Nucleoporins in U. maydis. (A) U. maydis homologues of nucleoporins were identified by BLAST search, and domain models were predicted by the SMART server. Note that Pom121 is no homologue of Pom152, but it may execute similar functions. Um, Ustilago maydis; Sc, S. cerevisiae; Hs, Homo sapiens; and Xl, Xenopus laevis. (B) GFP-fusion proteins of U. maydis Nup107, Nup214, and Pom152 localize to the nuclear envelope, which is visualized by monomeric RFP targeted to the ER. DNA is stained with DAPI. Cells were briefly fixed with 0.5% formaldehyde before observation. Bar, 1.5 μm. (C) Nup107, Nup214, and Pom152 proteins were either fused to GFP or monomeric RFP. Pom152 and Nup214 colocalize with Nup107, indicating that they are part of the same structures. Cells were fixed with 0.4% formaldehyde and 0.1% glutaraldehyde before observation. Bar, 1.5 μm. (D) Stoichiometry of nucleoporins. The signal intensities of Nup107-GFP, Pom152-GFP, and Nup214-GFP in the nuclear envelope were quantified. The nuclear envelope was identified by the presence ER-RFP (strains FB2N107_ER, FB2P152_ER, and FB2N214_ER). All values are mean value ± SD. (E) Distribution of NPCs, labeled with Nup107-GFP, in the nuclear envelope. Cells were fixed with 0.5% formaldehyde; images were taken in z-axis at 0.5-μm intervals. Image stacks were deconvolved, and the individual halves of the nucleus are presented as maximum projections in xy- or as yz-projection. Arrow points to an accumulation of Nup107-signals (yz-projection), which is found on one side of the nucleus. Bar, 1 μm. Also see Supplemental Material.

Localization of nucleoporins in mitosis. (A) In late prometaphase, the nuclear envelope falls back into the mother cell. Nup214-GFP leaves the envelope (214), whereas Pom152-GFP (152) disperses in the ER, with the most prominent signal in the old nuclear envelope (arrow). Nup107-GFP (107) first distributes into the cytoplasm, but it rapidly reappears in the daughter cell (arrowhead). The outline of the cell is indicated by a profile in the first image. D, daughter cell; M, mother cell. Bar, 3 μm. (B) Average signal intensities of Nup107-RFP and Nup214-RFP within the cytoplasm. Mitotic stages were identified by coexpression of histone 4-GFP (strains FB2N107R_H4G and FB2N214R_H4G). Values are mean ± SD. Sample size is indicated. (C) Average signal intensity of Nup107-GFP on the DNA over background cytoplasmic fluorescence in prometaphase and metaphase. Values are mean ± SD. Sample size is indicated. (D) Line-scan analyses of prometaphase and metaphase cells demonstrate that Nup107 associates with DNA after chromosome migration is completed and cells enter metaphase. The outline of the cells is indicated by a profile. D, daughter cell; and M, mother cell. Bar, 2 μm.

Nup107 subcomplex components colocalize in U. maydis metaphase. (A) The U. maydis homologues of Nup133 and Nup2 were identified by BLAST search, and the domain models were constructed according to SMART predictions (Pfam Nup133, BLAST value = 1.0 e⁻⁰⁶; and RanBD, BLAST value = 2.9 e⁻⁰⁹). Um, U. maydis; Sc, S. cerevisiae; Hs, H. sapiens. (B) Nup2-GFP localizes to the nuclear envelope, identified by ER-RFP, in a punctuate pattern like the other nucleoporin fusion proteins investigated. Bar, 1 μm. (C) The U. maydis homologue of Nup133 colocalizes with nuclear pore component Nup107 in interphase at the rim of the nucleus and in mitosis (metaphase) on the DNA in the bud. Bar, 1 μm.

Nup107 in U. maydis does not colocalize with kinetochores in mitosis. (A) In metaphase, a short spindle, lableded by YFP-αtubulin (Tub), is formed in the daughter cell that is surrounded by the condensed chromosomes, labeled by histone 4-CFP (H4). Nup107-YFP (107) also encompassed the metaphase spindle. Nup107-RFP (107) does not seem to colocalize with the kinetochore protein Mis12 (Mis), labeled with 3xGFP. Bar, 2 μm. (B) In anaphase A, the spindle (Tub) slowly elongates, and the chromosomes (H4) align along the spindle. Nup107-YFP (107) shows a similar distribution, indicating that the nucleoporin remains at the DNA during early anaphase. Nup107-RFP again does not colocalize with Mis12-3xGFP (Mis).

Nup107 rearrangement in anaphase. (A) As mitosis progresses through anaphase, the outer rims of the DNA masses (labeled by histone 4-CFP; H4) seem to collect ER membranes (marked by YFP with an ER retention signal) that are later incoporated into the newly forming nuclei (arrow). The outline of the cell is indicated by a profile in the first image. D, daughter cell; and M, mother cell. Time is given in minutes. Bar, 3 μm. Also see Supplemental Material. (B) Colocalization of Nup107-RFP (107) and histone 4-GFP-labeled chromosomes (H4) shows that the nucleoporin leaves the DNA and occurs at the outward edge of the separating chromosome masses during spindle elongation. In late anaphase B, Nup107 concentrates in small areas. Bar, 3 μm. (C) Line-scan analysis of an anaphase B spindle demonstrates that traces of Nup107-RFP are still found on the chromosomes (arrow), whereas most of the protein concentrates at the poles of the separating DNA. Bar, 3 μm. (D) In anaphase B, the new envelope begins to form at the outward rim of the chromosomes. Although Nup107 becomes incorporated into the newly forming NE, the other nucleoporins (Nup214, Pom152) are not yet concentrated at the new NE. Bar, 1.5 μm. Also see Supplemental Material.

Ordered reassembly of NPCs in telophase. (A) The new envelope closes in telophase (O'Donell and McLaughlin, 1984b). At this stage, Nup107 concentrates in the NE, whereas only traces of Nup214 and Pom152 are found. Note that low levels of Pom152 are present all over the ER. The nuclear marker NLS-3xRFP (nRFP) does not yet occur in the lumen of the small envelopes, indicating that protein import has not yet started. Bar, 1.5 μm. (B) As the chromosomes decondense, NLS-3xRFP occurs in the interior of the expanding nucleus, indicating that active protein import takes place. Nuclear import proceeds until cells begin to grow and form apical buds (integrated intensity; triangles), which indicates the beginning of G2 phase (Snetselaar and McCann, 1997; open symbols). Image series was taken from a single cell; the graph is based on the analysis of numerous nuclei whose circumference at a central plane was taken as an indication of their size and degree of chromosome decondensation. Elapsed time is given in minutes:seconds. Bar, 1.5 μm. (C) Quantitative analysis of the signal intensity of Nup107-GFP, Pom152-GFP, and Nup214-GFP (strains strains FB2N107G_ER, FB2N214G_ER, and FB2P152G_ER) in relation to the size of the nucleus, indicated by its circumference. An estimate of the timing and correlation to the cell cycle is presented above the graph. The amount of all nucleoporins continuously increases until cells begin to bud and reach G2 phase (open circles). (D) Colocalization of Pom152-GFP and Nup214-RFP in nuclei with a circumference of ∼4 μm shows that Pom152-GFP gives only a weak punctuate localization as indication of incorporation into NPCs, whereas the signal of Nup214-RFP is more pronounced (strain FB2P152G_214R), suggesting that Pom152 accumulates slightly before Nup214. Bar, 1 μm.

Role of the cytoskeleton in nuclear envelope reassembly. (A) Cells that express 2xRFP-Tub1 (Tub1) were placed on agarose cushions containing 30 μM benomyl. After 7 min (T = 7′), microtubules of the spindle are mainly disrupted (inset). D, daughter cell; and M, mother cell. Bar, 5 μm. (B) Anaphase cells are characterized by a cloud-like Nup107-GFP signal in the daughter cell that is divided by a cleft representing the spindle (see Figure 4). When placed on benomyl cushions (T = 0′), no ER membranes, marked by ER-RFP (ER), were surrounding the chromosomes. However, after 7-min treatment with benomyl (T = 7′), ER membranes surround the undivided chromosomes. These membranes are also decorated with a distinct Nup107-GFP signals, suggesting that they are newly formed NEs. Note that nuclear division and migration stopped in the absence of microtubules. Bar, 5 μm. (C) All nucleoporins tested reappear after 7 min of treatment with benomyl (T = 7′; strains FB2N133G_ER, FB2N214G_ER, and FB2P152G_ER). Bar, 2 μm. (D) In the absence of microtubules, chromosomes still decondese, and the Nup107-GFP containing nuclei enlarge. In agreement with the appearance of nucleoporins, protein import occurs, which is indicated by increasing levels of the NLS-3xRFP reporter protein (nRFP; T = 20′), suggesting that functional pores are formed. Time is given in minutes. Bar, 5 μm. (E) Treatment with 10 μM of the F-actin inhibitor latrunculin A did not have an obvious effect on NPC formation (Nup107) and protein import (nRFP; left, LatA), whereas simultaneous treatment with latrunculin and benomyl (right, LatA + Ben) abolished the formation of Nup107-labeled NPCs and nuclear import, suggesting that microtubules and F-actin cooperate in NE formation in U. maydis. Time is given in minutes. Bar, 2 μm.

Nuclear envelope behavior and NPC disassembly/reassembly in U. maydis. The interphase nucleus resides in the mother cell, and NPCs mediate communication between the cytoplasm and the interior of the nucleus. At the onset of mitosis, the spindle pole bodies become active, and astral microtubules pull them into the daughter cell. Chromosomes and some of the NPCs concentrate near the tip of this extension, whereas other NPCs already start to disassemble. In prometaphase, NPCs completely disassemble, and Nup107/Nup133 subcomplex and Nup214 disperse in the cytoplasm, whereas the integral Pom152 remains in the ER and the old envelope. In metaphase, the Nup107/Nup133 containing subcomplex partially returns to the DNA, where it also localizes during anaphase A. In late anaphase, the separating chromosomes begin to form new envelopes, in which Nup107 becomes concentrated. Nuclear envelopes close in telophase, and protein import starts after Nup214 and Pom152 are recruited. NPC assembly continues until G2 phase, when cells start to form a bud.