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Characterization of DIP1, a novel nuclear protein in Drosophila melanogaster
BBRC Biochemical and Biophysical Research Communications 307 (2003) 224–228 www.elsevier.com/locate/ybbrc
Characterization of DIP1, a novel nuclear protein in Drosophila melanogaster Bruna De Felice,a,* Robert Roy Wilson,b Paolo Mondola,c Gianfranco Matrone,c Simona Damiano,c Corrado Garbi,d Luigi Nezi,d and Tin Tin Sue a
d
Department of Life Sciences, University of Naples II, Via Vivaldi 43, 81100 Caserta, Italy b NOAA, 325 Broadway, Boulder, CO, USA c Department of Neuroscience, Section of Physiology, University of Naples “Federico II,” Naples, Italy Department of Cellular and Molecular Biology and Pathology, University of Naples “Federico II,” Naples, Italy e MCD Biology 347 UCB, University of Colorado, Boulder 80309-0347, USA Received 2 June 2003
Abstract We have recently identified in Drosophila melanogaster a new gene encoding a nuclear protein, DIP1. Here we report the developmental expression and the finding that DIP1 subcellular localization is in the nucleus and at the nuclear periphery during interphase in embryos. Interestingly, in humans, DIP1 antibody identified signals in nuclei from cultured cells and reacted with a rough 30 kDa protein in Western blotting experiments, demonstrating evolutionary conservation. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Nuclear protein; Drosophila; Humans; Nuclear periphery protein
The genomes of higher eukaryotes contain large amounts of simple and complex tandemly repeated DNA sequences, called satellite DNA. These are located primarily in centromeric heterochromatin. However, their role in possible functions of this region of the chromosome, such as chromosome pairing and segregation, is not very well understood yet. Dodeca satellite is a type of tandemly repeated DNA sequence that is located in the centromeric region of Drosophila melanogaster chromosome 3 and cross-hybridizes with DNA from other species including humans [1]. In human genome, dodeca satellite-like sequences were localized at the pericentromeric region of metaphase chromosome 9 and 15 and on the long arm of the Y chromosome. A homology between the dodeca-satellite consensus sequence and the human satellite 3 family sequence has been found. Also, Drosophila dodeca sequence showed a zipper-like motif (GGGA)2 similar to the human centromeric (TGGAA)n sequence [2].
In a previous work, using “one hybrid system assay” in yeast, we isolated a novel gene in D. melanogaster [3], using a double-strand dodeca-satellite sequence as a bait, encoding a protein, here renamed Dip1, of unknown function. The discovery of dodeca-satellite binding proteins could be biologically important, because they might be evolutionary conserved. To understand the role of this novel protein in D.m., we studied the Dip1 gene expression pattern and subcellular localization from embryos during embryonic cycle. Because of conservation of dodeca satellite in human genome, we asked whether Drosophila DIP1 has homologue proteins or a homologue protein domain in humans. Interestingly enough, in humans, DIP1 antibody also identified signals in nuclei from cultured cells and showed a rough 30 kDa protein in Western blotting experiments.
Materials and methods * Corresponding author. Fax: +39-823-274571. E-mail address: [email protected] (B. De Felice).
Quantitative reverse transcription-PCR. Total RNAs (5 lg) were isolated from flies from 0–2 h old embryos, 2–24 h old embryo, larvae,
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)01141-0
B. De Felice et al. / Biochemical and Biophysical Research Communications 307 (2003) 224–228 pupae, adult males, and females. The specific gene of interest was amplified with 1 ll (100 ng) of the total RNA in the presence of a mixture containing 5 pmol of specific primers (forward primer: 50 CTTCAATGAGTTTTGTCATGCTTTA-30 and reverse primer: 50 TTTCGATTCTCGTATTGCCTTACAT-30 ), 7.5 ll of a 2.7Cycler RNAMasterSYBR Green I solution (Roche), and 1.3 ll of Mn(Oac)2 stock solution. The mix was incubated for 30 min at 50 °C and the cDNA product was amplified by 35 cycles of PCR (30 s at 94 °C, 30 s at 55 °C, and 1 min at 72 °C). The incorporation of the dye into the amplified products was monitored by LightCycler (Roche), and the concentration of a specific transcript in the sample was analyzed by the associated software based on the standard curves predetermined with known amounts of target transcripts. Quantities of rp49 gene transcripts were used as a total-cDNA control. Results from three independent quantitative reverse transcription-PCR (RT-PCR) analyses were averaged. Immunostaining Drosophila embryos. Wild-type (Sevelen) embryos were collected on grape-agar plates. Chorions were removed by treatment with 50% bleach for 2 min followed by extensive rinsing in distilled water. Embryos were then fixed for 20 min in a two phase layer of heptane and PBS + 4% formaldehyde. The heptane layer and most of the aqueous layer were replaced with methanol and vitelline membranes were removed by vigorous shaking. Fixed embryos were blocked in PBT (PBS + 0.02% Tween 20) containing 3% normal goat serum, and were stained with the primary antibody (affinity-purified rabbit polyclonal generated by immunizing rabbits with the synthetic peptide: PAPVWEDQSDDVP) diluted 1:10 in blocking solution. Secondary antibody (anti-rabbit FITC; pre-absorbed) was used at 1:500 in blocking solution. The antibody incubations were for at least 2 h at room temperature. Embryos were also stained with 10 lg/ml Hoechst33258 (Sigma) in PBT for 4 min to visualize DNA and with 10 lg/ml Texas Red conjugated Wheat Germ Agglutinin (Molecular Probes) in PBT for 10 min to visualize the nuclear envelope. Stained embryos were mounted onto slides with Flourmount G (Southern Biotechnology Associates). Nuclear envelope preparation. Nuclei from Drosophila embryos and nuclear envelopes were isolated as described previously [4]. Cells. Human fibroblast cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 2 mM L -glutamine, 50 lg/ml streptomycin, and 50 IU/ml penicillin in 5% CO2 at 37 °C. Human neuroblastoma cells SK-N-BE (American Type Culture Collection) were grown in RPMI 1640 medium (Sigma, St. Louis, MO, USA) supplemented with 10% fetal calf serum (FCS, Sigma), 2 mM L -glutamine, 5 lg/ml streptomycin, and 50 IU/ml penicillin in 5% CO2 at 37 °C. Western blot experiments. The samples for gel electrophoresis, from human cells, were obtained after digestion of the nuclei by microccocal nuclease for 1 h on ice in (3.37 mM Tris–HCl, pH 7.4, 0.05 mM spermine, 0.125 mM spermidine, 20 mM KCl, and 1 mM CaCl2 ) and then adjusted to 2% SDS, 2% b-mercaptoethanol, and 10% glycerol. The amount of extract loaded per well of SDS–polyacrylamide gel corresponds to approximately 106 nuclei. Nuclear extract was electrophoresed on 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel. Standard procedures were used for Western blot experiments using enhanced chemoluminescence detection according to the manufacturer’s instructions (Amersham Life Science, UK). Primary rabbit anti DIP1 antibody was used at 1:1000 dilution, while the secondary antibody, a horseradish peroxidase-linked anti-rabbit IgG, was used at 1:200 dilution. Immunofluorescence microscopy. Immunofluorescence studies were performed on cells seeded onto 12-mm diameter coverslips. Cells were fixed for 15 min with 3% paraformaldehyde in PBS at room temperature, washed twice in PBS, and permeabilized with 0.1% Triton in PBS for 5 min. After two washes in PBS, the cells were incubated with the primary polyclonal anti-DIP1 for 4 h in humidified chamber, washed three times with PBS, and incubated with the secondary antibody
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(rhodamine-conjugated goat anti rabbit antibody) for additional 2 h at 37 °C in the same conditions. Cells were then washed three times with PBS and the coverslips were mounted on a microscope slide using a 50% solution of glycerol in PBS and examined with a Zeiss LSM 510 confocal microscope.
Results RT-PCR analysis In flies, RT-PCR analysis showed that a Dip1 transcript was maternally deposited and gradually decreased through embryogenesis. In flies, the amount of Dip1 transcripts increased during the early stages of pupation and reached the highest level during adulthood (Fig. 1). Thus, Dip1 expression appears to be correlated with those developmental stages that involve differentiating or terminally differentiated cells, with minimal transcription in proliferating cells. Subcellular localization of DIP1 during the cell cycle The DIP1 protein localization during embryonic division is detected by immunostaining of fixed Drosophila embryos. Fig. 2A shows cells from an embryo in the interphase of embryonic cycle 14. FITC signal is seen as dots, some of which are in the nucleus and others at the nuclear periphery. The FITC foci are seen in all interphase nuclei in embryos (Figs. 2C and D). Although all interphase cells exhibited nuclear DIP1 staining, cells undergoing mitosis off cycle 14 lack the FITC signals during metaphase (Fig. 2B). We conclude that this protein is nuclear during interphase and is degraded into the cytoplasm in metaphase when the nuclear envelope is disrupted.
Fig. 1. Real-time RT-PCR analysis of Dip1 gene. Primers specific to Dip1 were used to screen RNA from flies from 0–2 h old embryos (1), 2–24 h old embryo (2), larvae (3), pupae (4), adult males (5), and females (6). Results from three independent quantitative RT-PCR experiments were averaged.
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Fig. 3. Western blotting with anti-DIP1 antibody against nuclear extract prepared from human fibroblasts (A) and SK-N-BE human neuroblastoma cells (B).
antibody recognizes DIP1 present in the isolated nuclei, showing the expected 20 kDa signal [3]. The nuclei were then treated with nuclease and extracted with salt. This extracted fraction (Fig. 2G, lane 2) and the remaining fraction, which is enriched in nuclear envelope (Fig. 2G, lane 3), contain detectable DIP1. This demonstrates that DIP1 is a tightly associated component of the nuclear envelope as well as it is localized throughout the nucleus. Western blotting experiments in human cells A strong signal of about 30 kDa was detected in both fibroblasts and SK cells (Fig. 3) and this band was not detected using the preimmune sera (data not shown). We therefore assume that this 30 kDa protein could correspond to a putative homologue DIP1 protein in human. Immunostaining in human cells Fig. 2. (A) shows cells from an embryo in interphase of embryonic cycle 14. FITC signal is seen as dots some of which are in the nucleus (black arrow) and others at the nuclear periphery (white arrow). The nucleus indicated with an asterisk is shown with the FITC signal increased in inset to demonstrate that some FITC signal is also seen as diffused foci in the nucleus. (B) shows cells undergoing mitosis of cycle 14. Nuclear FITC foci are absent in these cells. Arrow points to a cell in metaphase. (C) and (D) show two different focal planes of the same field of nuclei from a cycle 14 embryo. Arrows point to nuclei that lack FITC foci in one focal plane (C) but not in the other focal plane (D). Such visualization on different focal planes reveals the presence of FITC foci in all interphase nuclei in these embryos. (E) and (F) show cells from an embryo in interphase 14. Primary antibody was omitted during the staining procedure. The image in (E) was processed as in (A) while the image in (F) is shown with the FITC signal increased as in (A-inset) to demonstrate the absence of both FITC dots and diffused foci. Scale bar: 11 lm. (G) Biochemical fractionation of nuclei from Drosophila embryos. Western blots of whole nuclei (lane 1), nuclease treated, salt extracted supernatant (lane 2), and highly enriched nuclear envelope fractions (lane 3) were probed for DIP1.
DIP1 is localized to the nuclear envelope at the interphase To obtain biochemical support for the association between DIP1 and nuclear envelope in D.m., we probed for its presence in nuclear envelope preparations from D.m. nuclei at the interphase. In Fig. 2G, lane 1 our
In both types of cultured cells the staining appeared to be located almost exclusively in the nucleus in the form of microscopically visible discrete foci (Fig. 4). This punctate pattern is best shown at higher magnification in c where a peculiar concentration of foci at the nuclear periphery is also evident. No signal was detected using preimmune serum (data not shown).
Discussion In a previous paper [3] we isolated in D.m. a novel gene, Dip1, encoding a protein of unknown function and we demonstrated a binding activity between dodeca-satellite sequence in D.m. and this novel protein. Using the Blast network service at NCBI, DIP1 protein produces significant alignments with gene products, in D.m. and Anopheles gambiae, respectively, of unknown function (Table 1). Our data from RT-PCR experiments and subcellular localization during cell-cycle in D.m. indicated that DIP1 is expressed mostly in differentiating or terminally differentiated cellular stage, with a reduced expression in embryos. Dip1 expression decreases in growing cells and increases in differentiating and/or terminally differentiated cellular stages such as other reported negative regulators of cell growth [5–7].
B. De Felice et al. / Biochemical and Biophysical Research Communications 307 (2003) 224–228
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Table 1 Alignment of Dip1 homologues
A Clustal alignment of Dip1 homologues from Drosophila melanogaster (Q9W080 and Q8SXW3), Anopheles gambiae (agCP3339). The region against which the peptide antibody was raised is underlined. The regions showing two putative protein kinase C phosphorylation sites are in boldface.
Interestingly, during interphase, such other nuclear proteins in eukaryotes [8–12], DIP1 is localized to the nuclear periphery and chromatin domain in all nuclei, but disappeared at the metaphase. Additionally, DIP1 protein shows two putative protein kinase C phosphorylation sites (Table 1), as well as numerous proteins in eukaryotes that are phosphorylated to promote the structural reorganization that accompanies the entry of the cells into mitosis [13,14]. Our interesting and unexpected finding is that DIP1 was found at the nuclear envelope during interphase. This association is not likely to be a transient, low affinity one, since it survives the strong extraction procedure used in isolating nuclear envelope. The significance of this finding is unclear, but it suggests that DIP1 may play different functions in the cell cycle, because the localization of a protein within a cell plays an important role in the function of that protein [8]. It has been known that centromeric regions
are found associated with the nuclear envelope [15,16] during interphase. Alternatively, DIP1 might be involved in regulating the nuclear transport of proteins required for entry into mitosis. Further experimentation could help one to determine the true role of DIP1 in D.m. An important aim of studying developmental control of cell-cycle regulation in organisms like Drosophila is to gain a similar understanding in vertebrates, although the latter seem to have more tiers of regulation [17]. A search of the GenBank database did not reveal any significant DIP1 homologue proteins in humans, but using anti-DIP1 antibody in Western blotting experiments from both fibroblasts and SK nuclear proteins we detected a strong signal of 30 kDa that was absent using the preimmune sera. In addition, staining these cells during interphase, we detected strong signals in nuclei and in nuclear periphery as well as in Drosophila. These
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References
Fig. 4. Subcellular localization of DIP1. Confocal microscopy images of human fibroblasts (A) and SK-N-BE human neuroblastoma cells (B,C). In both types of cultured cells the staining appeared to be located almost exclusively in the nucleus in the form of microscopically visible discrete foci. This punctate pattern is best shown at higher magnification in (C) where a peculiar concentration of foci at the nuclear periphery is also evident. Scale bar: (A,B) 5 lm and (C) 2.5 lm.
data indicate that the anti-DIP1 antibody binds to Drosophila nuclei as well as human nuclei. Therefore, we theorize that there is probably a DIP1 homologue protein in humans, demonstrating evolutionary conservation (see Figs. 3 and 4).
[1] J.P. Abad, M. Carmena, et al., Dodeca satellite: a conserved G + C-rich satellite from the centromeric heterochromatin of Drosophila melanogaster, Proc. Natl. Acad. Sci. USA 89 (1992) 4663–4667. [2] S.H. Chou, K.H. Chin, Quadruple intercalated G-6 stack: a possible motif in the fold-back structure of the Drosophila centromeric dodeca-satellite? J. Mol. Biol. 314 (2001) 139–152. [3] B. De Felice et al., Identification of a new gene encoding pericentromeric dodeca-satellite binding protein in Drosophila melanogaster, FEBS Lett. 455 (1999) 31–35. [4] R. Ashery-Padan, A.M. Weiss, et al., Distinct regions specify the targeting of otefin to the nucleoplasmic side of side of the nuclear envelope, J. Biol. Chem. 4 (1997) 2493–2499. [5] C.V. Dang, c-Myc target genes involved in cell growth, apoptosis, and metabolism, Mol. Cell. Biol. 1 (1999) 1–11. [6] A. Sommer et al., Identification and characterization of specific DNA-binding complexes containing members of the Myc/Max/ Mad network of transcriptional regulators, J. Biol. Chem. 273 (1998) 6632–6642. [7] S.B. Gim et al., Drosophila Med6 is required for elevated expression of a large but distinct set of developmentally regulated genes, Mol. Cell. Biol. 21 (2001) 5242–5255. [8] M.S. Campbell, K.T. Gordon, et al., Mitotic checkpoint proteins HsMAD1 and HsMAD2 are associated with nuclear pore complexes in interphase, J. Cell Sci. 114 (2001) 953–963. [9] S. Gehring et al., Inhibition of proliferation and apoptosis by the transcriptional repressor Mad1, J. Biol. Chem. 14 (2000) 10413– 10420. [10] A.E. Ikui et al., Control of localization of a spindle checkpoint protein, Mad2, in fission yeast, J. Cell Sci. 115 (2002) 1603– 1610. [11] T.T. Su, P.H. O’Farrell, Chromosome association of minichromosome maintenance proteins in Drosophila mitotic cycles, J. Cell Biol. 139 (1997) 13–21. [12] R. Padan et al., Isolation and characterization of the Drosophila nuclear envelope otefin cDNA, J. Biol. Chem. 14 (1990) 7808– 7813. [13] E.A. Nigg, Cyclin-dependent protein kinases: key regulators of the eukaryotic cell cycle, BioEssays 17 (1995) 471–480. [14] D.E. Smith, P.A. Fisher, Interconversion of Drosophila nuclear lamin isoforms during oogenesis, early embryogenesis, and upon entry of cultured cells into mitosis, J. Cell Biol. 260 (1989) 255– 265. [15] T. Haaf, M. Schmid, Centromeric association and non-random distribution of centromeres in human tumour cells, Hum. Genet. 81 (1989) 137–143. [16] Y. Li et al., MAD2 associates with the cyclosome/anaphasepromoting complex and inhibits its activity, Proc. Natl. Acad. Sci. USA 94 (1997) 12431–12436. [17] S.J. Vidwans, T.T. Su, Cycling through development in Drosophila and other metazoa, Nat. Cell Biol. 3 (2001) 35–39.
Characterization of DIP1, a novel nuclear protein in Drosophila melanogaster Bruna De Felice,a,* Robert Roy Wilson,b Paolo Mondola,c Gianfranco Matrone,c Simona Damiano,c Corrado Garbi,d Luigi Nezi,d and Tin Tin Sue a
d
Department of Life Sciences, University of Naples II, Via Vivaldi 43, 81100 Caserta, Italy b NOAA, 325 Broadway, Boulder, CO, USA c Department of Neuroscience, Section of Physiology, University of Naples “Federico II,” Naples, Italy Department of Cellular and Molecular Biology and Pathology, University of Naples “Federico II,” Naples, Italy e MCD Biology 347 UCB, University of Colorado, Boulder 80309-0347, USA Received 2 June 2003
Abstract We have recently identified in Drosophila melanogaster a new gene encoding a nuclear protein, DIP1. Here we report the developmental expression and the finding that DIP1 subcellular localization is in the nucleus and at the nuclear periphery during interphase in embryos. Interestingly, in humans, DIP1 antibody identified signals in nuclei from cultured cells and reacted with a rough 30 kDa protein in Western blotting experiments, demonstrating evolutionary conservation. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Nuclear protein; Drosophila; Humans; Nuclear periphery protein
The genomes of higher eukaryotes contain large amounts of simple and complex tandemly repeated DNA sequences, called satellite DNA. These are located primarily in centromeric heterochromatin. However, their role in possible functions of this region of the chromosome, such as chromosome pairing and segregation, is not very well understood yet. Dodeca satellite is a type of tandemly repeated DNA sequence that is located in the centromeric region of Drosophila melanogaster chromosome 3 and cross-hybridizes with DNA from other species including humans [1]. In human genome, dodeca satellite-like sequences were localized at the pericentromeric region of metaphase chromosome 9 and 15 and on the long arm of the Y chromosome. A homology between the dodeca-satellite consensus sequence and the human satellite 3 family sequence has been found. Also, Drosophila dodeca sequence showed a zipper-like motif (GGGA)2 similar to the human centromeric (TGGAA)n sequence [2].
In a previous work, using “one hybrid system assay” in yeast, we isolated a novel gene in D. melanogaster [3], using a double-strand dodeca-satellite sequence as a bait, encoding a protein, here renamed Dip1, of unknown function. The discovery of dodeca-satellite binding proteins could be biologically important, because they might be evolutionary conserved. To understand the role of this novel protein in D.m., we studied the Dip1 gene expression pattern and subcellular localization from embryos during embryonic cycle. Because of conservation of dodeca satellite in human genome, we asked whether Drosophila DIP1 has homologue proteins or a homologue protein domain in humans. Interestingly enough, in humans, DIP1 antibody also identified signals in nuclei from cultured cells and showed a rough 30 kDa protein in Western blotting experiments.
Materials and methods * Corresponding author. Fax: +39-823-274571. E-mail address: [email protected] (B. De Felice).
Quantitative reverse transcription-PCR. Total RNAs (5 lg) were isolated from flies from 0–2 h old embryos, 2–24 h old embryo, larvae,
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)01141-0
B. De Felice et al. / Biochemical and Biophysical Research Communications 307 (2003) 224–228 pupae, adult males, and females. The specific gene of interest was amplified with 1 ll (100 ng) of the total RNA in the presence of a mixture containing 5 pmol of specific primers (forward primer: 50 CTTCAATGAGTTTTGTCATGCTTTA-30 and reverse primer: 50 TTTCGATTCTCGTATTGCCTTACAT-30 ), 7.5 ll of a 2.7Cycler RNAMasterSYBR Green I solution (Roche), and 1.3 ll of Mn(Oac)2 stock solution. The mix was incubated for 30 min at 50 °C and the cDNA product was amplified by 35 cycles of PCR (30 s at 94 °C, 30 s at 55 °C, and 1 min at 72 °C). The incorporation of the dye into the amplified products was monitored by LightCycler (Roche), and the concentration of a specific transcript in the sample was analyzed by the associated software based on the standard curves predetermined with known amounts of target transcripts. Quantities of rp49 gene transcripts were used as a total-cDNA control. Results from three independent quantitative reverse transcription-PCR (RT-PCR) analyses were averaged. Immunostaining Drosophila embryos. Wild-type (Sevelen) embryos were collected on grape-agar plates. Chorions were removed by treatment with 50% bleach for 2 min followed by extensive rinsing in distilled water. Embryos were then fixed for 20 min in a two phase layer of heptane and PBS + 4% formaldehyde. The heptane layer and most of the aqueous layer were replaced with methanol and vitelline membranes were removed by vigorous shaking. Fixed embryos were blocked in PBT (PBS + 0.02% Tween 20) containing 3% normal goat serum, and were stained with the primary antibody (affinity-purified rabbit polyclonal generated by immunizing rabbits with the synthetic peptide: PAPVWEDQSDDVP) diluted 1:10 in blocking solution. Secondary antibody (anti-rabbit FITC; pre-absorbed) was used at 1:500 in blocking solution. The antibody incubations were for at least 2 h at room temperature. Embryos were also stained with 10 lg/ml Hoechst33258 (Sigma) in PBT for 4 min to visualize DNA and with 10 lg/ml Texas Red conjugated Wheat Germ Agglutinin (Molecular Probes) in PBT for 10 min to visualize the nuclear envelope. Stained embryos were mounted onto slides with Flourmount G (Southern Biotechnology Associates). Nuclear envelope preparation. Nuclei from Drosophila embryos and nuclear envelopes were isolated as described previously [4]. Cells. Human fibroblast cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 2 mM L -glutamine, 50 lg/ml streptomycin, and 50 IU/ml penicillin in 5% CO2 at 37 °C. Human neuroblastoma cells SK-N-BE (American Type Culture Collection) were grown in RPMI 1640 medium (Sigma, St. Louis, MO, USA) supplemented with 10% fetal calf serum (FCS, Sigma), 2 mM L -glutamine, 5 lg/ml streptomycin, and 50 IU/ml penicillin in 5% CO2 at 37 °C. Western blot experiments. The samples for gel electrophoresis, from human cells, were obtained after digestion of the nuclei by microccocal nuclease for 1 h on ice in (3.37 mM Tris–HCl, pH 7.4, 0.05 mM spermine, 0.125 mM spermidine, 20 mM KCl, and 1 mM CaCl2 ) and then adjusted to 2% SDS, 2% b-mercaptoethanol, and 10% glycerol. The amount of extract loaded per well of SDS–polyacrylamide gel corresponds to approximately 106 nuclei. Nuclear extract was electrophoresed on 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel. Standard procedures were used for Western blot experiments using enhanced chemoluminescence detection according to the manufacturer’s instructions (Amersham Life Science, UK). Primary rabbit anti DIP1 antibody was used at 1:1000 dilution, while the secondary antibody, a horseradish peroxidase-linked anti-rabbit IgG, was used at 1:200 dilution. Immunofluorescence microscopy. Immunofluorescence studies were performed on cells seeded onto 12-mm diameter coverslips. Cells were fixed for 15 min with 3% paraformaldehyde in PBS at room temperature, washed twice in PBS, and permeabilized with 0.1% Triton in PBS for 5 min. After two washes in PBS, the cells were incubated with the primary polyclonal anti-DIP1 for 4 h in humidified chamber, washed three times with PBS, and incubated with the secondary antibody
225
(rhodamine-conjugated goat anti rabbit antibody) for additional 2 h at 37 °C in the same conditions. Cells were then washed three times with PBS and the coverslips were mounted on a microscope slide using a 50% solution of glycerol in PBS and examined with a Zeiss LSM 510 confocal microscope.
Results RT-PCR analysis In flies, RT-PCR analysis showed that a Dip1 transcript was maternally deposited and gradually decreased through embryogenesis. In flies, the amount of Dip1 transcripts increased during the early stages of pupation and reached the highest level during adulthood (Fig. 1). Thus, Dip1 expression appears to be correlated with those developmental stages that involve differentiating or terminally differentiated cells, with minimal transcription in proliferating cells. Subcellular localization of DIP1 during the cell cycle The DIP1 protein localization during embryonic division is detected by immunostaining of fixed Drosophila embryos. Fig. 2A shows cells from an embryo in the interphase of embryonic cycle 14. FITC signal is seen as dots, some of which are in the nucleus and others at the nuclear periphery. The FITC foci are seen in all interphase nuclei in embryos (Figs. 2C and D). Although all interphase cells exhibited nuclear DIP1 staining, cells undergoing mitosis off cycle 14 lack the FITC signals during metaphase (Fig. 2B). We conclude that this protein is nuclear during interphase and is degraded into the cytoplasm in metaphase when the nuclear envelope is disrupted.
Fig. 1. Real-time RT-PCR analysis of Dip1 gene. Primers specific to Dip1 were used to screen RNA from flies from 0–2 h old embryos (1), 2–24 h old embryo (2), larvae (3), pupae (4), adult males (5), and females (6). Results from three independent quantitative RT-PCR experiments were averaged.
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B. De Felice et al. / Biochemical and Biophysical Research Communications 307 (2003) 224–228
Fig. 3. Western blotting with anti-DIP1 antibody against nuclear extract prepared from human fibroblasts (A) and SK-N-BE human neuroblastoma cells (B).
antibody recognizes DIP1 present in the isolated nuclei, showing the expected 20 kDa signal [3]. The nuclei were then treated with nuclease and extracted with salt. This extracted fraction (Fig. 2G, lane 2) and the remaining fraction, which is enriched in nuclear envelope (Fig. 2G, lane 3), contain detectable DIP1. This demonstrates that DIP1 is a tightly associated component of the nuclear envelope as well as it is localized throughout the nucleus. Western blotting experiments in human cells A strong signal of about 30 kDa was detected in both fibroblasts and SK cells (Fig. 3) and this band was not detected using the preimmune sera (data not shown). We therefore assume that this 30 kDa protein could correspond to a putative homologue DIP1 protein in human. Immunostaining in human cells Fig. 2. (A) shows cells from an embryo in interphase of embryonic cycle 14. FITC signal is seen as dots some of which are in the nucleus (black arrow) and others at the nuclear periphery (white arrow). The nucleus indicated with an asterisk is shown with the FITC signal increased in inset to demonstrate that some FITC signal is also seen as diffused foci in the nucleus. (B) shows cells undergoing mitosis of cycle 14. Nuclear FITC foci are absent in these cells. Arrow points to a cell in metaphase. (C) and (D) show two different focal planes of the same field of nuclei from a cycle 14 embryo. Arrows point to nuclei that lack FITC foci in one focal plane (C) but not in the other focal plane (D). Such visualization on different focal planes reveals the presence of FITC foci in all interphase nuclei in these embryos. (E) and (F) show cells from an embryo in interphase 14. Primary antibody was omitted during the staining procedure. The image in (E) was processed as in (A) while the image in (F) is shown with the FITC signal increased as in (A-inset) to demonstrate the absence of both FITC dots and diffused foci. Scale bar: 11 lm. (G) Biochemical fractionation of nuclei from Drosophila embryos. Western blots of whole nuclei (lane 1), nuclease treated, salt extracted supernatant (lane 2), and highly enriched nuclear envelope fractions (lane 3) were probed for DIP1.
DIP1 is localized to the nuclear envelope at the interphase To obtain biochemical support for the association between DIP1 and nuclear envelope in D.m., we probed for its presence in nuclear envelope preparations from D.m. nuclei at the interphase. In Fig. 2G, lane 1 our
In both types of cultured cells the staining appeared to be located almost exclusively in the nucleus in the form of microscopically visible discrete foci (Fig. 4). This punctate pattern is best shown at higher magnification in c where a peculiar concentration of foci at the nuclear periphery is also evident. No signal was detected using preimmune serum (data not shown).
Discussion In a previous paper [3] we isolated in D.m. a novel gene, Dip1, encoding a protein of unknown function and we demonstrated a binding activity between dodeca-satellite sequence in D.m. and this novel protein. Using the Blast network service at NCBI, DIP1 protein produces significant alignments with gene products, in D.m. and Anopheles gambiae, respectively, of unknown function (Table 1). Our data from RT-PCR experiments and subcellular localization during cell-cycle in D.m. indicated that DIP1 is expressed mostly in differentiating or terminally differentiated cellular stage, with a reduced expression in embryos. Dip1 expression decreases in growing cells and increases in differentiating and/or terminally differentiated cellular stages such as other reported negative regulators of cell growth [5–7].
B. De Felice et al. / Biochemical and Biophysical Research Communications 307 (2003) 224–228
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Table 1 Alignment of Dip1 homologues
A Clustal alignment of Dip1 homologues from Drosophila melanogaster (Q9W080 and Q8SXW3), Anopheles gambiae (agCP3339). The region against which the peptide antibody was raised is underlined. The regions showing two putative protein kinase C phosphorylation sites are in boldface.
Interestingly, during interphase, such other nuclear proteins in eukaryotes [8–12], DIP1 is localized to the nuclear periphery and chromatin domain in all nuclei, but disappeared at the metaphase. Additionally, DIP1 protein shows two putative protein kinase C phosphorylation sites (Table 1), as well as numerous proteins in eukaryotes that are phosphorylated to promote the structural reorganization that accompanies the entry of the cells into mitosis [13,14]. Our interesting and unexpected finding is that DIP1 was found at the nuclear envelope during interphase. This association is not likely to be a transient, low affinity one, since it survives the strong extraction procedure used in isolating nuclear envelope. The significance of this finding is unclear, but it suggests that DIP1 may play different functions in the cell cycle, because the localization of a protein within a cell plays an important role in the function of that protein [8]. It has been known that centromeric regions
are found associated with the nuclear envelope [15,16] during interphase. Alternatively, DIP1 might be involved in regulating the nuclear transport of proteins required for entry into mitosis. Further experimentation could help one to determine the true role of DIP1 in D.m. An important aim of studying developmental control of cell-cycle regulation in organisms like Drosophila is to gain a similar understanding in vertebrates, although the latter seem to have more tiers of regulation [17]. A search of the GenBank database did not reveal any significant DIP1 homologue proteins in humans, but using anti-DIP1 antibody in Western blotting experiments from both fibroblasts and SK nuclear proteins we detected a strong signal of 30 kDa that was absent using the preimmune sera. In addition, staining these cells during interphase, we detected strong signals in nuclei and in nuclear periphery as well as in Drosophila. These
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References
Fig. 4. Subcellular localization of DIP1. Confocal microscopy images of human fibroblasts (A) and SK-N-BE human neuroblastoma cells (B,C). In both types of cultured cells the staining appeared to be located almost exclusively in the nucleus in the form of microscopically visible discrete foci. This punctate pattern is best shown at higher magnification in (C) where a peculiar concentration of foci at the nuclear periphery is also evident. Scale bar: (A,B) 5 lm and (C) 2.5 lm.
data indicate that the anti-DIP1 antibody binds to Drosophila nuclei as well as human nuclei. Therefore, we theorize that there is probably a DIP1 homologue protein in humans, demonstrating evolutionary conservation (see Figs. 3 and 4).
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