout riphery (p), where it displayed a similar localization to interphase by nuclear location sequence (NLS)- SONBc Nup96 -GFP. Notably, N-NIMA constructs consis- mediated transport but is released from the nucleus

6 Current Biology 1978 Figure 5. Cell Cycle-Dependent Localization of NIMA at the Nuclear Periphery (A) Time-lapse imaging of NIMA-GFP (strain CDS151) after release from nimt23 G2 arrest. Time is after G2 release; n nuclei which fill with NIMA-GFP as they enter mitosis; p nuclei which display NIMA-GFP at the nuclear periphery. The scale bar represents 4 m. (See also Movie 9). (B) Time-lapse imaging of N-NIMA-mRFP and SONBc Nup96 -GFP. N-NIMA-mRFP is excluded from nuclei in G2 but accumulates at the nuclear periphery, defined by SONBc Nup96 - GFP, as cells enter mitosis. N-NIMA-mRFP was detectable at the nuclear periphery for over 10 min in this experiment. Nuclear accumulation of N-NIMA-mRFP is evident in the nucleus at 36 min, 40 s. The scale bar represents 5 m. specifically during mitosis [32]. We therefore examined HU and nocodazole. GFP-tubulin entered nuclei following the effect of NIMA expression on the localization of the NIMA induction (Figures 6C and 6D; see also Movie NLS of StuA labeled with DsRed [33](NLS-DsRed; a kind 10) but remained cytoplasmic in control cells not containing gift from Dr. Reinhard Fischer). Experiments were carried alca::nima (Figure 6D). out as above. NLS-DsRed localized to nuclei in S Collectively, these data indicate that NIMA expression phase-arrested cells (e.g., Figure 6A d ). After NIMA induction is sufficient to lead to changes in interphase NPC com- in the presence of HU, NLS-DsRed dispersed position and also to change interphase protein compart- throughout the cell (Figures 6A b and 6B). NLS-DsRed mentalization to resemble that of mitotic cells. remained nuclear in identically treated control cells that did not contain alca::nima (Figures 6A d and 6B). Impor- Aspergillus nidulans RanGAP Gains Access tantly, NLS-DsRed was not observed in nuclei that did to the Nucleus during Mitosis not display GFP-SONBn Nup98 at the nuclear periphery Fundamental to regulated nucleocytoplasmic transport during either normal mitosis or following NIMA induction is the cytoplasmic localization of RanGAP to help estab- (data not shown). lish a gradient of Ran-GTP across the nuclear envelope In A. nidulans, depolymerized tubulin is excluded from [6, 12]. We therefore examined the localization of the interphase nuclei but is able to enter mitotic nuclei [34]. single A. nidulans RanGAP during the cell cycle. As To test the effect of NIMA expression on the localization expected, An-RanGAP-GFP was cytoplasmic in the maof depolymerized tubulin, we used a strain containing jority of cells. However, occasionally cells displaying no alca::nima and -tubulin (tuba) tagged with GFP (GFPtubulin) nuclear exclusion of An-RanGAP-GFP were observed. [35]. Cells were first arrested in S phase, as Time-lapse imaging indicated that loss of An-RanGAP- above, and then microtubules were depolymerized by GFP cytoplasmic compartmentalization occurred during addition of nocodazole. Under these conditions, GFP- a small window of the cell cycle immediately preceding tubulin was excluded from the nuclei, which were evident nuclear division (Figure 7A; see also Movie 11). This as darker shadows within cells (Figure 6C, arrows). indicates that the compartmentalization of An-RanGAP NIMA expression was then induced in the presence of is modified during mitosis such that it is not excluded

7 Partial NPC Disassembly during A. nidulans Mitosis 1979 Figure 6. NIMA Modifies the NPC and Changes Nuclear/Cytoplasmic Compartmentalization When Expressed in S Phase Cells Cells were arrested in S phase with 25 mm HU and then expression of NIMA under control of the alca promotor was induced by exchange to inducing media still in the presence of HU. (A) Representative location of GFP-SONBn Nup98 and NLS-DsRed after exchange to inducing conditions. (A a and A b ) Induction of NIMA in strain CDS184 leads to dispersal of GFP-SONBn Nup98 from the NPC and loss of the nuclear compartmentalization of NLS-DsRed. (A c and A d )In identically treated control cells that do not contain alca::nima (strain CDS170), GFP-SONBn Nup98 remained at the NPC, and NLS-DsRed remained in the nucleus in 100% of cells. The scale bar represents 8 m. (B) Graph showing the percentage of cells retaining NLS-DsRed in nuclei with time after exchange to inducing media in control cells and cells inducibly expressing NIMA. (C) Representative location of GFP-tubulin depolymerized in the presence of nocodazole in S phase-arrested cells before and after NIMA induction in strain LY001. Depolymerized GFP-tubulin is excluded from nuclei (arrows) in S phase but gains access to nuclei following NIMA induction while maintaining the S phase arrest. The scale bar represents 8 m. See also Movie 10 for the complete time course and full field for this experiment. (D) Graph showing the percentage of cells with depolymerized GFP-tubulin in the nucleus 90 min after exchange to inducing media. GFPtubulin remained excluded from nuclei in identically treated control cells (strain L01029) containing no inducible NIMA. from the nuclei. Thus, it is anticipated that the transport tubulin [34], TINC-GFP and PlkA-GFP are cytoplasmic properties of the NPC are likely modified during mitosis during interphase but gain access to nuclei during mito- because the RanGTP gradient across the nuclear enve- sis [38, 39]. lope should be compromised. Consistent with this expectation, we have observed five additional examples of mitotic-specific changes in protein compartmental- Discussion ization. The PhoA-GFP and PhoB-GFP kinases [36] and a nuclear-localized domain of the PacC-GFP transcrip- In order to segregate DNA within the intact nuclear envelope tion factor [37] (a kind gift from Dr. Eduardo Espeso) are of a closed mitosis, tubulin and mitotic regulators released from nuclei during mitosis (data not shown). must gain access to the nucleus through the NPC. This Interestingly, a version of this PacC-GFP domain, which could be regulated by mitotic modification of specific is able to bind DNA, is only partially released from the nuclear transport pathways or by global modification of nuclei during mitosis, but a version containing a mutation the NPC structure and function during mitosis. Data in the DNA binding sequence equilibrated through- presented here demonstrate for the first time partial out the cell during mitosis (data not shown). Similar to disassembly of the NPC in an organism undergoing a

8 Current Biology 1980 formed by FG-repeat nucleoporins is likely to be less restrictive for diffusion of macromolecules. Second, the large reduction in available binding sites for karyopherins at the NPC may greatly reduce active transport mediated by these FG-repeat nucleoporins. In its extreme, partial NPC disassembly during mitosis may in fact allow equilibration of many proteins across the nuclear envelope. In this scenario, proteins could still localize to the nucleus or cytoplasm via localized binding sites, such as the spindle pole body in the case of nuclear tubulin. Recent studies in budding yeast have begun to define the compositional requirement for the FG-repeats of different nucleoporins. Remarkably budding yeast can tolerate deletion of over half the mass of their NPC FGrepeat domains [41]. Intriguingly, however, the only single and double FG-repeat deletions that are lethal in budding yeast involve Nup98 family members [41]. This suggests that the FG-repeat domains of Nup98-like nucleoporins play pivotal roles in NPC function, perhaps placing more significance on the mitotic dispersal of the A. nidulans Nup98 homolog SONBn Nup98. Further indicating mitotic modification of NPC transport properties, the predominantly cytoplasmic localization of An-RanGAP during interphase is lost during mitosis when it gains access to the nucleus. Compartmentalization of RanGAP to the cytoplasm helps establish a RanGTP gradient, which is required for active nuclear transport, across the nuclear envelope [6, 11, 12]. Therefore, the RanGTP gradient is likely compromised, which would affect regulated transport, during mitosis in A. nidulans. Indeed, experimentally localizing RanGAP to the nucleus has been demonstrated to affect transport across the nuclear envelope [42, 43]. Therefore, our data suggest that partial NPC disassembly during mitosis effects the selective barrier of the NPC which compromises the RanGTP gradient. This likely compromises active transport during mitosis, consis- tent with the observed equilibration of the refractive index between the cytoplasm and nucleus in A. nidulans during mitosis [44] and the mitotic specific changes in protein compartmentalization described here and elsewhere [15, 16, 32, 34]. The mitotic nuclear accumulation of many of these proteins such as tubulin [34] and the mitotic kinases NIMA [16], Cdk1 [15], and PlkA [39], is required for proper mitotic regulation. Similar changes in mitotic NPC function may also occur in other filamentous fungi in which cytoplasmic ZsGreen proteins, which are Figure 7. Loss of An-RanGAP Cytoplasmic Compartmentalization during Mitosis Time-lapse imaging of An-RanGAP-GFP in a cell progressing through mitosis. In G2, An-RanGAP-GFP localizes to the cytoplasm. This cytoplasmic compartmentalization is lost during mitosis but is reestablished following nuclear division. Images are a single confo- cal slice. The scale bar represents 5 m. (See also Movie 11). closed mitosis. The findings uncover a new mechanism by which closed mitosis may be regulated. Mitotic-Specific Changes in A. nidulans NPC Composition and Function At least five A. nidulans nucleoporins disperse from the NPC specifically during mitosis while three other nucle- during mitosis [45]. oporins remain at the nuclear periphery throughout nuclear division. This partial disassembly of the NPC during not regulated by active transport, gain access to nuclei mitosis has important implications for its function. Closed Mitosis An Open and Shut Case? SONBn Nup98, An-Nsp1, An-Nup42, and An-Nup159 are It is possible that evolutionary intermediates displaying all FG-repeat nucleoporins that disperse from the NPC aspects of both the closed mitosis of simple eukaryotes during mitosis. FG-repeat domains lack secondary and the open mitosis of vertebrates may exist. Indeed, structure [40] and are thought to form a selective barrier during mitosis in C. elegans and Drosophila syncytical to the passage of macromolecules through the NPC embryos, complete NEBD does not occur until after [6 9]. The selectivity of this barrier is likely based on metaphase [46, 47]. By definition, mitosis is closed in specific interactions between FG-repeat nucleoporins A. nidulans because it occurs inside an intact nuclear and karyopherin-cargo complexes [5, 6, 10]. We hypoth- membrane. However, multiple aspects of mitosis in A. nidulans esize that mitotic dispersal of SONA Gle2 and at least four have similarities to aspects of open mitosis sugesize FG-repeat nucleoporins from the NPC in A. nidulans gesting that A. nidulans mitosis may be another evolutionary effects NPC function on two levels. First, the barrier intermediate between completely closed and

9 Partial NPC Disassembly during A. nidulans Mitosis 1981 completely open mitoses. For example, the localization access to the nucleus during the closed mitosis of of An-RanGAP during mitosis is more analogous to ver- A. nidulans. Partial NPC disassembly may also have tebrate mitosis, in which compartmentalization of Ran- roles in higher eukaryotes in which NPC disassembly GAP is lost upon NEBD [48], than to budding yeast, occurs in a stepwise manner [61, 62]. In starfish oocytes, which maintain RanGAP in the cytoplasm during mitosis two phases of NEBD have been described [62]. Interest- [49]. In fact, mitosis in budding yeast may represent a ingly, Cdk1/cyclinB (MPF) begins to accumulate in the completely closed mitosis. The dispersal of nucleopor- nucleus during the first phase of NEBD, when peripheral ins from the NPC of budding yeast during mitosis has nucleoporins disperse from the NPC but the nuclear not been described and presumably does not occur in envelope remains intact [62]. In humans, translocation this intensively studied organism [50 53]. Rather, modi- of Cdk1/cyclinB to the nucleus also occurs prior to fication of specific transport pathways is likely important NEBD [63]. There is, therefore, a strong possibility that for mitotic regulation in budding yeast, as has recently nucleoporin phosphorylation leads to the onset of NPC been described for the Kap121 karyopherin transport disassembly, which may then allow nuclear accumulapathway [22]. At present, it is unclear if similar mitotic tion of Cdk1/cyclinB and entry into mitosis. This is how modification of specific nuclear transport pathways oc- we hypothesize that mitosis is regulated in A. nidulans, curs in A. nidulans. with NIMA promoting partial NPC disassembly. In this Mitotic disassembly of nucleoporins from the NPC is regard, it is noteworthy that expression of NIMA in mama hallmark of open mitoses. Our data suggest that An- malian cells leads to dispersal of the p62 nucleoporin, Pom152 and components of the A. nidulans Nup107- NEBD, and DNA condensation [31]. Therefore, it will be 160 complex (containing An-Nup133 and SONBc Nup96 ) of interest to see if any of the 11 human NIMA-related form a core NPC structure to which FG-repeat nucleokinases [64] play roles in NPC disassembly. porins assemble and disassemble. This has striking similarities to open mitoses, in which the Nup com- Conclusions plex is critical for post-mitotic NPC reassembly and may Our data demonstrate for the first time partial NPC disasbe part of a prepore structure to which FG-repeat nucleoporins are subsequently recruited [28, 54 56]. sembly during a closed mitosis and implicate the NIMA Given that A. nidulans represents a model genetic kinase in regulating this process. Regulated transport system displaying aspects of both closed and open mireduction in karyopherin binding FG-repeat domains at is modified during A. nidulans closed mitosis by the large tosis, further studies should provide interesting insights into mitotic regulation. the NPC and the consequent loss of cytoplasmic com- partmentalization of RanGAP. Our data are consistent NIMA Regulates Mitotic Changes in the NPC with the hypothesis that protein localization may be reg- A function for NIMA at the NPC during the G2/M transiactive ulated more by diffusion and localized binding than by tion was first suggested by genetic interactions [15, 18] transport during A. nidulans closed mitosis. This and is further supported here by localization and overexand may provide a mechanism to allow access of tubulin pression studies. Mitotic dispersal of SONBn Nup98 and mitotic regulators to the nucleus during mitosis. SONA Gle2 is dependent upon the activation of the NIMA kinase. Moreover, NIMA localization to the nuclear periphery Experimental Procedures at the G2/M transition indicates that it is in the General Techniques, Strains, and Plasmids right place at the right time to carry out mitotic modifica- Media and general techniques for culture of A. nidulans, protein tion of the NPC. Studies expressing dominant-negative extraction, Western analysis, transformation, immunofluorescence, NIMA constructs, which dwell at the NPC and cause G2 and DAPI staining were as previously described [14, 16, 29, 65]. delays, also support a role for NIMA at the NPC during Strains used in this study are listed in Table S1. Plasmid pcds24 the G2/M transition. contains NIMA-GFP under the control of the alca promotor. Plasmid pcds49 encodes amino acids of NIMA with a 5 HA tag Importantly, NIMA expression is sufficient to promote and 3 mrfp ( N-NIMA-mRFP) under control of the alca promotor release of SONBn Nup98 from the NPC out of cell-cycle in an AMA1-based autonomously replicating plasmid containing the phase. Similarly, the interphase compartmentalization pyroa selectable marker. Plasmid pal5b contains alca::nima [66]. of NLS-DsRed to the nucleus, and tubulin to the cyto- Plasmid pjh19 contains gpdp::stua C-term-DsRedT4 (NLS-DsRed) plasm, is disrupted by NIMA expression. These results and was a kind gift from Dr. Reinhard Fischer. Plasmid pcds28 was suggest that inappropriate expression of NIMA pro- generated by mutagenesis of plw26 [15], replacing the C-terminal motes changes in the S phase NPC such that nuclear/ HA tag with GFP. SONA Gle2 -HA immunoprecipitation and immu- cytoplasmic compartmentalization resembles that of noblotting was as described [18]. HA-SONBn NUP98 was specifically immunoprecipitated using EZview TM Red Anti-HA affinity gel (Sigma). mitosis. In this context it is noteworthy that similar to Phosphatase treatment of washed immunoprecipitates was with 40 Xenopus Nup98 [57, 58], SONBn Nup98 is phosphorylated U of lambda protein phosphatase (NEB) for 30 min at 30 C in the during mitosis. Moreover, this phosphorylation requires presence or absence of 1 mm sodium vanadate and 50 mm sodium activation of NIMA and SONBn Nup98 contains 29 sites fluoride as phosphatase inhibitors. corresponding to the minimal NIMA consensus sequence of FxxS/T [59, 60]. Generation of Constructs for C-Terminal Tagging with GFP or mrfp To identify A. nidulans nucleoporins, the appropriate S. cerevisiae A Novel Mechanism for Regulating Closed protein sequence was used to query the genome sequence of Mitosis in A. nidulans and Relevance A. nidulans (Aspergillus Sequencing Project. Center for Genome to Higher Eukaryotes Research: by us- Our data suggest that partial NPC disassembly is a novel ing BLAST searches. Putative orthologs identified in A. nidulans mechanism by which tubulin and mitotic regulators gain were then used to query the genome of S. cerevisiae to confirm the

10 Current Biology 1982 identification of each nucleoporin. The predicted stop codon of each Oakley (Ohio State University) and Dr. Eduardo Espeso (Centro de gene was confirmed by sequencing of 3 RACE-PCR products. The Investigaciones Biologicas CSIC, Madrid, Spain) for strains and Dr. following genes were tagged with GFP by gene replacement: An- John Donnan (John Innes Centre, UK), Dr. Reinhard Fischer (Max- Pom152 (AN3454.2), An-Nup42 (AN4595.2), An-Nsp1 (AN4499.2), Planck-Institut, Marburg, Germany), and Dr. Greg May (The UniversonBc Nup96 (AN5627.2), An-Nup133 (AN4293.2), An-Nup159 sity of Texas, MD Anderson Cancer Center) for plasmids. This work (AN2086.2), and An-RanGAP (AN4862.2). Gene-specific tagging was supported by the National Institutes of Health grant GM constructs containing the pyrg A.f.(Aspergillus fumigatus pyrg) selectable marker were generated by a fusion-pcr method as described [67]. Homologous recombination following A. nidulans Received: August 27, 2004 transformation results in gene replacement encoding a C-terminally Revised: September 28, 2004 GFP-tagged protein of interest. Homologous integration was con- Accepted: September 29, 2004 firmed by PCR using primers flanking the stop codon of the gene Published: November 23, 2004 of interest or flanking the entire targeting construct. Expression of proteins of the predicted size was confirmed by Western blotting using GFP specific antibodies. The growth of strains containing References GFP- or mrfp-tagged proteins was compared to wild-type to en- 1. Heath, I.B. (1980). Variant mitosis in lower eukaryotes: Indicators sure no growth defects at 32 C. of evolution of mitosis? Int. Rev. Cytol. 64, Wente, S.R. (2000). Gatekeepers of the nucleus. Science 288, Imaging Cells were grown in 35 mm glass-bottom Microwell dishes (MatTeck) 3. Rout, M.P., and Aitchison, J.D. (2001). 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