Cell Motility and the Cytoskeleton 19:1-8 (1991)
A Monoclonal Antibody Recognizing a Common Antigen on Drosophila Embryos and Human Fibroblasts Giuliano Callaini and Maria Giovanna Riparbelli Department of Evolutionary Biology, University of Siena, Siena, Italy We used a monoclonal antibody specific for vimentin from human fibroblasts to stain whole mounts of Drosophilu embryos. In immunofluorescenceobservations this antibody cross-reacts with an antigenic determinant localized throughout mitosis at the nuclear boundary. Double fluorescence observations with the Rb188 antibody that specifically recognizes a centrosomal protein of the Drosophilu embryo [Whitfield et al., 19881 showed that the anti-vimentin antibody crossreacts with an antigen localized in the centrosomal region. Key words: anti-vimentin antibody, nuclear envelope, centrosomes, Drosophila early embryogenesis
The Drosophila embryo is a convenient tool to examine the spatial order during mitosis and the contribution of the cytoskeletal proteins to its maintenance. The nuclear divisions which lead to the formation of the blastoderm occur in very rapid succession without the formation of cellular membranes. After the first nine mitotic divisions, which occur synchronously in the central region of the egg, the majority of the nuclei migrate to the periphery and become easily visible in the whole mounts. The following four nuclear divisions lead to the formation of the complete blastoderm. During the last mitotic events (cycles 10, 11, 12, and 13) the synchrony as observed during intravitelline divisions is lost and mitotic waves may be observed (for details of this process see Zalokar and Erk [ 19761 and Foe and Alberts [ 19831). The Drosophilu embryo thus offers the possibility of following different mitotic stages at the same time. Although the distribution and dynamics of two of the major cytoskeletal systems, microfilaments and microtubules, have been extensively examined in early Drosophila embryos [Warn, 1986; Karr and Alberts, 1986; Kellogg et al., 19881, the presence of cytoplasmic intermediate filaments in Drosophila is still uncertain. Walter and Alberts  examined in Drosophila embryos the distribution of an antigenic determinant crossreacting with a monoclonal antibody raised to a major cytoplasmic 46 kda protein isolated by Falkner et al. 0 1991 Wiley-Liss, Inc.
 from Drosophila cultured cells and claimed to be an IF-like protein on the basis of several typical features [Walter and Biessmann, 1984a,b]. Here we report the results obtained in early Drosophila embryos with a monoclonal antibody raised to human fibroblast vimentin. This antibody does not seem to recognize a cytoplasmic filamentous network as visualized by Walter and Alberts [ 19841, but it apparently cross-reacts with antigenic determinants localized at the nuclear boundary. MATERIALS AND METHODS
For immunofluorescence microscopy the Drosophila embryos were dechorionated in a 50% commercial bleach solution, washed with distilled water, and fixed according to Warn and Warn [ 19861. After washing in PBS the embryos were placed for 1 hr in PBS containing 0.1% bovine serum albumin, washed again in PBS, and incubated overnight at 4°C with a commercially available monoclonal antibody against vimentin (Sclavo, Siena). This antibody was made in mouse after immunization with purified vimentin from human cultured fibroblasts. Received July 3 1, 1990; accepted December 21, 1990. Address reprint requests to Dr. Giuliano Callaini, Department of Evolutionary Biology, University of Siena, Via P.A. Mattioli 4, 53100 Siena, Italy.
Callaini and Riparbelli
Fig. 1 . Immunofluorescence staining of a CHO cell with the anti-vimentin antibody
After rinsing in PBS the material was incubated for 30 min with fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG serum (Miles). To stain the nuclei the embryos were incubated 3 min with 1 pgiml of the DNA-specific dye Hoechst 33258 in PBS. The embryos were then washed in PBS and mounted in 90% glycerol containing n-propyl-gallate [Giloh and Sedat, 19821. Some embryos were incubated with the second antibody to ensure the validity of the results; in no case was the fluorescence significant. Chinese hamster ovary cells (CHO) were fixed for 5 min each with methanol and acetone at -20°C. After washing in PBS the incubation with antibodies for indirect immunofluorescence was carried out as described above. Fluorescence observations were carried out with a Leitz Aristoplan microscope equipped with fluorescein and UV filters. Photomicrographs were taken with Kodak Tri-X pan film. RESULTS lmmunof luorescence With the Anti-Vimentin Antibody
When the anti-vimentin antibody was employed to stain Drosophila embryos and vertebrate cells, quite different immunofluorescence patterns were obtained. In interphase CHO cells that solely contain vimentin as intermediate filament cytoskeleton [Cabral et al., 19811 the antibody shows a network of intermediate filaments throughout the cytoplasm (Fig. 1). On the contrary, in Drosophila embryos the monoclonal antibody does not stain a similar cytoplasmic array of filamentous structures, but apparently reacts with an antigenic determinant that is localized at the periphery of the nucleus. In sur-
face views of whole mounts of Drosophila syncytial blastoderm the monoclonal antibody staining reveals thin fluorescent rings with two opposite circular regions where the fluorescence is more intense (Fig. 2a). Hoechst stain (Fig. 2b) and simultaneous light microscope observation using Nomarski optics (not shown) demonstrates that the localization of the antibody is restricted to the nuclear periphery and to the poles of the spindle. A feeble fluorescent punctate staining is also observable in the cytoplasm among the nuclei (Fig. 2a). During prophase, when the chromatin condenses, the antibody staining pattern is similar to that observed in the previous stage, except for the gradual elongation of the fluorescent structures, which become oval-shaped (Fig. 2c,d). When the chromosomes are aligned at the metaphase plate, the antibody binding pattern is similar to that observed in the previous stage, except for a further elongation of the structures which surround the chromatin region (Fig. 2e,f). As the chromosomes approach the poles during anaphase the fluorescent structures elongate further, becoming elliptical, and the pole staining decreases (not shown). During late anaphase, when the spindle elongates, there is a further elongation of the fluorescent structures and the staining at the poles diffuses over a large region (Fig. 3a,b). In suitable images we can see that the fluorescent structures are composed of thin fibrils running between the poles; a short zone of intense fluorescence is visible in the middle of these structures (Fig. 3a). Transition from anaphase to telophase is marked by the further evolution of the antigen binding pattern.
Common Antigens in Drosophila and Vertebrate Cells
Fig. 2. Fluorescence micrographs of whole mounts of Drosuphilu embryos stained with monoclonal antibody against human vimentin (a,c,e) and Hoechst dye (b,d,€). a,b) interphase; c,d) prophase; e,f) metaphase. Bar = 20 km.
Callaini and Riparbelli
Fig. 3. Fluorescence micrographs of whole mounts of Drosophih embryos stained with monoclonal antibody against human vimentin (a,c,e) and Hoechst dye (b,d,f). a,b) anaphase; c,d) telophase; e,f) early interphase. Bar = 20 p n .
Common Antigens in Drosophila and Vertebrate Cells
When the condensed chromatin expands the antibody recognizes elongated structures which are more fluorescent at their extremities, where cap-like formations are observed (Fig. 3c,d). The fluorescence in the equatorial region becomes diffuse and an irregular bright body can be detected in the probable region of the mid-body (Fig. 3c). At the end of telophase the daughter nuclei are reformed and the antigen binding sites are distributed in clearly fluorescent ring-shaped structures (Fig. 3e,f). Punctate fluorescence is visible in the cytoplasm among the nuclei. Relation Between Anti-Vimentin and Anti-Centrosome Staining Because the antibody staining is also observed throughout mitosis at the pole regions (Fig. 4a,b), we performed double labeling with the monoclonal anti-vimentin antibody and the Rb 188 rabbit antiserum that recognizes a centrosomal-associated antigen in the Drosophila embryo [Whitfield et al., 19881, to compare centrosome position and nuclear pole staining. The relation between the pole stain and centrosome position is clearly visible during interphase, prophase, and metaphase, when the fluorescence at the poles is localized in circular structures, but it becomes less apparent when the pole stain is more diffuse at anaphase and telophase. Figure 4c is a detail of some interphase nuclei in which the circular structures recognized by the anti-vimentin antibody are visible in frontal and sagittal views. Centrosomes, as recognized by the Rb188 antibody, are apparently localized in correspondence with the bright rings (Fig. 4d). When we measured the distance between rings and centrosomes in the same nucleus, we found that the distance between the centrosomes was larger than that between the rings. Double exposure confirms this observation and shows that the centrosomes are at the tips of the fluorescent rings during the first phases of mitosis, immersed in diffuse fluorescence during anaphase (Fig. 4e,f) or there is no apparent relationship between the position of the centrosomes and antivimentin stain at telophase (Fig. 4g,h). DISCUSSION In this paper we have shown by indirect immunofluorescence the presence in early Drosophila embryos of a protein component sharing an epitope with human fibroblast vimentin. Although cytoplasmic intermediate filaments have been documentated in vertebrate cells, their occurrence and distribution in invertebrates is supported by sparse evidence (for review see [Goldman et al., 1986; Franke, 1987; Steinert and Roop, 1988; Bartnik and Weber,
1989; Klymkowsky et a]., 19891). Falkner et al.  isolated two major cytoplasmic proteins of 40 and 46 kda from cultured Drosophila cells. These proteins seem to be related to the intermediate filament class of proteins. On the basis of several biochemical and immunological properties Walter and Biessmann [ 1984a,b] suggested that the 46 Kda protein was a homologue of vertebrate vimentin. The monoclonal antibody raised against this 46 kda protein recognizes a cytoskeletal network of vimentin-type in vertebrate cells [Walter and Biessmann, 1984al. Recently, Cervera et al.  isolated several polypeptides capable of polymerizing into filaments of intermediate type from Drosophila flies. Our antibody strongly stains vimentin cytoskeleton in vertebrate cells but in Drosophila embryos it does not give fluorescence images comparable either to the IF distribution pattern of CHO or to the diffuse cytoplasmic staining described by Walter and Alberts [ 19841. We have shown that this antibody principally stains components of the nuclear boundaries in early Drosophila embryos and gives a punctate stain in the cytoplasm during late telophase and interphase. Since this study is mostly based on immunofluorescence techniques and immunoelectron microscope studies have yet to be performed with the Drosophila material, no conclusion can be reached about the exact localization of the antigenic determinants cross-reacting with the antibody against fibroblast vimentin. However the localization of the staining on the nuclear boundaries suggests cross reactivity with some components of the nuclear envelope. Nuclear envelope polypeptides comparable to vertebrate lamins have been described in Drosophila [Risau et al., 1981; Fisher et al., 1982; Fuchs et al., 1983; McKeon et al., 1983; McKeon et al., 1984; Smith and Fisher, 1984; Filson et al., 1985; Frash et al., 1986, 1988; Smith et al., 1987; Fisher, 19881. However, immunofluorescence microscopy shows that the antibody against fibroblast vimentin strongly stains the nuclear envelope at interphase and throughout the mitotic cycle. This is in contrast to the behavior of antibodies directed against the nuclear lamina of embryonic Drosophila cells, which also give strong staining of the nuclear envelope at interphase, but the stain is lost during mitosis because the antigens, likely lamins A and C in vertebrate cells [Gerace and Blobel, 19801, seem to be dispersed in the cytoplasm [Fuchs et al., 1983; Smith et al., 1987; Frash et al., 1986, 19881. Only lamin B, which remains associated with vesicles presumably derived from the fragmentation of the nuclear envelope, is conserved during the mitotic cycle in vertebrate cells [Gerace and Blobel, 1980; Burke and Gerace, 19861, but this protein has never been identified in Drosophila. The antigen binding pattern observed in Drosoph-
Callaini and Riparbelli
Fig. 4. Interference contrast (a) and immunofluorescence with anti-vimentin (b,c,e,g) and Rb188 (d,f,h) antibodies of early Drosophila embryos. a,h,c,d) prophase; arrows in a and b mark the poles of the same spindle, arrowheads in c and d indicate the fluorescent rings recognized by the anti-vimentin antibody and the centrosomes at the periphery of the same nuclei. Bar = 20 )*m.
Common Antigens in Drosophila and Vertebrate Cells
ila embryos is similar to the staining obtained with an antibody against otefin, a polypeptide localized on the nuclear envelope throughout mitosis in both tissue culture cells and embryos of Drosophila [Miller et al., 1985; Harel et al., 19891. However, immunofluorescence observations reveal slightly different staining patterns. Otefin labeling fades throughout mitosis and there is an increase in the diffuse cytoplasmic background, suggesting partial solubilization of the protein. Our antibody gives nearly constant staining throughout mitosis and during late telophase and interphase a diffuse punctate fluorescence appears in the cytoplasm in addition to the staining of the nuclear envelope. The persistence of the fluorescent staining agrees with electron microscope observations showing that the nuclear envelope remains essentially intact throughout mitosis, but that the nuclear pore complexes are lost and a second layer of closely adherent fenestrated cisternae is formed [Stafstrom and Staehelin, 19841. Like otefin, which appears associated with the spindle regions during prometaphase [Harel et al., 19891, the anti-vimentin antibody stains the spindle poles throughout mitosis. Double immunofluorescence showed that the centrosomal material is localized in correspondence with the polar anti-vimentin labeling and the pattern of pole staining varies according to centrosome shape. When the centrosomes are compact spheres, during interphase, prophase, and metaphase, pole staining as observed with the anti-vimentin antibody, is concentrated in bright rings; but when the centrosomes enlarge in anaphase and telophase, the fluorescence becomes diffuse. These observations, indicating a close relation between centrosomes and nuclear envelope, agree with previous findings suggesting the association of the nuclear envelope with the centrosomes [Bornens, 1977; Fais et al., 19841. The structural basis for this interaction is unclear. Although certain findings deny the association between intermediate filaments and centrosomes, the findings of Katsuma et al.  indicate that the centrioles are frequently observed within a dense network of intermediate filaments which appear to radiate from a juxtanuclear position. It has indeed been demonstrated that the monoclonal antibody against the 46 kda protein from Drosophila Kc cells obtained by Falkner et al. [ 19811 and Walter and Biessmann [ 1984al cross-reacts with an antigen of 68 kda in sea urchin eggs and embryos, apparently related to the centrosomal region stained in immunofluorescence experiments [Schatten et al., 19871. Buendia et al.  showed that a monoclonal antibody raised against centrosomes isolated from human lymphocytes recognizes the pericentriolar material and intermediate filaments in the same cells, suggesting a relationship between intermediate filaments and centrosomal antigens. Moreover, Sullivan et al.
[ 19901 reported that a mammalian anti-neurofilament antibody reacts strongly with Drosophila centrosomes. The monoclonal antibody raised against human fibroblast vimentin may be useful tool for following nuclear dynamics during mitosis in early Drosophila embryos and for studying the relationship between centrosomes and nuclear envelope.
We are indebted to Dr. W. Whitfield of the Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, for his generous gift of the Rb188 antibody and to Dr. R. Vignani of the Department of Environmental Biology, Siena, for providing CHO cells. This work was supported by a 40% grant from the Minister0 della Pubblica Istruzione.
REFERENCES Bartnik, E., and Weber, K. (1989): Widespread occurrence of intermediate filaments in invertebrates: Common principles and aspects of diversion. Eur. J . Cell Biol. 50:17-33. Bornens, M. (1977): Is the centriole bound to the nuclear membrane? Nature 27O:SO-82. Buendia, B., Antony, C., Verde, F., Bornens, M., and Karsenti, E. (1990): A centrosomal antigen localized on intermediate filaments and mitotic spindle poles. J . Cell Sci. 97:259-271. Burke, B., and Gerace, L. (1986): A cell free system to study reassembly of the nuclear envelope at the end of mitosis. Cell M:63Y -6.52.
Cabral, F., Gottesman, M.M., Zimmerman, S.B., and Steinert, P.M. (198 1): Intermediate filaments from Chinese hamster ovary cells contain a single protein. J. Biol. Chem. 256:1428-1431. Cervera, M., Domingo, A , , Vinos, J . , and Marco, R. (1987): Drosophila melanogaster contains a set of polypeptides capable of polymerizing into intermediate-like filaments. Biochem. Biophys. Res. Com. 144:1043-1048. Fais, D., Nadezhdina, E., and Chentsov, Y. (1984): Evidence for the nucleus-centriole association in living cells obtained by ultracentrifugation. Eur. J. Cell Biol. 33: 190-196. Falkner, F.G., Saumweber, H., and Biessmann, H. (1981): Two Drosophila melanogaster proteins related to intermediate filament proteins of vertebrate cells. J . Cell Biol. 91:17.5-183. Filson, A.J., Lewis, A., Blobel, G., and Fisher, P.A. (1985): Monoclonal antibodies prepared against the major Drosophila nuclear matrix-pore complex-lamina glycoprotein bind specifically to the nuclear envelope in situ. J. Biol. Chem. 260:31643172.
Fisher, P.A. (1988): Karyoskeletal proteins of Drosophila. In Adoph, K.W. (ed): “Chromosome and Chromatin Structure, Vol. 3.” Boca Raton, FL: CRC Press, pp. 119-150. Fisher, P.A., Bemos, M., and Blobel, G . (1982): Isolation and characterization of a proteinaceous subnuclear fraction composed of nuclear matrix, peripheral lamina, and nuclear pore complexes from embryos of Drosophila rnelanogaster. J . Cell Biol. 92: 674-686.
Foe, V.E., and Alberts, B.M. (1983): Studies of nuclear and cytoplasmic behaviour during the five mitotic cycles that precede
Callaini and Riparbelli
gastrulation in Drosophila embryogenesis. J. Cell Sci. 61:3170. Franke, W. W. (1987): Nuclear lamins and cytoplasmic intermediate filament proteins: A growing multigene family. Cell 48:3-4. Frash, M., Glover, D.M., and Saumweber, H. (1986): Nuclear antigens follow different pathways into daughter nuclei during mitosis in early Drosophilu embryos. J . Cell Sci. 82:155-172. Frash, M., Paddy, M., and Saumweber, H. (1988): Developmental and mitotic behaviour of two novel groups of nuclear envelope antigens of Drosophila melanoguster. J. Cell Sci. 90:247-263. Fuchs, J.P., Giloh, H., Kuo, C.H., Saumweber, H., and Sedat, J. (1983): Nuclear structure: Determination of the fate of the nuclear envelope in Drosophila during mitosis using monoclonal antibodies. J. Cell Sci. 64:331-349. Gerace, L., and Blobel, G. (1980): The nuclear envelope lamina is reversibly depolymerized during mitosis. Cell 19:277-287. Giloh, I.I., and Sedat, J.W. (1982): Fluorescence microscopy: Reduced photobleaching of rhodamine and fluorescein protein conjugates by n-propyl gallate. Science 217: 1252-1255. Goldman, R.D., Goldman, A.E., Green, K.G., Jones, J.C.R., Jones, S.M., and Yang, H.-Y. (1986): Intermediate filament networks: Organization and possible functions of a diverse group of cytoskeletal elements. J. Cell Sci. Suppl. 5:69-97. Harel, A., Zlotkin, E., Nainudel-Epszteyn, S . , Feinstein, N., Fisher, P.A., and Gruenbaum, Y . (1989): Persistence of major nuclear envelope antigens in an envelope-like structure during mitosis in Drosophilu melanogaster embryos. J. Cell Sci. 94:463470. Karr, T.L., and Alberts, B.M. (1986): Organization of the cytoskeleton in early Drosophilu embryos. J. Cell Biol. 102:14941509. Katsuma, Y . , Swierenga, S . H . H . , Marceau, N . , and French, S.W. (1987): Connections of intermediate filaments with the nuclear lamina and the cell periphery. Biol. Cell. 59:193-204. Kellogg, D.R., Mitchison, T.J., and Alberts, B.M. (1988): Behaviour of microtubules and actin filaments in living Drosophila embryos. Development 103:675-686. Klymkowsky, M.W., Bachant, J.B., and Domingo, A. (1989): Functions of intermediate filaments. Cell Motil. Cytoskel. 14:309331. McKeon, F.D., Tuffanelli, D.L., Fukuyama, K., and Kirschner, M. W. (1983): Autoimmune response against conserved determinants of nuclear envelope proteins in a patient with linear scleroderma. Proc. Natl. Acad. Sci. USA 80:4347-4378. McKeon, F.D., Tuffanelli, D.L., Kobayashi, S . , and Kirschner, M.W. (1984): The redistribution of a conserved nuclear envelope protein during the cell cycle suggests a pathway for chromosome condensation. Cell 36:83-92. Miller, K.G., Karr, T.L., Kellogg, D.R., Mohr, I.J., Walter, M., and Alberts, B.M. (1985): Studies on the cytoplasmic organization of early Drosophila embryos. Cold Spring Harbor Symp. quant. Biol. 50:79-90.
Risau, W . , Saumweber, H., and Symmons, P. (1981): Monoclonal antibodies against a nuclear membrane protein of Drosophila. Exp. Cell Res. 133:47-54. Schatten, H., Walter, M., Mazia, D., Biessmann, H . , Paweletz, N., Coffe, G., and Schatten, G. (1987): Centrosome detection in sea urchin eggs with a monoclonal antibody against Drosophila intermediate filament proteins: Characterization of stages of the division cycle of centrosomes. Proc. Natl. Acad. Sci. USA 84:8488-8492. Smith, D.E., and Fisher, P.A. (1984): Identification, developmental regulation, and response to heat shock of two antigenically related forms of a major nuclear envelope protein in Drosophila embryos: Application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots. J. Cell Biol. 99:20-28. Smith, D.E., Gruenbaum, Y., Benios, M . , and Fisher, P.A. (1987): Biosynthesis and interconversion of Drosophila nuclear lamin isoforms during normal growth and in response to heat shock. J. Cell Biol. 105:771-790. Stafstrom, J.P., and Staehelin, L.A. (1984): Dynamics of the nuclear envelope and of nuclear pore complexes during mitosis in the DrosophiEa embryo. Eur. J. Cell Biol. 34:179-189. Steinert, P.M. and Roop, D.R. (1988): Molecular and cellular biology of intermediate filaments. Ann. Rev. Biochem. 57:593-625. Sullivan, W., Minden, J.S., and Alberts, B.M. (1990): daughterlessabo-like, a Drosophila maternal-effect mutation that exhibits abnormal centrosome separation during the late blastoderm divisions. Development 110:311-323. Walter, M., and Alberts, B.M. (1984): Intermediate filaments in tissue culture cells and early embryos of Drosophila melanogasfer. In Davidson, E.H., and Firtel, R.A., (eds.): “Molecular Biology of Development. UCLA Symposium on Molecular and Cellular Biology”. New York: Alan R. Liss, Inc., pp. 263272. Walter, M.F., and Biessmann, H. (1984a): Cross-reaction of a monoclonal antibody with intermediate filament-like proteins from vertebrate and nonvertebrate organisms. J. Mol. Cell Biochem. 60199-108. Walter, M.F., and Biessmann, H. (1984b): Intermediate-sized filaments in Drosophila tissue culture cells. J. Cell Biol. 99:14681477. Warn, R.M. (1986): The cytoskeleton of the early Drosophila embryo. J. Cell Sci. Suppl. 5:311-328. Warn, R.M., and Warn, A. (1986): Microtubule arrays present during the syncytial and cellular blastoderm stages of the early Drosophila embryo. Exp. Cell Res. 163:201-210. Whitfield, W.G.F., Millar, S.E., Saumweber, H., Frash, M., and Glover, D. (1988): Cloning of a gene encoding an antigen associated with the centrosome on Drosophila. J. Cell Sci. 89x467-480. Zalokar, M., and Erk, I. (1976): Division and migration of nuclei during early embryogenesis of Drosophilu melanogasrer. J. Microsc. Biol. Cell. 25:97-106.