We identified Nudel as a protein associated with the kinetochore protein mitosin in a yeast two-hybrid screen by using the kinetochore-binding regions of mitosin as bait (data not shown) (40
). To study its biological functions, we generated specific polyclonal antibodies in chickens by using an MBP fusion containing residues 39 to 345 of Nudel as immunogen. Specific IgY was purified by affinity chromatography (10
). Purified IgY specifically recognized bacterial Nudel but not MBP (data not shown). Nudel and NudE are highly related proteins in mammals, with an identity of ~53%. As shown in Fig. , the IgY antibody actually recognized both FLAG-Nudel (lane 3) and FLAG-NudE (lane 4).
FIG. 1. Biochemical properties of cellular Nudel and NudE. (A) Specificity of anti-Nudel IgY. HEK293T cells expressing either FLAG-Nudel (lanes 1 and 3) or FLAG-NudE (lanes 2 and 4) were lysed and subjected to SDS-10%PAGE and immunoblotting with the indicated (more ...)
To identify cellular Nudel and NudE, CV1 lysate was immunoprecipitated and then immunoblotted by using purified IgY. A doublet of ~40 kDa was detected (data not shown). To confirm that the doublet was related to Nudel, two aliquots of IgY were incubated with 10 μg of MBP or the immunogen, respectively. As shown in Fig. , preincubation with MBP did not affect recognition of the doublet (lane 1); in contrast, treatment with the immunogen completely blocked the antibody, as expected (lane 2). Since human Nudel is just 10 residues longer than NudE, the doublet appeared to correspond to both proteins.
Purified IgY recognized protein bands of ~40 kDa in a variety of cell lines, including CV1, human hepatoma SMMC-7721, human embryonic kidney HEK293, human astrocytoma U251, mouse neuroblastoma and rat glioma hybrid cell line NG108-15, and human neuroblastoma SK-N-SH (Fig. ). Nudel and NudE were therefore widely distributed, with relatively low levels in HEK293 (Fig. , lane 3). Further studies with mouse tissues indicated that the proteins were highly expressed in brain tissue (Fig. , lane 5), which was in agreement with other reports (20
). Nudel and NudE were also expressed in heart (Fig. , lane 3), skeletal muscle (lane 6), and lung (lane 7) tissue. In contrast, they were almost undetectable in spleen (Fig. , lane 1), stomach (lane 2), kidney (lane 3), and liver (lane 8) tissue.
Phosphorylation of Nudel and NudE in M phase.
To investigate the expression patterns of the Nudel family proteins during the cell cycle, SMMC-7721 cells were synchronized at G1
/S, S, M, and G1
phase, respectively (40
). The extent of synchrony was monitored by flow cytometry. The sample synchronized at the G1
/S boundary by using hydroxyurea was estimated to be 53.1% in G1
, 30.2% in S, and 16.7% in G2
/M. An S-phase sample was collected after release from the G1
/S boundary for 4 h; its cell cycle profile was 71.5% in S, 19.9% in G1
, and 8.6% in G2
/M. An M-phase sample was collected by mitotic shake-off after hydroxyurea and nocodazole blocking; its cell cycle distribution was 91.8% in G2
/M, 2.1% in G1
, and 6.1% in S. A G1
-phase sample was prepared by releasing a portion of the M-phase sample for 6 h; its cell cycle profile was 59.0% in G1
, 10.0% in S, and 30.0% in G2
/M. Microscopic examination of 4′,6′-diamidino-2-phenylindole (DAPI)-stained cells indicated that, in this G1
sample, the mitotic index was less than 5%. Cells in this G1
sample were indeed mainly in interphase.
Immunoblotting indicated a distinct pattern of Nudel and NudE in the cell cycle. In SMMC-7721 cells, only a single band was detected in interphase (Fig. , lanes 1 to 3). The protein level in S phase was two- to threefold higher than those in G1
/S. Detection of a doublet in CV1 but of only a single band in SMMC-7721 might suggest differential distribution of both proteins (9
). In M phase, however, multiple bands appeared (Fig. , lane 4). Their slower migration in SDS-polyacrylamide gel electrophoresis (PAGE) excluded the possibility of degradation but favored posttranslational modification. A homologue of Nudel in Xenopus
, MP43, has been shown to be phosphorylated by mitotic extract in vitro but not by interphase extract (30
). Since phosphorylation of MP43 also results in similar migration changes (30
), we reasoned that the band shift of Nudel and NudE in M phase might also be due to phosphorylation.
FIG. 2. Phosphorylation of Nudel and NudE in M phase. (A) Specific modification of Nudel and NudE in M phase. Human hepatoma SMMC-7721 cells were synchronized at the indicated stages of the cell cycle prior to lysis. Nudel and NudE were immunoprecipitated from (more ...)
To test if Nudel and NudE were indeed phosphorylated, the proteins were immunoprecipitated from SMMC-7721 cells synchronized in M phase and were treated with calf intestinal alkaline phosphatase (CIAP). After CIAP treatment the protein band migrated to the same position the interphase Nudel and NudE did (Fig. , lanes 1 and 2). In contrast, the migration of mock-treated Nudel was not affected (Fig. , lane 3). The slower migration bands were therefore phosphorylated forms of Nudel and NudE.
To distinguish Nudel from NudE, FLAG-tagged Nudel and NudE were expressed in HEK293T cells and were immunoblotted with anti-FLAG M2 antibody. In cells treated with nocodazole to enrich M-phase population, the phosphorylated forms characterized by their slower migration rates were readily detected for both FLAG-Nudel (Fig. , lane1) and FLAG-NudE (lane 5) in addition to unphosphorylated forms due to the existence of interphase cells (Fig. , compare lanes 2 and 6). Both proteins were therefore phosphoproteins in M phase. Furthermore, when the threonine and serine resides in all S/TP motifs of Nudel were mutated into valine or alanine, the phosphorylated form disappeared (Fig. , lane 3). As a control, the percentages of mitotic cells expressing FLAG-Nudel and FLAG-Nudelmt5 were comparable after IIF staining of the sample cells. The major phosphorylation sites of Nudel in M phase were therefore located in the S/TP motifs.
Phosphorylation by Cdc2 and Erk2.
Cdc2 is a major kinase controlling G2
/M transition (19
). Erk forms of MAPK, on the other hand, are important for meiosis progression in addition to their roles in G0
). Although their functions in somatic cell mitosis are less understood, association of activated Erk1 and -2 with kinetochores and asters implies their involvement (37
Since Nudel and NudE were both phosphorylated in M phase, we first checked if they were substrates of Cdc2 in vitro. As shown in Fig. , incubation of FLAG-tagged NudE (lane 1) and Nudel (lane 2), but not Nudelmt5 (lane 3), with purified Cdc2 (New England Biolabs) resulted in incorporation of [32P]phosphate groups. Since three of the five S/TP motifs in Nudel are typical MAPK sites (PXS/TP), we also performed similar assays by using purified Erk2. As shown in Fig. , activated Erk2 (New England Biolabs) also phosphorylated both Nudel (lane 2) and NudE (lane 4) but not Nudelmt5 (lane 5). Phosphorylation was not detected in the absence of exogenous kinase (lanes 1 and 3), excluding the possibility of kinase contamination in the immunoprecipitates. The phosphorylation sites of Erk2 therefore also fell into the S/TP motifs.
FIG. 3. Phosphorylation by Cdc2 and Erk2. (A) Phosphorylation by Cdc2 in vitro. FLAG-tagged NudE, Nudel, and Nudelmt5 were immunoprecipitated with anti-FLAG beads and were treated with purified Cdc2 in the presence of [γ-32P]ATP. (B) Phosphorylation by (more ...)
To further determine the detailed phosphorylation sites, we purified five FLAG-tagged mutants, NudelS1 to NudelS5. Each mutant contained only one distinct S/TP motif. When these mutants were incubated with purified Cdc2, only NudelS2, NudelS4, and NudelS5 were phosphorylated (Fig. ). Phosphorylation sites of Erk2 were determined in the same way (Fig. ). In this case, only NudelS2 and NudelS5 were phosphorylated. Therefore, T219, S242, and T245 of Nudel were phosphorylation sites of Cdc2 in vitro. In contrast, Erk2 only phosphorylated T219 and T245. These two sites, with surrounding sequences such as PATP from residues 217 to 220 and PLTP from 243 to 246, respectively, are indeed typical MAPK sites.
To get further clues for kinases responsible for Nudel phosphorylation in M phase, we performed kinase assays by using mitotic extracts prepared from SMMC-7721 cells (Fig. ). As expected, the extracts phosphorylated histone H1 (Fig. , lane 1), and the phosphorylation was completely abolished in the presence of 50 μM olomoucine (lane 2), a potent Cdc2 inhibitor (50% inhibitory concentration, 7 μM), and less-potent Erk1 inhibitor (50% inhibitory concentration, 25 μM) (32
). The extracts also phosphorylated FLAG-Nudel (Fig. , lane 4) but not FLAG-Nudelmt5
(lane 3). Similarly, phosphorylation of FLAG-Nudel was eliminated in the presence of olomoucine (Fig. , lane 5).
Association with the centrosomes and mitotic spindles.
Both Nudel and NudE are centrosome proteins, although noncentrosomal fractions always exist in the cytoplasm (9
). Our IgY also consistently recognized centrosomal Nudel and NudE (Fig. , panel 1), which colocalized with the microtubule-organizing center (MTOC) (panel 2).
FIG. 4. Association of Nudel and NudE with centrosomes and mitotic spindles. Arrows and arrowheads point to positions of centrosomes and spindle poles, respectively. Scale bar, 10 μm. (A) Confocal images showing the centrosomal staining of anti-Nudel (more ...)
To further explore their roles in the cell cycle, we expressed GFP-Nudel and GFP-NudE in HEK293 cells so that localization of exogenous proteins could be directly visualized through GFP autofluorescence. High-level expression of either protein resulted in bright punctate distribution in the cytoplasm (data not shown). In contrast, cells expressing moderate to low levels of GFP fusion proteins featured bright juxtanuclear fluorescence dots or speckles (Fig. , panel 1). Some cells had one or two closely spaced dots colocalized with γ-tubulin (Fig. , panels 2 and 3), a centrosome marker (38
). The rest showed multiple fluorescence dots clustered together (Fig. , panel 1). In such cells, at least one dot colocalized with γ-tubulin (Fig. , panels 2 and 3), indicating that the dot clusters are located at the centrosome regions. During M phase, the centrosomal association of GFP-Nudel was largely reduced. Instead, a fraction of the protein associated with the mitotic spindle, whereas the rest dispersed in the cytoplasm (Fig. , panels 4 to 6). GFP-NudE behaved in a manner similar to that of GFP-Nudel (Fig. , inlets). Their association with spindles was usually weak but was clearly visible over the background of cytoplasmic fluorescence. As a control, GFP alone failed to target to either the centrosome or the spindle (Fig. , panels 1 to 4).
Regulation of the subcellular localization by phosphorylation.
The coincidence between phosphorylation of Nudel and NudE and the reduced spindle pole localization in M phase implies that phosphorylation may regulate their distribution during the cell cycle. In addition to Nudelmt5
, we created another mutant, Nudelpmt5
, in which the serine and threonine residues in all S/TP motifs were mutated into glutamic acids to hopefully mimic phosphorylation (7
). Effects of phosphorylation on subcellular localization of Nudel were explored by comparing behaviors of the mutants with that of the wild type.
We found that both GFP-Nudel and GFP-Nudelmt5 formed bright centrosomal foci in interphase cells (Fig. and B, panels 1 and 2). In contrast, GFP-Nudelpmt5 only weakly localized to the centrosomes (Fig. , panels 1 and 2). For quantitative purposes, their centrosomal fluorescence was measured from 10 cells with comparable GFP fluorescence for each protein. Although the values varied from cell to cell, the centrosomal intensities of the first two proteins were generally six- to eightfold higher than those of GFP-Nudelpmt5. From metaphase to anaphase, while a fraction of GFP-Nudel associated with mitotic spindles (Fig. , panels 3 to 6), GFP-Nudelmt5 bound preferably to spindle poles (Fig. , panels 3 to 8). GFP-Nudelpmt5 behaved like GFP-Nudel during this period (Fig. , panels 3 to 6). In telophase, while the spindle pole localization of GFP-Nudel became apparent (Fig. , panels 7 and 8), GFP-Nudelpmt5 still failed to show such localization clearly (Fig. , panels 7 and 8). Phospho-Nudel and Nudelpmt5, which simulated phosphorylation, thus correlated with stronger distribution on spindles at metaphase (Fig. , panels 3 and 4) and weak association with the MTOC. In contrast, unphosphorylated Nudel in interphase cells and Nudelmt5, which was incapable of phosphorylation, exhibited stronger localization at the MTOC.
FIG. 5. Phosphorylation regulates localization of Nudel at the centrosomes and mitotic spindles. Typical HEK293T cells expressing GFP-tagged Nudel (A), Nudelmt5 (B), or Nudelpmt5 (C) are shown for comparison. DNA was stained by DAPI. Panels 1 and 2, interphase (more ...)
To further corroborate these findings, we fractionated centrosomes from HEK293T cells transfected to express each of the three fusion proteins. Immunoblotting indicated that GFP-Nudel was indeed cosedimented with the centrosomal fractions indicated by γ-tubulin (Fig. , lanes 4 to 6), and so were GFP-Nudelmt5 and GFP-Nudelpmt5 (data not shown). Moreover, although the levels of Nudel and mutants were similar in total cell lysates (Fig. , lanes 1 to 3), GFP-Nudelpmt5 exhibited much weaker association with centrosomes (Fig. , lane 3). In contrast, GFP-Nudel and GFP-Nudelmt5 exhibited comparable levels in the centrosome fractions (lanes 1 and 2).
FIG. 6. Association of GFP-Nudel and mutants with centrosome fractions. (A) Copurification of GFP-Nudel with centrosome fractions. Centrosomes were fractionated from HEK293T cells expressing GFP-Nudel as described in Materials and Methods. Fractions with odd (more ...) Convergence to the spindle poles following depletion of cellular ATP.
Nudel and NudE appear as regulatory factors for cytoplasmic dynein through direct interaction (9
), although more direct evidence is still required for better understanding of their functional relationship. In mitotic PtK1 cells, the spindle checkpoint proteins, such as Mad2, Bub1, and CENP-E, are constantly transported from kinetochores to spindle poles along spindle microtubules by cytoplasmic dynein (13
). This dynamic process, which contributes to inactivation of the spindle checkpoint essential for proper anaphase onset, can be visualized by treating cells with azide to reduce cellular ATP to 5 to 10% of normal levels (ATP inhibitor assay). Such a treatment results in depletion of these proteins, including dynein, from kinetochores and subsequent accumulation at spindle poles in a microtubule-dependent manner (13
). ATP is important for dissociation of dynein from microtubules (22
). Reduction of cellular ATP levels after azide treatment probably impairs this process, leading to accumulation of dynein and its partners or cargos at the extreme minus ends of microtubules or the MTOC. A continuous transport process can therefore be visualized easily. If Nudel and NudE are functional partners of dynein in M phase, they should comigrate with it in the ATP inhibitor assay.
We found that Nudel and NudE were indeed transported to spindle poles after azide treatment in mitotic HEK293T cells. As shown in Fig. , the pattern of GFP was not altered without (panel 1) or with (panel 2) azide treatment. However, GFP-NudE exhibited strong spindle pole localization in azide-treated cells (Fig. , panels 3 to 6) in contrast to mock-treated ones (panels 1 and 2). Star-shaped structures around spindle poles were especially visible in cells at prometaphase (Fig. , panels 3 and 4), suggesting association of GFP-NudE with aster microtubules. GFP-Nudel also showed the similar phenotypes (data not shown). In mock-treated cells, a fraction of dynein exhibited its typical spindle localization (Fig. , panel 2) (14
), a pattern resembling that of GFP-Nudel (Fig. , panel 1). Thirty minutes after azide treatment, both proteins were concentrated at the spindle poles (Fig. , panels 1 and 2). Such poleward congression was also observed in cells from anaphase (Fig. , panels 4 to 6) to early telophase (data not shown). At late telophase, however, dynein no longer displayed dramatic accumulation at poles after azide treatment. Instead, the majority of it concentrated in regions around the midbody, where the remains of interdigitated spindle microtubules resided (Fig. , panel 8). A substantial fraction of GFP-Nudel, despite it being in aggregation form, was also distributed in these areas (Fig. , panel 7). Interestingly, GFP-Nudel and GFP-NudE frequently aggregated into numerous foci in both mitotic cells (Fig. and E) and interphase cells (see Fig. ) following azide treatment, although the number of foci varied from cell to cell. Such aggregation, however, was not observed in cells expressing GFP alone (Fig. ). The above patterns clearly differed from those seen with mock-treated cells (Fig. , panels 4 to 9). These data reinforced the point that Nudel and NudE bound to cytoplasmic dynein and migrated with it to spindle poles.
FIG. 7. Transport of Nudel and NudE to spindle poles along microtubules. HEK293T cells transfected with appropriate plasmids for 36 h were treated with phosphate-buffered saline (saline) or phosphate-buffered saline containing Na-azide and DOG (Az/DOG) for 30 (more ...)
FIG. 9. Effects of Nudel phosphorylation on azide-induced convergence to centrosomes and Lis1-binding activity. (A) Time-lapse images for interphase cells expressing the indicated proteins. Images were recorded live every 3 min following ATP inhibitor assays. (more ...)
To further corroborate these results, we treated cells with nocodazole for 3 h to disassemble microtubules prior to ATP inhibitor assay in the presence of nocodazole. As a control, the majority of cellular GFP-Nudel was transported to spindle poles in cells without nocodazole treatment (Fig. ). The presence of nocodazole completely blocked the polar transport of Nudel in M phase, indicating that the transport process is indeed microtubule-dependent (Fig. ). Aggregation of Nudel following azide treatment, however, was not affected by nocodazole (Fig. ).
Involvement in cytoplasmic dynein-mediated poleward transport.
To clarify if Nudel and NudE were simply passengers of the dynein motor or active players for dynein function, we created a deletion mutant, GFP-NudelN20
, which lacked amino acids 114 to 133 in the Lis1-binding domain (Fig. ) (25
). Such deletion completely disrupted Nudel-Lis1 interaction (Fig. ) but not Nudel homodimerization (data not shown). Moreover, since the dynein-binding domain between residues 256 to 345 was intact, this mutant should still interact with dynein (25
). Time-lapse microscopy showed that, compared to that of wild-type GFP-Nudel (Fig. ), accumulation of this mutant at spindle poles after azide treatment was dramatically reduced (Fig. ), suggesting impairment of its poleward transport. In either untreated or mock-treated mitotic cells, GFP-NudelN20
did not exhibit clear spindle localization (Fig. , panel 1, and F, panel 1) (data not shown). The typical distribution of dynein on the spindle (Fig. , panel 2) was also disrupted in cells expressing GFP-NudelN20
(Fig. , panel 2). As controls, in the surrounding untransfected mitotic cells of both mock-treated and azide-treated samples, dynein still displayed its typical localization (data not shown). After azide treatment, cytoplasmic dynein also failed to fully congregate at spindle poles (Fig. , panels 4 to 6). Rather, the majority of dynein was trapped on the spindle (Fig. , panel 5), indicating that the poleward movement of dynein was at least partially disrupted by NudelN20
FIG. 8. Involvement of Nudel in dynein-mediated poleward transport. Arrowheads point to spindle poles. Scale bar, 10 μm. (A) Diagram of NudelN20. BD, binding domain. (B) Loss of interaction between NudelN20 and Lis1. HEK293T cells coexpressing HA-Lis1 (more ...)
We further examined if overexpression of GFP-NudelN20
impaired the poleward transport of spindle checkpoint proteins by dynein. Bub1 is one of the spindle checkpoint proteins transported from kinetochores to spindle poles (14
). Due to a lack of appropriate antibodies for IIF, we coexpressed FLAG-Bub1 with GFP-Nudel in HEK293T cells. In intact or mock-treated metaphase cells, a portion of FLAG-Bub1 showed distribution on the spindle similar to that of GFP-Nudel (Fig. , panel 1 to 3 and data not shown). Its kinetochore localization was hardly visible at this stage. After azide treatment both Bub1 and Nudel displayed strong accumulation at spindle poles as discrete speckles (Fig. , panels 4 to 6). However, coexpression with GFP-NudelN20
led to inhibition of the poleward transport of FLAG-Bub1 (Fig. , panels 4 to 6). Although some proteins were accumulated at spindle poles, a substantial amount of FLAG-Bub1 was distributed on the entire spindle (Fig. , panel 5), resembling the pattern of dynein (Fig. , panel 5). In addition, the spindle localization of FLAG-Bub1 was also disrupted in control cells (Fig. , panel 2). These data indicated a clear role of Nudel for the activities of the dynein motor during M phase.
Effects of Nudel phosphorylation on its dynein-mediated transport and Lis1-binding activity.
To further explore how phosphorylation affected Nudel functions, we first examined if phosphorylation affected accumulation of Nudel at the MTOC. Time-lapse microscopy revealed dramatic intensification of centrosomal fluorescence of GFP-tagged Nudel, Nudelmt5, and Nudelpmt5 following the time of azide treatment in both mitotic cells (data not shown) and interphase cells (Fig. ), indicating that the phosphorylation status of Nudel is not critical for dynein function. In mitotic cells, their average accumulation rates were sequentially 3,495 ± 536/min (means ± standard deviations) (n = 4), 1,553 ± 354/min (n = 5), and 1,935 ± 403/min (n = 8) according to our studies with time-lapse microscopy. For the interphase cells shown in Fig. , the quantitative data for their centrosomal fluorescence intensities are listed in Fig. . According to these results, we propose that phosphorylated Nudel exhibits a higher dissociation rate from the MTOC and therefore weaker localization at spindle poles (see Discussion).
For clues at the molecular level, we performed coimmunoprecipitation to check interactions between Nudel, Lis1, and dynein. As shown in Fig. , FLAG-Nudelpmt5 reproducibly pulled down much more endogenous Lis1 than FLAG-tagged mt5 mutant or wild-type Nudel did in interphase (lanes 2 to 4), although their protein levels were constant in the total cell lysates (data not shown). To further corroborate these results, transfected cells were treated with nocodazole to enrich M-phase population. Microscopic examination indicated that about 50% of transfectants on average were arrested in prometaphase after 12 h of treatment. Since poor attachment of HEK293T cells to culture dishes precluded shake off of the mitotic cells, whole populations were assayed. In this case, both the pmt5 mutant and Nudel brought down more Lis1 than the mt5 mutant did (Fig. , lanes 5 to 7). DIC was detected in the immunocomplexes as faint bands only after extensive incubation (overnight) during immunoprecipitation (Fig. ). These data indicate that phospho-Nudel binds to Lis1 more efficiently. This conclusion is also consistent with the slightly stronger association of Lis1 with FLAG-Nudel than with the mt5 mutant (Fig. , lanes 3 and 4), because a small fraction of Nudel was phosphorylated (lane 4, upper panel) due to the existence of mitotic cells in the population.