Persistence of major nuclear envelope antigens in an envelope-like ...

Persistence of major nuclear envelope antigens in an envelope-like structure during mitosis in Drosophila melanogaster embryos

AMNON HAREL1, EFRAT ZLOTKIN 1 , SANDRA NAINUDEL-EPSZTEYN1, NAOMI FEINSTEIN 1 , PAUL A. FISHER2 and YOSEF GRUENBAUM1-* ' Department of Genetics, The Life Sciences Institute, The Hebrew University ofJerusalem, Jerusalem, Israel 91904 and 2The Department of Pharmacological Sciences, Health Sciences Center, State University ofNezv York at Stony Brook, Stony Brvok, t\Y 11794-8651, USA * Author for correspondence

Summary Using monoclonal antibodies, we followed the fate of three different nuclear envelope proteins during mitosis in Drosophila early embryos by indirect immunofluorescence microscopy. Two of these proteins, lamin and otefin, a newly characterized nuclear envelope polypeptide with an apparent Mr of 53 000, are apparently present in an envelope-like structure that is present throughout mitosis. Immunoelectron microscopy of interphase nuclei indicates that otefin, like lamin, is not a component of nuclear pore complexes. In contrast with lamin and otefin, gpl88, a putative pore complex component, was completely redistributed through the surround-

ing cytoplasm during prophase in comparable early embryo specimens and was present in an envelope only in interphase. Together with previous morphological studies by other workers, these data suggest that the entire mitotic apparatus including condensed chromosomes and spindle is enclosed by an envelope throughout mitosis during early embryogenesis in Drosophila. This 'spindle envelope', as it has been named by others, contains both lamin and otefin but probably not pore complex proteins.

Introduction

Efforts from several laboratories have focused on the fate of vertebrate nuclear envelope components during mitosis (Ely et al. 1978; Gerace et al. 1978; Jost and Johnson, 1981; Krohne et al. 1978; Ottaviano and Gerace, 1985). Immunocytological studies performed in conjunction with cell fractionation experiments showed that during prophase, the polymeric lamina disintegrates into lamin oligomers that are dispersed uniformly in the cytoplasm. This pattern of protein distribution persists until telophase, at which time the lamina begins to reform at the centrosomal regions until it fully envelopes the chromatin in each of the daughter nuclei. Similar results were obtained with antibodies directed against vertebrate nuclear pore complex proteins including p62 (Davis and Blobel, 1986), gpl90 (Gerace et al. 1982) and a number of other pore complex glycoproteins (Snow et al. 1987). These results suggest that during mitosis in vertebrates, both lamina and pore complexes disassemble, coincident with the breakdown of the nuclear envelope observed microscopically.

The nuclear envelope is composed of inner and outer lipid bilayer membranes, which are separated by the perinuclear space. The two membranes join at the pore complexes, presumed passageways for the exchange of macromolecules between the nucleus and cytoplasm (e.g. see Feldherre^a/. 1984; Dworetzky and Feldherr, 1988). Underlying the inner membrane is the nuclear lamina (Franke, 1974), a proteinaceous layer of intermediate filament-like fibrils (Aebi et al. 1986). Major polypeptide components of the nuclear lamina, termed lamins, have been identified in a number of organisms (for reviews, see Gerace, 1986; Krohne and Benavente, 1986). In higher eukaryotes, the nuclear envelope breaks down at the onset of mitosis, only to be re-formed in each of the two daughter cells as mitosis is completed. This process has been termed open mitosis (see Franke, 1974). In contrast, lower eukaryotes such as yeast and myxomycetes, undergo mitotic chromosome segregation within an essentially intact nuclear envelope (see Heath, 1980). Nuclear division is accomplished by karyokinesis, a process not unlike cytokinesis in higher organisms. This has been termed closed mitosis. Journal of Cell Science 94, 463-470 (1989) Printed in Great Britain © The Company of Biologists Limited 1989

Key words: Drosophila, nuclear envelope, mitosis.

Drosophila melanogaster is an organism well-suited to the study of nuclear envelope structure and function. Homologs of many of the well-characterized vertebrate nuclear envelope proteins have been identified (for a 463

review, see Fisher, 1988). Moreover, the rapid early phases of Drosophila embryogenesis provide a unique perspective from which to view mitosis and the events surrounding it. The first 13 nuclear division cycles in the embryo occur rapidly and nearly synchronously within a syncitium. Each of the first nine cycles lasts approximately 10 min; the last four cycles are each approximately 15min in length (Zalokar and Erk, 1976). Initial reports on the fate of the nuclear lamina during mitosis in Drosophila early embryos showed that, in prophase, the lamina apparently invaginated from both sides towards the central plane of mitosis. During metaphase, the lamina started to break down and lamins were apparently dispersed in the cytoplasm in irregularly shaped particles (Fuchse? al. 1983). On the other hand, detailed electronmicroscopic analysis of the nuclear membranes in Drosophila early embryos (prior to cellularization) revealed that, during mitosis, the nuclear membranes broke at the spindle poles but remained fully in evidence elsewhere (Stafstrom and Staehelin, 1984). However, nuclear pore complexes associated with the envelope during interphase apparently disassembled and were lost during mitosis, leaving behind numerous fenestrations in the membrane. It was also shown in this study that in mitosis, a second layer of closely adherent membranous cisternae was acquired just outside the original membranes (Stafstrom and Staehelin, 1984). To elucidate further the fate of the Dmsophila nuclear envelope during mitosis in early embryos, we followed the fates of three different nuclear envelope proteins by indirect immunofluorescence. We show that at least a portion of both otefin (which is a transliteration of the Hebrew word meaning literally, 'envelopes'), a 53K (K=10 3 M r ) protein that we localized to the inner nuclear membrane and/or lamina, and lamin* are retained in a structure that envelopes the entire mitotic apparatus including condensed chromosomes and spindle, while gpl88, a putative pore complex protein, is apparently released from the envelope during prophase and reintegrates into the envelope during interphase. These results confirm the suggestion of Stafstrom and Staehelin (1984) that during the rapid early phases of embryogenesis, Drosophila nuclei undergo what may in effect be a form of closed mitosis similar to that seen in lower eukaryotes.

Materials and methods Antibodies Monoclonal anti-Drosophila lamin antibody, 611A3A6 and monoclonal anti-otefin antibody 618A207 (Miller et al. 1985) were gifts from Dr Bruce Alberts. Monoclonal anti-Drvsophila gpl88 antibodies AGP-26 and AGP-78 (Filson et al. 1985) were * Only a single nuclear lamin has thus far been identified in Dmsophila melanogaster. Several isoforms, distinguishable on the basis of one-dimensional SDS-PAGE mobility, are derived from a single primary translation product through post-transational modification (Smith et al. 1987; Gruenbaum et al. 1988). All are apparently recognized equally well by the monoclonal anti-lamin antibodies used in this study. 464

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purified from tissue-culture supernatants and concentrated by ammonium sulfate precipitation as previously (McConnell et al. 1987). 5-nm gold-conjugated goat anti-mouse IgG was from Janssen Pharmaceutica (Piscataway, NJ). Affinity-purified Texas Red-conjugated rabbit anti-mouse IgG and rhodamineconjugated goat anti-mouse IgG were from Jackson Laboratories (West Grove, PA). Cell fractionation, SDS-PAGE and immunoblot analysis About 5X108 Schneider cells were washed once in RSB (10 mMNaCl, 3mM-MgCl2, lOmM-Tris-HCl, pH7.4, 0.5M-phenylmethylsulfonyl fluoride (PMSF)), incubated for 10min on ice and Dounce homogenized (50 strokes with a tight pestle). Following centrifugation (750 g, 10min, 4°C), the supernatant was collected (fraction SFI). The pellet was resuspended in RSB, loaded on a cushion of RSB containing 1.9M-sucrose and centrifuged (VTI 50.1 rotor, 24000revsmin~', 30min, 4°C). The isolated nuclei were washed once in RSB containing 0.25 M-sucrose (RSBS), resuspended in 2ml RSBS and an equal volume of cold solution containing 2M-NaCl, lOmMEDTA, lOmM-Tris-HCl, pH7.4, was then added to the tube. Following 10 min incubation at room temperature, the tube was centrifuged (4500£, 15 min, 4°C), the supernatant was collected (fraction SFII) and the pellet was subjected to two washes in RSBS (fraction P). Protein lysates from the different subcellular fractions were prepared as described (McConnell et al. 1987). SDS-PAGE was according to Laemmli (1970). Blots were first incubated for 2h with 618A207 anti-otefin antibody (diluted 1:2), followed by 2h incubation with l25I-labeled sheep anti-mouse IgG antibody (Amersham, England) and exposed to X-ray film. Immunofluorescence microscopy Collection, permeabilization and fixation were essentially as described by Karr and Alberts (1986). Drosophila melanogaster (Canton-S) embryos (0-3 h old) were collected at 25°C, rinsed in 0.4% NaCl, 0.03% Triton X-100 and dechorionated in a half-strength solution of commercial bleach. Dechorionated embryos were washed with 0.4 % NaCl, 0.03 % Triton X-100 to remove all traces of sodium hypochlonte and transferred to a 50 ml round-bottom flask containing 2 ml of 60mM-KCl, 15mM-NaCl, 0.15 mM-spermine, 0.5 mM-spermidine, 15 mM 2-mercaptoethanol, 15 mM-Tris-HCl, pH 7.4 (buffer A of Wallace et al. 1971), plus 8 ml of heptane. Taxol was added to a final concentration of 0.5 ;tM, and after less than 30 s of vigorous shaking, 1 ml of freshly prepared 37 % formaldehyde was added. Shaking was continued for an additional 10min. For devitellinization, the fixed embryos were collected, rinsed with PBS and transferred to a 50 ml round-bottom flask, previously cooled to —70°C, which contained 5 ml of heptane, 4.5 ml of methanol and 0.5 ml of 50mM-EGTA, pH7.0. After vigorous shaking for 10 min, the temperature was rapidly raised to approximately 23 °C by swirling the flask under a stream of hot tap water (Mitchison and Sedat, 1983). Devitellinized embryos were then rinsed with a methanol: EGTA solution minus the heptane and rehydrated by passage through solutions of methanol: PBS (4:1, 1:4 (v/v)) and finally, PBS alone. Indirect immunofluorescent staining of whole fixed embryos was also essentially as described by Karr and Alberts (1986). All incubations were done at room temperature, using gentle rotation in a humid chamber. Embryos were transferred into deep-depression slides containing PBS with 1 % bovine serum albumin (BSA) and 0 . 1 % Triton X-100 (PBSBT). Embryos were then incubated with primary antibody for 2h. When hybridoma tissue culture supernatant was used, it was diluted 1: 1 (v/v) with PBSBT. Otherwise antibodies were diluted to a

concentration of about 5 jUgml ' in PBSBT. Following a 3-h rinse in PBSBT, embryos were stained for 2 h with either rabbit or goat anti-mouse IgG coupled to Texas Red or rhodamine, respectively. Embryos were then rinsed for 2h in PBSBT. DNA was stained with 4,6-diamidino-2-phenylindole (DAPI) for 5 min in a 1 jUgml"1 solution of the dye in PBS. Following a 10 min rinse in PBS, the embryos were transferred successively into solutions of 1: 4, 2: 3, 3: 3 and 4: 1, glycerol: PBS (v/v) and finally mounted on a microscope slide in a solution of 2 % H-propyl gallate in glycerol (Giloh and Sedat, 1982). Drosophila embryos were viewed in a Zeiss Universal microscope equipped with epifluorescence illumination and a 100x/l.3 NA Planapo objective. Photographs were taken on Kodak Technical Pan 2415 film and developed with D19 or HC110 developer. Immunoelectron microscopy Drosophila melanogaster (Canton-S) embryos (0-3.Sh old) were collected, dechorionated, fixed, devitelhmzed and reacted with monoclonal antibody as described above. All incubation steps were performed at room temperature. Following a 2-h incubation with antibody, the embryos were rinsed for 3 h in PBSBT and then incubated with 5-nm colloidal gold-conjugated goat anti-mouse IgG diluted in PBS with 1 % BSA (PBSB). Embryos were next rinsed for 3 h with PBSB, then for 1 h in PBS, and then post-fixed in a solution containing PBS with 2 % glutaraldehyde and 2% formaldehyde for 30min. Following rinsing for 30 min with PBS, embryos were next fixed with 1 % OSO4. The post-fixed embryos were dehydrated in ethanol/propylene oxide and embedded in Epon. From the embedded embryos, 70 nm thick sections were cut, stained with uranyl acetate and lead citrate and viewed with a Phillips 300 transmission electron microscope at 60 kV.

Results Otefin is a nuclear envelope protein Initial experiments with monoclonal antibody 618A207 suggested that it specifically recognized a 53K polypeptide, here termed otefin, that was localized to the periphery of the nucleus by indirect immunofluorescence (Miller et al. 1985). We confirmed these observations in both tissue culture cells (not shown) and embryos (see Fig. 3, for example). lmmunoblot analysis performed on various developmental stages of Drosophila revealed that otefin is present in relatively large amounts during the embryonic stages and during pupation (Fig. 1A). We extended the analysis to include cell fractionation experiments and immunoelectron microscopy. These results are shown in Figs IB and 2, respectively. Following homogenization of Drosophila Schneider tissue-culture cells most or all of the otefin present in the crude homogenate (Fig. IB, lane T) was recovered in the isolated nuclei (Fig. IB, lane N). Also, most of the otefin in isolated nuclei was resistant to 1 M-NaCl extraction (Fig. IB, lanes P and SFII). Identical results have previously been reported for the Drosophila lamins (Filson et al. 1985). Results of immunoelectron microscopic experiments (Fig. 2) added to impressions obtained previously from indirect immunofluorescence analyses (Miller et al. 1985). Drosophila embryos from both the syncitial and cellular stages were fixed with formaldehyde in the presence of taxol (Karr and Alberts, 1986), incubated

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Fig. 1. SDS-PAGE and immunoblot analysis of otefin in various developmental stages of Drosophila (A), and its distribution during subcellular fractionation of Drosophila Schneider 2 tissue-culture cells (B). SDS-PAGE was on a 10% polyacrylamide gel and proteins were blot-transferred to nitrocellulose. The blots were incubated with 618A207 anti-otefin antibody (diluted 1:2) as the primary antibody, followed by incubation with 125I-labelled sheep anti-mouse IgG. A. Equivalent amounts of material (4 units; see Fisher et al. 1982) were loaded in each lane. The developmental stage from which the lysates were prepared is marked above the lane. The two higher molecular mass bands in the 0-2 h old embryos lane were not detected in several other independent blots (data not shown). B. The different lanes were loaded with whole lysate (T); nuclear lysate (N); nuclear pellet that was obtained following the incubation of the isolated nuclei with 1 M-NaCl (P); supernatant of the saltextracted nuclei (SFII). In lane P, nuclear lysate that corresponds to about twice the amount of cells (compared to all the other lanes in B) was loaded. Marker positions on the left apply to both A and B. Drosophila nuclear envelope antigens

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first with monoclonal anti-otefin antibody and then with 5-nm gold-conjugated goat anti-mouse IgG. After appropriate washing, the embryos were post-fixed with glutaraldehyde and OSO4, embedded in Epon and sectioned for electron microscopy. Upon examination of hundreds of nuclei in several independent experiments, we found gold particles restricted to the inner membrane of the nuclear envelope (Fig. 2A and B). This result is consistent with localization either to the membrane itself or to the underlying structures of lamina and pore complexes. Tangential views of the nuclear envelope showed that the nuclear pore complexes were essentially unreactive with the anti-otefin antibody apparently ruling out pore complex localization (Fig. 2C). Probing of similar specimens with gold-conjugated goat anti-mouse IgG alone failed to reveal any nuclear envelope staining (not shown). The fate of otefin during mitosis in early embryos Indirect immunofluorescence microscopy was used to investigate the fate of otefin during the rapid mitotic cycles characteristic of early embryogenesis in Drosophila. These results are shown in Fig. 3. To determine the different stages of mitosis in the specimens being examined, we used the DNA-specific dye, DAPI. DAPIstained specimens are shown in the left-hand panels; immunofluorescence micrographs of the same fields are shown on the right. In interphase, otefin was confined to the nuclear periphery and labeling of the nuclear envelope appeared to be diffusely granular (Fig. 3A). During prophase, otefin remained in a round envelope (Fig. 3B) and this morphology persisted through prometaphase (Fig. 3C). At this stage, spindle poles on opposite sides of the nucleus were also labeled, suggesting that some otefin may be associated with the spindle poles. At metaphase, a dramatic change in the immunofluorescence staining pattern obtained with anti-otefin antibody was observed. The envelope-like structure defined by immunofluorescent staining elongated perpendicular to the metaphase plate, enveloping the entire mitotic apparatus including the condensed chromosomes, spindle and spindle poles (Fig. 3D). During anaphase, this envelope-like structure was first elongated further and then became barbell-

shaped (Fig. 3E). In telophase, the barbell shape was lost as otefin was distributed into each of the two daughter nuclei. As the embryonic cell entered mitosis there was a reduction in the signal intensities and, in addition to the envelope-like staining seen with the anti-otefin antibody, there was an increase in the diffuse cytoplasmic background staining. This, in conjunction with the fact that envelope-like staining during mitosis was reduced relative to nuclear envelope staining seen with anti-otefin antibody during interphase, suggests that a portion of the otefin may be solubilized and redistributed during mitosis while a second portion remains associated with an envelope-like structure. Taxol is required during fixation in order to preserve otefin in an envelope-like structure during mitosis In comparing our current data on otefin localization during mitosis in early embryos with results of similar experiments performed with anti-lamin antibodies (Fuchs et al. 1983), we were struck by the apparent difference between the two antigens. As noted in the Introduction, Fuchs et al. (1983) reported that during mitosis the lamins were apparently dispersed in granular patches through the surrounding cytoplasm. There was no evidence for the persistence of an envelope-like structure surrounding the mitotic apparatus on the basis of staining with anti-lamin antibodies. In comparing our results for otefin with those of Fuchs et al. (1983) for lamin, one methodological difference between our current analysis and that of Fuchs et al. (1983) was noted, i.e. that we included taxol in our fixation step. We therefore repeated the staining of early embryos with anti-otefin antibody without inclusion of taxol at any point in the procedure. The results of this experiment are shown in Fig. 4. Without taxol present during fixation, the results obtained with anti-otefin antibody were similar to those that had previously been reported for the lamin (Fuchs et al. 1983). During prophase, the envelope as revealed by otefin staining started to invaginate (Fig. 4A). During metaphase, the majority of the antigen was redistributed

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Fig. 2. Immunoelectron microscopic localization of otefin. Specimens were probed with monoclonal anti-otefin antibody 618A207. A,B. Section cut perpendicular to the plane of the nuclear envelope; C, tangenital view. The nucleoplasmic side is designated Nu. Arrow designates a selected pore complex. The bar in C represents 200 nm and applies to all panels. X 62 500.

in the cytoplasm (Fig. 4B) and in telophase, most of the antigen was located over the centrosomes (Fig. 4C). The fate of lamin during mitosis in early embryos: a reinvestigation after fixation in the presence of taxol In light of the effects of taxol on the immunofluorescence staining pattern revealed with anti-otefin antibodies, we set about to reinvestigate the distribution of lamin during mitosis in early embryos. Embryos were fixed, permeabilized and probed with monoclonal anti-lamin antibodies exactly as described above for the experiments with anti-otefin shown in Fig. 3. Results of these analyses shown in Fig. 5 are essentially identical with those seen with anti-otefin antibody. During interphase, typical nuclear envelope staining was observed (Fig. SA). In prophase, when the chromosomes started to condense, the anti-lamin staining remained in a rounded structure (Fig. 5B), which became elongated at metaphase perpendicular to the mitotic plate and included the spindle pole area (Fig. 5C). Early in anaphase, further elongation was observed (Fig. 5D), which become more pronounced during late anaphase (Fig. 5E). In addition to the persistent envelope-like staining revealed with anti-lamin antibodies during mitosis, we also noted an increase in diffuse cytoplasmic background staining. This is again consistent with the notion that only a portion of the lamin remained in an envelope-like structure during mitosis while the remainder was redistributed through the surrounding cytoplasm. The fate of gpl88 during mitosis in early embryos Both lamin and otefin are apparently associated with the inner membrane of the nuclear envelope and neither is found in nuclear pore complexes. On the other hand, gpl88 is the Drosophila homolog of mammalian gpl90, a transmembrane glycoprotein localized to the rat liver nuclear pore complex by Gerace et al. (1982). Pore complex localization for gpl88 in Drosophila salivary gland nuclei has recently been confirmed (M. Berrios, personal communication). We therefore set about to investigate the distribution of gpl88 during mitosis in Drosophila early embryos using two monoclonal anti-

Fig. 3. Indirect immunofluorescence localization of otefin at different stages of mitosis. Drosophila melanogaster embryos were at stages 12-13 (Foe and Alberts, 1983) and were probed with monoclonal anti-otefin antibody 618A207. DAPI staining, left; anti-otefin staining, right, side-by-side. A. Interphase; B, early prophase; C, prometaphase; D, metaphase; E, late anaphase. The bar in the immunofluorescence-stained portion of E represents 20;