Two proteins that cycle asynchronously between centrosomes and nuclear structures: Drosophila CP60 and CP190

In animal cells, centrosome-nucleated microtubule arrays function in a wide variety of cellular processes including cell division and chromosome segregation, directed cell movement, and interphase cytoplasmic organization (for reviews see Mazia, 1987; Vorobjev and Nadezhdina, 1987; Kellogg et al., 1994). Studies using electron microscopy have shown that centrosomes consist of a pair of centriolar cylinders surrounded by a cloud of pericentriolar material (PCM) that is the source of the nucleated microtubules (Gould and Borisy, 1977; Keryer et al., 1984; Rieder and Borisy, 1982; Vorobjev and Chentsov, 1982). The microtubule nucleating activity of the PCM is due to the presence of γ-tubulin containing ring complexes (γ-TuRCs) that can nucleate microtubules when isolated in vitro and that appear to be anchored within the PCM of intact centrosomes when visualized by EM tomography (Moritz et al., 1995; Zheng et al., 1995).

The centrosome is structurally very dynamic, changing continuously in a cell cycle-dependent manner. The centriolar cylinders duplicate once per cell cycle in concert with changes in the surrounding PCM that are thought to provide the structural basis for the very different interphase and mitotic microtubule assemblies. In interphase tissue culture cells, the PCM contains small electron-opaque aggregates, or satellites, that surround the parent centriole. During prophase, the satellites disappear and are replaced by a large mitotic ‘halo’ of lighter staining material that surrounds the parent centriole (Robbins et al., 1968; Rieder and Borisy, 1982; Vorobjev and Chentsov, 1982). The nucleating capacity of centrosomes also changes in a cell cycle dependent fashion; in one study, mitotic centrosomes were found to nucleate about five times as many microtubules as interphase centrosomes (Kuriyama and Borisy, 1981). What molecular changes underlie these architectural transitions?

At a molecular level, very little is known about either the protein composition of the PCM or the cell cycle-specific regulation of its structure and nucleating capacity, although phosphorylation has been proposed to play a role in the mitotic maturation of centrosomes (Buendia et al., 1992; Vandre and Borisy, 1989; Bailly et al., 1989; Centonze and Borisy, 1990; Engle et al., 1988). A small number of proteins have been identified that are able to maintain their centrosomal localization in the absence of microtubules, suggesting that they are core components of the PCM: these include γ-tubulin, pericentrin, centrosomin, CP190 and CP60 (Raff et al., 1993; Doxsey et al., 1994; Li and Kaufman, 1996; Oegema et al., 1995).

Like the centrosome, the nucleus is a complex and dynamic organelle. It is clear that the nucleus is a highly ordered structure in which chromosomes are arranged in specific configurations (Comings, 1980). It has been proposed that a protein-based nuclear skeleton exists which gives the nucleus its structure and organizes both chromosomes and enzyme complexes involved in DNA replication (Hozak et al., 1993), transcription (Carter et al., 1993), and RNA processing and transport (Spector, 1993; Meier and Blobel, 1992). Electron microscopic preparations using special resinless embedding methods have revealed a scaffold-like protein network within the nucleus (Capco et al., 1982), although such networks have not been seen using more conventional preparations, and could be artifactual. Likewise, several different biochemical preparations have been described in which isolated nuclei are extracted with special detergents and solvents, and in some cases heattreated, leaving behind an insoluble protein residue (Capco et al., 1982; Fisher et al., 1982; Lebkowski and Laemmli, 1982). These nuclear scaffold or matrix preparations may reflect an actual connected structure present in living cells, or they may simply reflect an aggregation of proteins induced by the preparation procedure. Thus, a major question of nuclear architecture has been to what extent is there a coherent protein skeleton within the nucleus in vivo? The key to this question is to identify protein components of such a skeleton which can themselves be visualized in living cells, and to ask if the dynamics of these proteins is consistent with their assembly into large-scale structures within the nucleus in vivo.

An understanding of the dynamic structural changes that occur at centrosomes and within nuclei will require the isolation of well defined centrosomal and nuclear components and the study of their dynamics at centrosomes and in nuclei. CP190 and CP60 are two nuclear components that are also components of the core PCM, shuttling between nuclei and centrosomes in a cell-cycle dependent manner (Frasch et al., 1986; Whitfield et al., 1988, 1995; Kellogg et al., 1995; Raff et al., 1993; Oegema et al., 1995).

The cloning and sequencing of CP190 revealed a novel protein of 1,096 amino acids, containing a cluster of four putative zinc fingers located in the middle of the predicted protein (Whitfield et al., 1995). The regions of CP190 responsible for its nuclear and centrosomal localizations have been identified (Oegema et al., 1995).

CP60 was identified by immunoaffinity chromatography on columns constructed from anti-CP190 antibodies (Kellogg and Alberts, 1992). Like CP190, CP60 localizes to nuclei and to centrosomes in a cell cycle-dependent manner (Kellogg et al., 1995). CP60 has been cloned and sequenced and shares no significant amino acid homology with any other known protein; it contains 6 consensus cdc2 phosphorylation sites and is phosphorylated in vivo (Kellogg et al., 1995).

To begin to get a molecular handle on the cell cycledependent changes that occur at centrosomes and in nuclei, we have compared the localizations of CP190 and CP60 both by high resolution light microscopy (in fixed embryos) and by quantitative 3-D wide-field fluorescence time-lapse microscopy (in live embryos following injection of the fluorescently labeled proteins). We find that, although CP60 was identified by its biochemical association with CP190, the two proteins have very different temporal and spatial localization patterns in Drosophila embryos. Surprisingly, the nuclear envelope is not required for the nuclear retention of CP60, and the localization pattern of CP60 suggests that it binds to a novel nuclear structure that persists into mitosis. Because we are able to visualize this CP60 network directly in living cells, we conclude that some nuclear structure independent of chromosomes exists in vivo. The presence of a protein in this structure that is also a component of the pericentriolar material could suggest a link between the centrosome and the nucleus.

The expression, purification and fluorescent labeling of the CP190 and CP60 fusion proteins were performed as described previously, as were embryo injection and confocal microscopy (Oegema et al., 1995).

Three-dimensional images of living embryos were obtained by widefield fluorescence microscopy (Hiraoka et al., 1991) using an Olympus ×60 objective with a numerical aperture of 1.4. The computer-controlled stage was stepped in vertical increments of 0.75 μM, and at each vertical position, one 256×256 pixel optical section was acquired for each wavelength, using a cooled CCD. A total of 16 optical sections in each wavelength (fluorescein and rhodamine) were collected to form a single 3-D multiwavelength image. The stage was then reset to the initial position and the process repeated continuously, to form a time-lapse series. Using this scheme, one two-wavelength 3-D image was collected every 52 seconds. The fixed embryos were examined the same way, except that 512×512 pixel optical sections were taken. For the high resolution micrographs of fixed material optical sections were taken every 0.2 μm. Following image acquisition, out of focus blur was removed using constrained iterative deconvolution (Agard et al., 1989).

In order to quantify changes in total fluorescence in centrosomes and nuclei, all centrosomes and nuclei were defined by tracing their outlines using an interactive modeling program (Chen et al., 1995). The outlines thus defined were used to classify each pixel in the image as nuclear, centrosomal, or cytoplasmic background. In each section, the average cytoplasmic background was calculated and then subtracted from each nuclear pixel in order to reduce the contribution of scattered light to the measurements. Because out-of-focus light from the nuclei occasionally overlapped a centrosome even after deconvolution, the average intensity of all pixels falling outside a centrosome but within 0.35 μm of the centrosomal boundary was computed and subtracted from each pixel in the centrosome. The total intensity contributed by all centrosomal and nuclear pixels was then calculated separately for each wavelength.

Embryos were fixed in 37% formaldehyde as described (Theurkauf, 1992). The rabbit antibodies to CP60 and to amino acids 385-508 of CP190 have been described (Kellogg et al., 1995; Oegema et al., 1995). The goat anti-CP190 antibody used was prepared by immunizing a goat with a total of 4 mg of a maltose binding protein-fusion with CP190 amino acids 606-870, a fragment of CP190 previously described as 190c (Kellogg and Alberts, 1992). Immunizations and bleeds were carried out by the Berkeley Antibody Company (Richmond, CA). Antibodies were affinity purified as described (Kellogg and Alberts, 1992). Secondary antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc (West Grove, PA). For the immunofluorescence in Fig. 4, rabbit anti-CP60 and rabbit anti-CP190 were directly labeled with N-hydroxy succinimidyl fluorescein (Molecular probes, Eugene, OR) and N-hydroxy succinimidyl Cy-5 (Amersham, Arlington Heights, IL), respectively, according to the manufacturer’s instructions.

Fluorescence in situ hybridization (FISH) with a digoxygenin-labeled rDNA probe (Dernberg et al., 1996a) was carried out in fixed embryos using a modified version (Dernburg et al., 1996b) of a previously published method (Hiraoka et al., 1993). Following FISH, embryos were washed four times in 2× SSCT (0.3 M CaCl, 0.03 M Na₃ citrate, 0.1% Tween-20), blocked with 6 mg/ml normal goat serum (Jackson Immunoresearch Laboratories, West Grove, PA) in 2× SSCT for 4 hours, and were then incubated overnight with rabbit anti-CP60 antibody at 1.3 μg/ml in 2× SSCT. The embryos were then washed four times in 2× SSCT (1 hour per wash), incubated for 4 hours with fluorescein rat anti-digoxygenin (1:8,000) and rhodamine donkey anti-rabbit (diluted 1:200) in 2× SSCT. Embryos were washed three times for 10 minutes and then overnight in 2× SSCT before washing for 30 minutes in 2× SSCT and staining for 10 minutes in 0.5 μg/ml DAPI in 2× SSCT. Embryos were then washed two times in 50 mM Tris-HCl, pH 8.5, and mounted in antifade mounting medium (Vectashield, Vector Laboratories, Inc., Burlingame, CA).

Schneider cells were cultured at 25°C in D22 insect medium (Sigma Chemical Co.) supplemented with 10% fetal calf serum. Approximately 6.5× 10⁸ cells were pelleted in a clinical centrifuge for 3 minutes at 860 g. The pellet was washed once in PBS and then resuspended in 50 ml of 10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 5 mM MgCl₂, 1/1,000 protease inhibitor stock, 0.1 mM PMSF and allowed to swell for 5 minutes at room temperature before repelleting. (Protease inhibitor stock is 1.6 mg/ml benzamidine HCl and 1 mg/ml each phenanthroline, aprotinin, leupeptin and pepstatin A.) The cell pellet was resuspended in 10 ml of 0°C lysis buffer (15 mM Tris-HCl, pH 7.4, 80 mM KCl, 5 mM MgCl2, 0.1% digitonin, 1/100 protease inhibitor stock, 1 mM PMSF), immediately transferred to a 15 ml glass dounce and homogenized by 10-15 strokes with a tight pestle. The lysate was spun at 1,000 g for 10 minutes at 4°C. Pelleted nuclei were washed twice with 40 ml of 5 mM Tris-HCl, pH 7.4, 2 mM KCl, 5 mM MgCl₂, 0.1% digitonin, 1/100 protease inhibitor stock, 1 mM PMSF. Pelleted nuclei were resuspended in 1 ml of digestion buffer (10 mM Tris-HCl, pH 7.4, 50 mM NaCl, 300 mM sucrose, 3 mM MgCl₂, 1 mM Na₃EGTA, 1 mM PMSF, 1/100 protease inhibitor stock and 0.5% (v/v) Triton X-100) and digested at 37°C for 20 minutes in the presence of 100 units/ml RNase free DNase (BoehringerMannheim Biochemicals, Indianapolis, IN). Ammonium sulfate was added at 25°C from a 1 M stock solution to a final concentration of 0.25 M. The nuclei were pelleted as above and resuspended in 500 μl of digestion buffer. NaCl was added to a final concentration of 2 M from a 4 M NaCl stock in digestion buffer. Nuclei were held on ice for 10 minutes before pelleting the matrices and resuspending them in 800 μl of digestion buffer.

To prepare samples for western blotting, 50 μl samples were taken of nuclei, nuclear matrices, total lysate and cytoplasm. CaCl₂ was added to 2 mM, 1/20 volume of 2 mg/ml micrococcal nuclease was added and the samples were held on ice for 30 minutes. Load buffer (60 mM Tris-HCl, pH 6.8, 2% SDS, 20 mM DTT) was added to 1 ml and the samples were heated at 90°C for 3 minutes. Samples were precipitated by the addition of 0.2 volumes of 100% trichloracetic acid and were resuspended in sample buffer and sonicated for 1 minute in a waterbath sonicator. Samples of lysate, cytoplasm, nuclei and matrices corresponding to 9 μg of crude lysate were separated on an 8.5% (CP190, CP60 and topo II) or a 10-15% (histones) polyacrylamide gradient gel and were western blotted for Topo II, CP190, CP60 and histones. Western blotting was performed as described (Kellogg et al., 1989) using mouse monoclonal antibodies to topoisomerase II (Swedlow et al., 1993) or histones (mAb 052, Chemicon Industries), or the affinity purified rabbit anti-CP60, and goat anti-190 antibodies described earlier. Signals were detected by Enhanced Chemiluminescence (Amersham).

In order to directly compare the behavior of CP190 and CP60 in living embryos, we co-injected embryos with fluoresceinlabeled CP60 and rhodamine-labeled CP190 fusion proteins. The embryos were then followed during the surface syncytial divisions (nuclear cycles 10-13) using confocal microscopy. A time lapse series of confocal images of such an embryo, beginning in interphase of nuclear cycle 12, is shown in Fig. 1A (the times to the left of each panel are relative to nuclear envelope breakdown). Although both CP190 and CP60 shuttle between nuclei and centrosomes, the timing of their localizations is significantly different. In interphase of nuclear cycle 12 (Fig. 1A, 0 seconds), both CP190 and CP60 are localized to nuclei. Upon nuclear envelope breakdown (0 seconds), the CP190 immediately begins to move to centrosomes and, by +31 seconds, CP190 centrosomal fluorescence has reached nearly maximal intensity; CP190 remains at centrosomes throughout mitosis until telophase, when it begins to accumulate in reforming nuclei (311 seconds). CP60, in contrast, accumulates at centrosomes primarily during anaphase and telophase (see Fig. 1A, 222, 256 and 311 seconds).

To characterize the localizations of CP190 and CP60 relative to nuclear envelope breakdown and reformation, embryos were co-injected with either rhodamine labeled CP60 or CP190 and fluorescein labeled 40,000 MW dextran. The dextran is excluded from nuclei with intact nuclear envelopes, and it therefore provides a marker for nuclear envelope breakdown and reformation (Kalpin et al., 1994). Shown in Fig. 1B is a time lapse series of confocal images taken of a CP60/dextran injected embryo. In interphase (−40 seconds), the labeled dextran is excluded from nuclei, making them visible as black circles against a background of cytoplasmic dextran. Between nuclear envelope breakdown and late metaphase, CP60 remains attached to residual nuclear structures with very little accumulation at centrosomes (Fig. 1B, 0 and 51 seconds). CP60 accumulates dramatically at centrosomes during anaphase and telophase (Fig. 1B, 196 seconds and 327 seconds) and remains at centrosomes at reduced intensity, into early interphase (485 seconds). During interphase, CP60 gradually moves into nuclei (1,146 seconds); unlike CP190, CP60 never completely disappears from centrosomes during interphase, although its centrosomal localization becomes very weak.

Using the same approach, we were able to show that accumulation of CP190 at centrosomes is coincident with nuclear envelope breakdown and its accumulation in nuclei begins coincident with nuclear reformation in telophase (data not shown).

To quantitate the localization patterns of the CP190 and CP60 fusion proteins in live embryos, time-lapse three-dimensional images of living embryos were collected using wide-field fluorescence microscopy after injection of fluorescein labeled CP190 and rhodamine-labeled CP60 6XHis fusion proteins. One two-wavelength 3-D image was collected every 52 seconds. A stereo pair of one such image, taken of an embryo in metaphase of nuclear cycle 12, is shown in Fig. 2A. After computational processing to remove out of focus information, all of the centrosomes and nuclei in each field were manually outlined in every focal plane of each timepoint. Total centrosomal and nuclear fluorescence for the field was then calculated for each timepoint. We quantitated centrosomal and nuclear fluorescence for two embryos and obtained identical results. The results for one of these embryos, followed between prometaphase of cycle 12 and the beginning of cycle 14, are graphed in Fig. 2B.

As expected, the temporal localizations of CP190 and CP60 are asynchronous both at centrosomes and in nuclei. CP190 centrosomal fluorescence peaks during prometaphase immediately following nuclear envelope breakdown and remains high throughout mitosis (Fig. 2B, top panel timepoints 0-5 and 1825). Some CP60 accumulates at centrosomes between nuclear envelope breakdown and metaphase (Fig. 2B, top panel, time points 0-2 and 18-21), but the majority of the centrosomal CP60 accumulates during anaphase and telophase (timepoints 3-5 and 22-25). CP60 fusion protein remains at centrosomes well into interphase; CP190, in contrast, was not detectable at centrosomes during most of interphase. CP190 nuclear fluorescence begins to increase in telophase and reaches maximal intensity early in interphase (Fig. 2B, timepoints 5-18); in interphase of cycle 13, CP60 nuclear fluorescence reaches peak levels only just before nuclear envelope breakdown.

The CP190 and CP60 fusion proteins had been bacterially expressed, purified and labeled with fluorophores, any of which could alter their properties compared to the native proteins. In addition, the 6XHis CP190 fusion protein contained only the C-terminal 85% of the protein (addition of the N-terminal 166 amino acids makes the protein insoluble in bacteria). We therefore wanted to determine if the endogenous CP60 and CP190 behave in the same manner as the injected fusion proteins.

Fixed embryos were incubated with goat anti-CP190 and rabbit anti-CP60, and were processed in a single batch for immunofluorescence. Three-dimensional images were obtained from the fixed embryos in a manner identical to that used for the live embryos except larger (512×512 pixel) optical sections were taken. We collected data from 2 embryos for each stage of the cell-cycle in cycle 12 and from 5 embryos for each stage of the cell-cycle in cycle 13. To avoid bias in embryo selection, embryos were selected and classified as being in interphase, metaphase, anaphase or telophase by their DAPI staining patterns. A portion of a single optical section for one field of nuclei from an embryo representative of each cell cycle state between telophase of cycle 12 and telophase of cycle 13 is shown in Fig. 3A. We found that the localization patterns of the endogenous CP190 and CP60 were qualitatively similar to the localization patterns obtained with the injected labeled fusion proteins. For example, native CP190 is present in telophase nuclei (see Fig. 3A) when no CP60 is detectable in nuclei, and endogenous CP60 shows relatively weak centrosomal localization in metaphase embryos.

In Fig. 3B we plot the average values for centrosomal fluorescence as a function of cell cycle state. Although the time resolution is poor, the fixed data confirms our live results. The CP190 centrosomal fluorescence reaches peak levels by metaphase whereas the centrosomal fluorescence of CP60 increases substantially between metaphase and anaphase, peaking at telophase. Although we did not quantitate the changes in nuclear fluorescence during interphase, the nuclear staining in the fixed images was consistent with our live data. To determine whether the asynchronous accumulation of CP190 and CP60 was limited to the syncytial embryonic divisions, we characterized the localization pattern of CP190 and CP60 in the cells of the developing embryonic brain (Fig. 4) and in cultured Schneider S2 cells (data not shown). During these cellular divisions, CP190 localizes to centrosomes throughout mitosis. CP60 localizes to centrosomes only weakly, if at all, during metaphase but prominently during anaphase and telophase, suggesting that the asyncronous localization of CP190 and CP60 to centrosomes is a general phenomenon.

Although the localization patterns described above for CP190 and CP60 are similar in all of the syncytial blastoderm nuclear cycles (nuclear cycles 10-13) there are some differences. During the shorter nuclear cycles 10 and 11, there appears to be at least some CP190 at centrosomes throughout the entire cell cycle. In the longer cycles, 12 and 13, CP190 completely disappears from centrosomes during interphase as judged by our inability to detect centrosomal CP190 in fixed embryos double labeled for another centrosomal marker, such as γtubulin or CP60 (data not shown).

A second difference observed seems more profound. A timelapse series of confocal images of an embryo in nuclear cycle 11 that had been co-injected with CP60 and 40,000 MW fluorescein dextran is shown in Fig. 5A. Whereas CP60 appears to accumulate in nuclei before nuclear envelope breakdown in the later nuclear cycles, CP60 localizes to nuclei simultaneous with nuclear envelope breakdown in nuclear cycles 10 and 11. (Compare the nuclear CP60 in cycle 11 before (−35 seconds), and after (+30 seconds) nuclear envelope breakdown in Fig. 5A with the nuclear CP60 in cycle 12 before (−40 seconds) and after (+50 seconds) nuclear envelope breakdown in Fig. 1B.)

Results in fixed embryos are consistent with the live data in this regard (Fig. 5B). The fact that CP60 is able to rapidly diffuse into and concentrate in nuclei upon nuclear envelope breakdown in cycles 10 and 11 suggests that the localization of CP60 to mitotic nuclei is due to attachment to large structures in the region of the nucleus.

The in vitro association observed between CP190 and CP60 encouraged us to look at their nuclear localizations at higher resolution to determine if these two proteins co-localize in nuclei. Appropriate sections from interphase and mitotic cycle 13 nuclei stained for CP190, CP60 and DNA are shown in Fig. 6.Both CP190 and CP60 appear ‘fibrous’ within the nucleus but, although there are some regions of overlap, CP190 and CP60 do not co-localize extensively within nuclei. In addition, neither CP190 nor CP60 visibly co-localizes with DNA. During mitosis, the patterns of CP190 and CP60 are similar in character to their patterns during interphase and both proteins appear to be excluded from the region around the chromosomes. Taken together with the results obtained in live embryos that CP60, and to some extent CP190, remain localized in the nucleus well after nuclear envelope breakdown, these results suggest that CP190 and CP60 are binding to large residual nuclear structures that persist into mitosis.

Previous work has shown that although both CP60 and CP190 will bind to microtubules in vitro, neither protein colocalizes along the lengths of microtubules in the region of the spindle. In addition, both CP190 and CP60 will accumulate at centrosomes with normal kinetics in the absence of microtubules (Oegema et al., 1995). To determine if microtubules are required for the nuclear localization of CP60 during mitosis, embryos were treated briefly with colchicine and then fixed and processed for immunofluorescence. We found that depolymerization of the microtubule spindle had no effect on the localization of CP60 to residual nuclear structures during mitosis (data not shown).

In embryos older than cycle 14 (post-cellular blastoderm), CP190 and CP60 have been reported to co-localize to spots within the nucleus (Kellogg et al., 1995). High resolution optical sections of a region from such an embryo stained for CP190, CP60 and DNA are shown in Fig. 7A,B. The nuclear localization of much of the CP190 and CP60 is similar to that in Fig. 6, except some of the CP60 and CP190 now co-localize to 1-3 prominent spots within each nucleus. These spots do not correspond to DNA that we can detect by DAPI staining. To determine if these spots are associated with the nucleolus, embryos were fixed and stained for CP190 and CP60 simultaneously with hybridization of a probe recognizing ribosomal DNA (Fig. 7C). We conclude that the CP190/CP60 spots are also not co-incident with the nucleolus.

The fibrous localization patterns of CP190 and CP60 within nuclei, combined with the fact that CP60 (and CP190 to a lesser extent) localizes to the nucleus even after nuclear envelope breakdown, suggested that CP190 and CP60 might be associated with structural elements within the nucleus. To determine if CP190 and CP60 are components of the conventionally defined ‘nuclear matrix’ fraction, nuclei were isolated from Drosophila tissue culture cells, treated with DNase and extracted with high salt to remove the DNA and associated proteins. Western blots following the procedure show that, while histones are completely extracted, CP190 and CP60 remain in the nuclear matrix fraction along with the expected topoisomerase II (Fig. 8). Immunofluorescence of isolated nuclei and nuclear matrices stained for either CP190 or CP60 and DNA confirmed that the DNA was completely removed by our extraction procedure whereas CP60 and CP190 appeared unaffected (data not shown). We obtained similar results for nuclei isolated from Drosophila embryos (data not shown). This ‘nuclear matrix’ preparation contains both CP60 and CP190, which do not colocalize, and topoisomerase II, which colocalizes with chromatin (Swedlow et al., 1993), indicating that the conventional ‘nuclear matrix’ preparation contains proteins corresponding to at least several distinct nuclear structures.

Although CP60 was initially identified by virtue of its biochemical association with CP190 and both CP60 and CP190 shuttle between nuclei and centrosomes (Kellogg et al., 1992, 1995), our results show that the timing of their localizations is significantly different. In addition, within the nucleus, CP190 and CP60 have different spatial localization patterns.

CP190 and CP60 cycle asynchronously between nuclei and centrosomes. Experiments in both live and fixed embryos demonstrate that CP190 is most prominent at centrosomes between nuclear envelope breakdown and telophase, whereas centrosomal CP60 is at peak levels between anaphase and early interphase.

The centrosome cycle in Drosophila embryos has been described in detail (Callaini and Riparbelli, 1990) and is slightly different from that of somatic tissue culture cells in which centrosomes divide during interphase. In Drosophila embryos, the centriolar cylinders lose their perpendicular orientation in late metaphase, consistent with preparation for centrosome division; the centrosomes then become visibly less compact in early anaphase, forming ovoid plates by late anaphase. During telophase, the duplicated centrosomes physically separate. CP190 is present at centrosomes immediately following nuclear envelope breakdown when centrosomes make the transition from their interphase functions next to the nuclear envelope to their new roles at spindle poles. The concentration of CP60 at centrosomes between anaphase and early interphase coincides with the period of centrosome duplication and separation, making possible some role for CP60 in these processes.

The accumulation of CP60 in nuclei also lags that of CP190. Whereas CP190 accumulates in nuclei as they reform in telophase, reaching peak levels early in interphase, CP60 accumulates in nuclei gradually during interphase reaching peak levels just before nuclear envelope breakdown (in nuclear cycles 12 and 13). In the earlier, shorter nuclear cycles, 10 and 11, CP60 accumulates in nuclei simultaneous with nuclear envelope breakdown. The failure of CP60 to accumulate in nuclei during interphase in the earlier nuclear cycles could be due to the shorter amount of time spent in interphase in these cycles or to the different timing of a regulatory event. Because CP190 always accumulates before CP60, both at centrosomes and in nuclei, it is possible that CP190 has a role in the recruitment of CP60 to centrosomes, to nuclei, or to both structures or that CP190 and CP60 function in a sequential process that occurs both at centrosomes and in nuclei.

By high resolution 3-D microscopy in fixed embryos, CP190 and CP60 appear ‘fibrous’ within the interphase nucleus. The majority of CP60 and CP190 do not co-localize in nuclei, nor does either protein co-localize extensively with the DNA visible by DAPI staining. Association with DNA is a possibility since CP190 contains 4 putative zinc fingers and antibodies to CP190 and CP60 were found to recognize bands on isolated salivary gland chromosomes (Whitfield et al., 1995). Our results cannot eliminate the possibility that CP190 and CP60 interact with DNA at discrete sites.

Since the fibrous patterns of CP60 and CP190 in the nucleus persist into mitosis, when chromosomes are completely separated from the CP190 and CP60 networks, the fibrous appearance of their staining cannot be due to a simple exclusion from regions in the nucleus occupied by interphase chromatin. The fact that CP190 and CP60 do not co-localize within nuclei is further evidence for some structure in the nucleus of a fibrous nature.

In older embryos (post syncytial blastoderm) CP190 and CP60 co-localize to a few prominent spots within nuclei (see Fig. 7). These spots do not co-localize with blocks of heterochromatin, visible by DAPI staining, or with the nucleolus; they thus define a novel sub-nuclear structure.

Interestingly, the localization patterns of CP190 and CP60 in nuclei during metaphase are remarkably similar to their localization patterns during interphase, suggesting that CP190 and CP60 may be associating with unknown nuclear structures that persist into mitosis. The fact that CP60 is able to rapidly concentrate in nuclei upon nuclear envelope breakdown in cycles 10 and 11 is especially striking in this regard. Consistent with previous work demonstrating that CP190 and CP60 do not co-localize with microtubules in the region of the spindle (Oegema et al., 1995), treatment of embryos with colchicine to depolymerize the microtubule spindle does not affect the nuclear localization of CP60 during mitosis.

In spite of their in vitro association, our results clearly show that CP190 and CP60 have different temporal and spatial localization patterns, suggesting that the interaction between CP190 and CP60 is complex and likely to be regulated in a cell cyclespecific manner. Studies on CP190 and CP60 in extracts are consistent with the observed complexity of their localization patterns. CP60 contains 6 consensus cdc2 phosphorylation sites and western blotting of extracts reveals the existence of multiple phosphorylated forms in vivo (Kellogg et al., 1995). In addition, a kinase present in eluates from anti-CP190 immunoaffinity columns can phosphorylate CP60 in vitro (Kellogg et al., 1995), suggesting that the phosphorylation of CP60 could be in part regulated by its association with CP190. Bacterially expressed CP60 forms a higher order oligomer, which is also formed by a poorly phosphorylated form of CP60 predominant in concentrated Drosophila extracts (M. Moritz, K. Oegema and B. M. Alberts, unpublished data). One speculative possibility, for example, is that dephosphorylation of CP60 during anaphase releases it from its attachment to residual nuclear structures and allows CP60 to oligomerize at centrosomes where it may interact with microtubules.

We have shown that CP190 and CP60 are two proteins that localize in an alternating asynchronous fashion to the pericentriolar material and to the nucleus. In addition, we demonstrate that CP60 (and probably CP190) associate with a intranuclear network present in living embryos. CP190 and CP60 may function sequentially in a process that occurs at centrosomes and could be sequestered in nuclei when they are not needed; alternatively CP190 and CP60 could function both at centrosomes and in nuclei. Genetic studies will hopefully result in clarification of the functions of CP190 and CP60 in embryos.

We thank Arshad Desai and Doug Kellogg for critical reading of the manuscript. This work was supported by grants from the National Institutes of Health to B.M.A (GM23928) and to J.W.S (GM-22510116). W.F.M. was additionally supported by a Howard Hughes Medical Institute predoctoral fellowship.