Microinjection of an Antibody to the Ku Protein Arrests Development in Sea Urchin Embryos.
RUTH M. EMPSON 
HOWARD RASMUSSEN [1,2][*]
Abstract. Ku is the regulatory subunit of the DNA-dependent protein kinase (DNA-PK). This enzyme plays a role in DNA repair, recombination, and transcription. It is composed of a large catalytic subunit (p460), and a regulatory heterodimer, the Ku protein, which consists of 86-kDa and 70-kDa subunits. These various components of the enzyme have been found in both eggs and embryos of the sea urchin. When variable amounts of a specific monoclonal antibody to the Ku protein (Ku 162) were injected into one cell of a 2-cell embryo of Lytechinus pictus, they caused a dose-dependent developmental arrest of the injected cell. The non-injected cell continued to develop normally. In contrast, injection of an antibody (N3H10) raised against the 70-kDa subunit of the Ku protein had no effect on development when injected into 2-cell-stage embryos. Co-injection of purified DNA-PK with the antibody reversed the antibody-mediated inhibition of development. In the fertilized egg and during the early stages of development, the DNA-PK was localized largely in the cytoplasm, but in later developmental stages, it assumed a nuclear location. On the basis of these results, we postulate that the injection of the Ku antibody either prevents the translocation of the DNA-PK into the nucleus or interferes with its enzymatic activity either in the nucleus or in the cytoplasm. In either case, the results suggest that DNA-PK plays an important role in regulating the early stages of embryogenesis in this primitive organism.
The DNA-dependent protein kinase, DNA-PK, was first detected in the nuclei of mammalian cells (Walker et al., 1985; Carter et al., 1990). Studies in the last several years have provided evidence that this enzyme is involved in DNA repair, and in the activation of specific transcription factors (reviewed by Anderson and Lees-Miller, 1992; Weaver, 1995). It may also be involved in regulating DNA synthesis (Brush et al., 1994), Ku-associated ATP-dependent helicase activity (Tuteja et al., 1994), and DNA-dependent ATPase activity (Vishwanatha and Baril, 1990; Cao et al., 1994). The enzyme consists of a large catalytic subunit, p460, and a heterodimeric regulatory component, the Ku protein. The latter consists of subunits of 86 and 70 kDa (Anderson, 1993; Gottlieb and Jackson, 1993). The Ku protein possesses a DNA-binding domain, and enzymatic activity is thought to be induced when this domain binds double-stranded DNA, (ds)DNA (Anderson and Lees-Miller, 1992; Dvir et al., 1992; Anderson, 1993; Gottlieb and Jacks on, 1993). However, the catalytic subunit of DNA-PK can be activated by another DNA-binding protein, heat shock transcription factor 1 (Peterson et al., 1995). Additional complexity of control may be involved in its catalytic function, because the p86 subunit of Ku is a somatostatin receptor protein that can regulate the activity of protein phosphatase 2A (Le Romancer et al., 1994). In addition, it has recently been reported that in cultured mammalian cells, a significant amount of the Ku protein is present in the cytoplasmic portion of the cell (Fewell and Kuff, 1996). These findings have led to the proposal that one or both of the Ku subunits may function in ways other than through the activation of nuclear DNA-PK, or that DNA-PK can be activated by factors other than (ds)DNA and function to regulate events in the cytoplasmic domain.
More recent work has shown that the enzyme is present in lower organisms, and particularly in the oocytes, eggs, and embryos of several marine invertebrates (Walker et al., 1985; Kanungo et al., 1996a, b) as well as in mice, frogs, and Drosophila (Finnie et al., 1995). Thus, DNA-PK may have one or more evolutionarily conserved functions. Transgenic mice that are deficient in the Ku 86 subunit exhibit severe combined immunodeficiency (Nussenzweig et al., 1996; Zhu et al., 1996). Such mice are considerably smaller (40%-80%) than their normal littermates, and cultured cells derived from these mice display variable degrees of delay in cell cycle progression (Nussenzweig et al., 1996).
That high concentrations of DNA-PK exist in the eggs of sea urchins (Kanungo et al., 1996a, b) and frogs (Kanungo et al., 1997) raises the interesting possibility that this enzyme may also play a regulatory role in early embryogenesis in these species. To explore this possibility, we initially undertook studies of DNA-PK localization and activation before and after fertilization of the eggs of Arbacia punctulata, the purple sea urchin. The unfertilized egg of this organism contains considerable amounts of both Ku and p460, as measured by western analysis, and by immunocytochemistry (Kanungo et al., 1996b). These enzyme subunits are located largely, if not entirely, in the cytoplasmic compartment of the egg. Nonetheless, in the unfertilized egg, the DNA-PK cannot be activated by the addition of (ds)DNA to cytoplasmic extracts; i.e., the enzyme is present in the cytoplasm in some cryptic form (Kanungo et al., 1996b). Within minutes of fertilization, and without evidence of new protein synthesis, the enzyme is still in the cytoplasmic fraction, but can now be activated when (ds)DNA is added to this fraction. Of equal interest is that, if one coats Protein A Sepharose (PAS) beads with a specific monoclonal antibody to the Ku protein, Ku 162, and then uses these beads to isolate the Ku proteins from the cytoplasmic extracts of fertilized eggs, the beads bind the holoenzyme, and in this bound form the enzyme is active in the absence of (ds)DNA.
As the fertilized egg develops, there is a progressive shift of the various protein components of DNA-PK from a cytoplasmic to a nuclear location within the cell (Kanungo et al., 1996b). The enzyme is located exclusively in the nuclear domain from the blastula stage onward, but in embryos at the 2- and 4-cell stages the enzymatic activity remains largely in the cytoplasm (Kanungo et al., 1996a, b). In this report, we demonstrate that microinjection of the antibody, Ku 162, into one cell of a 2-cell embryo of Lytechinus pictus, the white sea urchin, inhibits the further development of the injected cell, but has no effect on the non-injected cell. This species, rather than Arbacia punctulata, was employed in the present experiments because the microinjection experiments were more easily performed in its embryos.
Materials and Methods
Collection of eggs and embryos. Male and female Lytechinus pictus were obtained from the Marine Resources Department of the Marine Biological Laboratory, Woods Hole, Massachusetts. Shedding of eggs and sperm was induced by injecting 0.5 ml of 0.5 M potassium chloride into the coelom. Batches of eggs were inseminated by mixing them with diluted sperm. Embryos were collected at scheduled times.
Kinase assay. Cytoplasmic extracts from the 2-cell embryos of L. pictus were prepared following the procedures already described (Ballinger et al., 1984; Kanungo et al., 1996a). Twenty-five units of purified human DNA-PK (Promega) or extracts prepared from L. pictus 2-cell embryos (from 250 embryos) was added to the antibody-coated PAS beads (Pharmacia) in a 500-[micro]l volume made up by kinase assay buffer, and incubation was carried out at 4[degrees]C with constant mixing for 2 h. The PAS beads were washed six times with kinase assay buffer. The assay consisted of 12 [micro]l of kinase assay buffer containing DTT (dithiothreitol) at a final concentration of 1 mM, 200 [micro]M of peptide substrate (EPPLSQEAFADLWKK) (Anderson, 1993), 2 mM of [MgCl.sub.2], 130 mM of ATP, and 10 [micro]Ci of gamma-[32P]ATP (3000Ci/mmol) (NEN, Du Pont). The assay was carried out at 25[degrees]C for 30 min and stopped by adding glacial acetic acid to a final concentration of 30%. The reaction product was spotted onto p81 phosph ocellulose discs (Whatman). Several washes with 15% acetic acid following a 30-min wash with 30% acetic acid were carried out. The discs were finally washed for 5 min in acetone, air-dried, and the uptake of radioactivity was assessed by scintillation counting. The counts obtained from the control beads, not coated with any IgG but treated with either the purified human DNA-PK or extracts of L. pictus embryos, were used as a means of measuring nonspecific background radioactivity. These values were subtracted from the counts obtained from reactions using mouse IgG- and Ku 162-coated PAS beads.
Immunoprecipitation. DNA-PK holoenzyme was immunoprecipitated with PAS beads that had been coated with the Ku antibody as follows: PAS beads were pre-swollen in kinase assay buffer (50 mM HEPES, pH 7.4; 10 mM EGTA, 40 mM NaC1, 100 mM potassium acetate, 8.5 mM [CaCl.sub.2], 2.29 mM [MgCl.sub.2], 277 mM glycerol). Two micrograms of preimmune mouse IgG or of a monoclonal antibody to human Ku (Ku 162) that recognizes a conformational epitope of the Ku protein (Neomarkers, CA) was added to a 10-[micro]l packed volume of PAS beads. After 1 2-h incubation at 4[degrees]C with constant mixing, the beads were washed four times with kinase assay buffer.
Immunoblotting. The immunoprecipitates from PAS beads were eluted by boiling in SDS-PAGE sample buffer and resolved on a 7.5% SDS-PAGE column (Laemmli, 1974). Duplicate gels were run. One was employed to prepare autoradiographs. The proteins from the other gel were transferred to nitrocellulose membrane and immunoblotted (Towbin et al., 1979) with anti-p460 antibody as previously described (Kanungo et al., 1996b).
Preparation of antibodies for microinjection. The IgG antibodies (Ku 162 and N3Hl0) and the control IgGs were used for the microinjection experiments (Wang et al., 1993). They were a generous gifts from Dr. Westley Reeves of the University of North Carolina, Chapel Hill. Antibodies against RNAP II were obtained from Promega, and preimmune mouse IgG was purified using a Pierce immunoglobulin purification kit (Cat # 44667). The purified IgGs were dialyzed against [Ca.sup.2+]-, [Mg.sup.2+]-free PBS (Silver, 1986) and concentrated to 3 [micro]g/[micro]l. The antibody-enzyme complex was prepared using 6 [micro]g of Ku 162 antibody and 10 [micro]g of purified human DNA-PK (Promega) and incubated on ice with intermittent mixing for 2 h. The mixed aliquot was diluted to 1 ml with [Ca.sup-2+]-, [Mg.sup.2+]-free PBS, then dialyzed (4 X 2 liters) overnight. The dialyzed antibody-enzyme complex was collected and concentrated using an Amicon filter concentration unit. The final concentration was brought back to the origi nal volume of the antibody solution used (10 [micro]l), giving a final concentration of 600 ng/[micro]l Ku 162 and 1000 ng/[micro]l DNA-PK.
Microinjection. Specimens of L. pictus were obtained from Marinus Inc. (Long Beach, CA). To induce a female to shed eggs, about 0.5 ml of 0.5 M KCI was injected into the intracoelomic cavity. The eggs were then passed through an 80-[micro]M diameter Nitex membrane to remove the jelly that surrounded them and washed once in [Ca.sup.2+]-containing seawater. A 0.5-ml sample of eggs was placed onto a glass coverslip previously coated with poly-L-lysine, and the eggs were exposed to sperm. Low densities of activated sperm (1/40,000 dilution of dry collected sperm) were used to prevent the occurrence of polyspermy. if fertilization proceeded with an efficiency of at least 90%, we continued the experiment. After fertilization, the dishes were covered and left at 17[degrees]-19[degrees]C for 1 h, at which time the embryos begin first division. Solutions of antibodies that had been dialyzed and concentrated to known protein concentrations (3 [micro]g/[micro]l in the pipette, estimated as a final amount of 15 X [10.su p.-12] g in the cell) were microinjected into one cell of the 2-cellstage embryo shortly after first cleavage and before aster formation of the second cleavage. We typically injected 1%-2% of the volume of the embryo. An occasional embryo was damaged by the microinjection procedure; such clearly damaged embryos were discarded. The injected embryos were again covered and remained on the microscope stage for a further 2 h; we then determined the incidence of continued or arrested division. In most experiments, digital images of the eggs were recorded using either an integrating cooled CCD camera (Hamamatsu, USA) or an intensified CCD camera (Photonics Science, Robertsbridge, UK). The camera was attached either to a Metamorph (Universal Imaging, USA) system controlled by a Pentium PC or an lonvision (Improvision, Coventry, UK) system run by a Macintosh. In all experiments we used the uninjected cell as an internal control.
Immunocytochemistry. Embryos at the 2-cell stage immediately after antibody injection were fixed in 3% formaldehyde (Kanungo et al., 1996b) and processed for indirect immunofluorescence by incubating with fluorescein conjugated anti-mouse IgG (Sigma Cat # F 0257). Embryos were then whole-mounted onto glass coverslips and viewed with epifluorescence and appropriate filters using a Zeiss 135 inverted microscope (Zeiss, Oberkochen, Germany). Pictures were recorded using a cooled CCD camera and collected digitally with Metamorph (Universal Imaging, West Chester, PA). The DNA of some of the injected cells arrested as a consequence of Ku 162 administration were counter-stained with a DNA stain, Hoechst 3342 (0.001 mg/ml, Molecular Probes, Oregon) to determine the structure of the DNA, and specifically to answer the question of whether the failure of these cells to undergo cell division could be due to the induction of apoptosis as a result of the antibody.
Results and Discussion
To determine whether DNA-PK plays a unique role during early embryogenesis, we microinjected two anti-Ku antibodies, both raised against human Ku, into one cell of 2-cell embryos, and examined their effects on subsequent development. For technical reasons, we employed Lytechinus pictus in these studies rather than Arbacia punctulata. Hence, it was necessary to demonstrate that the fertilized eggs and early embryos of this species would express DNA-PK, and that this enzyme could be activated by the same means as those employed in the Arbacia. The first monoclonal antibody against the Ku protein, Ku 162, is an IgG that recognizes a conformational epitope on the Ku heterodimer. It has been used successfully to immunodeplete the DNA-PK activity from Xenopus oocytes (Kanungo et al., 1997). When PAS beads are coated with this antibody, the enzyme in cytoplasmic extracts of L. pictus 2-cell embryos associates with the beads and is catalytically active in the absence of (ds)DNA (Fig. lA). This antibody could not be employed in western analysis because it detects the Ku heterodimer, but neither of its subunits. However, a western blot of the proteins eluted from the PAS beads after labeling with [[P.sup.32]]-gamma ATP shows that the interacting catalytic subunit of DNA-PK (p460) is present and can undergo autophosphorylation (Fig. 1B). An immunoblot of the immunoprecipitates made with a monoclonal antibody raised against the human p460 shows that a polypeptide identical to p460 is co-immunoprecipitated by Ku 162 antibody from the cytoplasmic extracts prepared from 2-cell embryos of L. pictus (Fig. lB, lanes 5 and 6).
Based on these results, initial experiments were carried out to determine the effect of the injection of the antibody, Ku 162, or purified DNA-PK, into one cell of a 2-cell embryo. The injection protocol is illustrated in Figure 2A. A concentration of Ku 162 was chosen (2.5 pg) that completely arrested cell division in about one-half of the injected cells in 2-cell embryos 1 h after fertilization (n = 17). At that time, most of the uninjected embryos were at the 16/32 cell stage of development. On the other hand, further progression of the injected cell was completely arrested in one-half of the embryos (n = 17) injected with 2.5 pg of Ku 162 (Fig. 2B). Some cells were injected with a standard, but not maximally effective, concentration of purified DNA-PK either in the presence or absence of Ku 162. Injection of purified DNA-PK (the holoenzyme) alone had no effect on the development of the injected cells (n = 12, data not shown). However, when the holoenzyme was co-injected with Ku 162, the normal Ku 162-dep endent inhibition of cell development was overcome, and 90% (n = 10) of the co-injected cells developed normally (Fig. 2C). In other cells, injection of either 0.5 M KC1 (n = 13) or anti-RNA polymerase II antibody (Promega) (n = 15) had no effect on the ability of the antibody, Ku 162, to induce developmental arrest.
The further effects of the antibody, Ku 162, on embryonic cell development were analyzed in several ways: serial dilutions of Ku 162 were used to construct a dose-response curve; the effect of the second antibody, N3H10, was determined; and that of preimmune mouse IgG was examined. In addition, the possibility that Ku 162 was inhibiting cell division by causing cell necrosis or apoptosis was evaluated.
As shown in Figure 3A, the injection of standard aliquots of serially diluted Ku 162 antibody solution into one cell of a 2-cell embryo produced two effects on cell development. First, the number of cells that are completely arrested decreased as the amount of injected Ku 162 decreased (Fig. 3A, B, C). Second, at the highest concentration of Ku 162 (5 pg/cell) the development of all injected cells (n = 21) was completely arrested at the single-cell stage (Fig. 3A). The injection of 2.5 pg/cell of the Ku 162 antibody completely arrested development of 40% of the cells (n = 18) (Fig. 3A, right), and the remaining 60% of injected cells (n = 17) displayed variable rates of development (Fig. 3B, left). The injection of 1 pg of Ku 162 had no discernible effect on 50% of cells; i.e., there was no evidence of developmental arrest (Fig. 3C, left). In this case, only 10% of cells showed a complete inhibition of cell division, and 40% displayed variable degrees of retarded development (Fig. 3C, right). To determine if Ku 162 antibody acts by inhibiting cell division, and not by causing cell necrosis or apoptosis, a number of the cells of embryos (n = 12) that displayed complete arrest were counter-stained with Hoechst 33342. Evidence of apoptosis was not seen in any of the arrested cells (Fig. 3A, right).
In contrast to the results with the Ku antibody 162, injection of comparable amounts of N3H10, a Ku antibody directed against the 70-kDa subunit of Ku, had no apparent effect on the development of the injected cell of a 2-stage embryo (n = 15). It is of interest that one of the subunits (Ku 86) of Ku, but not the other (Ku 70), affected the postfertilization cleavage in sea urchin. This differential function specific to Ku 86 is consistent with the observation that potential functions of DNA-PK, such as X-ray sensitivity, is restored by Ku 86 even when Ku 70 is absent (Smider et al., 1994; Taccioli et al., 1994). An obvious question is whether these two Ku antibodies have different effects on the activity of DNA-PK. On the one hand, we have found that Ku N3H10, when put on PAS beads, is incapable of immunoprecipitating DNA-PK in an active form. What is not yet clear is whether either antibody inhibits DNA-PK in situ or in cytoplasmic extracts of sea urchin embryos. Attempts to address this question by using cytoplasmic extracts have proven difficult for several technical reasons, so at present we have no answer.
The present results show that a sufficient concentration of an antibody specific against the Ku heterodimer, Ku 162, when injected into the cytoplasm of one cell of a two-cell sea urchin embryo, completely blocked later cell divisions of the injected cell (Fig. 3). The simultaneous injection of the holoenzyme rescued the injected cells from developmental arrest (Fig. 3C). At intermediate doses, the effects of the Ku 162 antibody were attenuated; i.e., the number of cells completely arrested was reduced, and a number of cells were delayed, but not completely arrested, in their development (Fig. 2B, C). Although the simultaneous injection of the holoenzyme rescued the cells injected with Ku 162 from developmental arrest, injection of the holoenzyme itself had no consistent effect. In these rescue experiments, however, the purified holoenzyme, which is quite labile, appears to be acting as an antigen rather than as an active enzyme. In this light, the lack of effect when the holoenzyme is injected should not be taken as evidence that endogenous active enzyme has no effect on cell cycle progression. Additional experiments will be needed to test this issue in a critical fashion.
An inhibition of mitosis in sand dollar embryos follows the microinjection of antibodies against the calcium transport enzyme of muscle (Silver, 1986). And a similar inhibition occurs in sea urchin embryos after the microinjection of antibodies against kinesin-like proteins (Wright et al., 1993), the mitotic calcium transport system (Hafner and Petzelt, 1987), or a 62-kDa mitotic apparatus protein (Dins more and Sloboda, 1989). On the other hand, a controlled translocation of other nuclear enzymes, like DNA polymerase (Loeb et al., 1970), from cytoplasmic stores to the nucleus occurs during sea urchin embryogenesis. Recently, it has been reported that the catalytic activity of DNA-PK does not require Ku (Yaneva et al., 1997; Hammerstein and Chu, 1998). The simplest hypothesis to account for our result is that the Ku 162 antibody interferes with a temporally controlled transport of DNA-PK from a cytoplasmic store to the nucleus as development proceeds. Alternatively, this antibody may inhibit the activity of the enzyme in either a nuclear or a cytoplasmic location. In any case, our results suggest that Ku plays an important role in the control of cell cycle events during early embryogenesis.
We thank Westley Reeves for the generous gift of the Ku antibodies used in these studies and Timothy Carter for the anti-p460 antibody. We thank Judith Venuti, Richard Cameron, and John Hardin for helpful discussions, Krishna Patel for help with some of the experiments, and Rudi Rotten-fusser and Ivan Burba of Carl Zeiss, Inc., for their generous help in providing microscopic facilities that allowed this work to be performed. Support was provided by NIH grant DK-19813-20 to H. R. and by funds from the Medical College of Georgia; R.M.E. is supported by the Wellcome Trust.
(1.) Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912, and (2.) Marine Biological Laboratory, Woods Hole, Massachusetts 02543; (3.) University Department of Pharmacology, University of Oxford, Oxford, OXI 3QT, UK
Received 24 March 1999; accepted 16 August 1999.
(*.) Author to whom correspondence should be addressed. E-mail: email@example.com
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|Author:||KANUNGO, JYOTSHNA; EMPSON, RUTH M.; RASMUSSEN, HOWARD|
|Publication:||The Biological Bulletin|
|Date:||Dec 1, 1999|
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