- Email: [email protected]
Enolase is present at the centrosome of HeLa cells
EXPERIMENTAL
CELL
RESEARCH
202,4X3-463
(19%)
Enolase Is Present at the Centrosome of HeLa Cells SHARONA. JOHNSTONE,DAVIDMORTONWAISMAN,ANDJ. Department
of Medical Biochemistry,
*Department
of Anatomy,
Antibodies raised against the C-terminus and N-terminus region of yy enolase, as well as a polyclonal antibody raised against bovine brain yy enolase, were used to study the distribution of this glycolytic enzyme during the cell cycle in HeLa cells. Enolase was found to be present throughout the cytoplasm of both interphase and dividing cells. In addition, a portion of cellular enolase was detected at the centrosome throughout the cell cycle. The capacity of glycolytic enzymes to play a structural as well as a glycolytic role suggests that the presence of enolase at the centrosome may be correlated with the organization of both the interphase cytoskeleton and the mitotic spindle. o 1992 Academic PWSS, IIIC.
INTRODUCTION The centrosome of animal cells functions as the site for the organization of both the interphase cytoskeleton and the spindle of dividing cells. Ultrastructurally, the centrosome in most vertebrate cells has two distinct domains, one containing the centrioles and the other the pericentriolar material. It is this material that appears to serve as the major site for the organization of cytoskeletal elements and is composed of a complex set of proteins [l]. A survey of known centrosomal proteins indicates that they fall into two groups: those associated with the centrosome throughout the cell cycle (e.g., tubulin) and those that associate with the centrosome only during the period of cell division (e.g., ~34”~“~). One role of the centrosome may be to act as a focal point for the concentration of many ubiquitous cellular proteins, since many centrosomal proteins are also found throughout the cytoplasm [l]. The sera of patients with systemic rheumatic disease contain autoantibodies to many cellular components including the centrosome [2-41. These autoantibodies react with the centrosome throughout the cell cycle and with the basal bodies of some cell types 151. Recently, it has been demonstrated that autoantibodies responsible for centrosome reactivity in some patients react with
1 To whom dressed.
correspondence
0014.4827/92 $5.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
and reprint
requests
should
University
of Calgary, Calgary, Alberta,
Canada T2N 4Nl
determinants present on enolase and specifically the yy isotype [6]. Enolase (2-phospho-D-glycerate hydrolase) is a glycolytic enzyme that is present as three homodimerit isozymes in eukaryotes [7-lo]. A muscle-specific form, /3& has been demonstrated, as has a nonneuronal isozyme, OXX.This form is also present in a variety of tissues including glial cells of adult neuronal tissue. The third isozyme of enolase, yy, is also referred to as the neuronal-specific form of the enzyme [9], although several reports have indicated that the yy isotype is present in nonneuronal tissue [6, 11-151. This isotype has also been shown to serve as a neuronal survival factor [16]. Although the glycolytic pathway is well defined at the biochemical level, the dynamic and spatial organization of glycolysis in living cells has not been well characterized. While it has been generally assumed that these enzymes are free “floating,” several recent studies have suggested that they may also be localized to specific domains within the cell. Clarke et al. [17] have presented the concept that glycolytic enzymes are capable of dual functions, playing both a structural and catalytic role. Structurally, some glycolytic enzymes have been shown to function in vitro in the bundling of actin and tubulin. When these proteins bind their substrate, their ability to participate in the bundling of actin or tubulin is attenuated. In this study we have investigated the relationship between the distribution of enolase and the centrosome. Using a series of specific antibodies, we show that a subset of enolase localizes to the centrosome throughout the cell cycle. Since both a structural and catalytic role have been ascribed to the glycolytic enzymes [17], our study suggests that the presence of enolase at the centrosome may be related to the role of the centrosome as a microtubule organizing center.
EXPERIMENTAL PROCEDURES Antibody production. Antibody production was based on the procedures described in Harlow and Lane [B]. Peptides corresponding to the N-terminal (SZ-SVVEQEKLDNLMC-93) and C-terminal (321-VTNPKRIERAVEEC-333) regions of yy enolase were chemically synthesized, purified by reverse-phase high-performance liquid chromatography, and coupled to keyhole limpet hemocyanin via an additional cysteine residue attached to the C-terminus of each pep-
be ad-
458 Inc. reserved.
B. RATTNER*'~
ENOLASE AT THE CENTROSOME OF HeLa CELLS
123456789 FIG. 1. Immunoblot of anti-enolase antibodies reacted against 2 pg of purified yy enolase (lanes 1, 4, 7) or total HeLa mitotic cell protein (lanes 2,3, 5, 6,8, 9). Antibodies used in these reactions were Northern (lanes i, Z), CEl (lanes 4, 5), GGl (lanes 7, S), and preimmune sera form NE1 rabbits (lane 3), CEl rabbits (lane 6), and GGl rabbits (lane 9). Antibody dilutions were l:lOO, 1:250, and l:lOOO, respectively. Left arrows, origin (top) and dye front (bottom); right arrow denotes the position of enolase.
tide [19]. New Zealand white rabbits were injected with 250 pg of antigen in Freund’s incomplete adjuvant. Rabbits were bled 10 days subsequent to boosting. Antibodies to yy enolase were generated using yy enolase purified from frozen bovine brain as described by Tokuda et al. (201. Antisera were purified by affinity chromatography on Sepharoseimmobilized antigen. One milligram of peptide or protein was coupled to 1 ml of A&Gel 10 (Bio-Rad) in 10 mM Mops, pH 7.4, according to the manufacturer’s instructions. Chromatography was carried out according to the procedure of Perlman et al. [21], using the pellet obtained after ammonium sulfate fractionation (50% saturation) of whole sera. Tissue culture and indirect immunofluorescence. Monolayer cultures of HeLa cells (American Type Tissue Collection, Bethesda, Maryland) were grown in Joklik’s suspension medium supplemented with 10% fetal calf serum. Forty-eight hours prior to use, cells were seeded onto coverslips. To prepare cells for indirect immunofluorescence (IIF), cells were washed in PBS and fixed in 100% methanol at -20°C for 10 min and air dried. We also prepared cells using different fixation protocols including paraformaldehyde and methanol-acetone. These procedures had no effect on the ability to detect enolase antibody reactivity at the eentrosome. Some fixation procedures (paraformaldehyde) resulted in a reduced cytoplasmic signal, facilitating the visualization of centrosome reactivity. Methanol-fixed cells were rehydrated in PBS and incubated for 1 h in primary antibody at 37°C. After three washes in PBS, the coverslips were incubated with fluorescein-conjugated anti-rabbit IgG + IgM + Ig (H + L) (Dimension Labs) at a dilution of 1:ZO. After 1 h at 37’C the coverslips were washed three times in PBS and mounted in 90% glycerol containing paraphenylenediamine and observed using a Nikon Optophot fluorescent microscope. Images were recorded on Ilford HP-5 film. Double-label experiments were preformed using HeLa cells grown on coverlips or centrosomal preparations prepared as described below and deposited on coverslips according to the procedure of Mitchison and Kirschner [22]. After fixation (3.5% paraformaldehyde), the preparations were incubated in a mixture of a human autoimmune serum reactive with the centrosome [6] and the appropriate anti-enolase antibody (see text). Tie samples were processed as described above using the following secondary antibodies: TRIT-conjugated anti-hu-
459
man IgG + IgM + IgA (H + L) and a FITC anti-rabbit IgG + IgM + IgA (H + L) (Dimension Labs). Western blotting. Immunoblotting was performed on nitrocellulose strips containing proteins separated by 12% SDS-polyacrylamide gel electrophoresis according to the procedures of Tobwin et al. [23] and Laemmli 1241. The proteins used for these blots included mitotie cell protein from cells obtained by selective mitotic detachment from monolayer cultures of HeLa cells, mterpbase centrosomal proteins prepared by the procedure of Mitcbison and Kirschner [22], and spindle proteins isolated according to the procedure of Kuriyama et al. [25]. Antibody binding to nitrocellulose strips was detected using peroxidase-conjugated anti-rabbit IgG + I& t IgM (II + L) at the manufacturer’s recommended dilution (1:fOOO) for 2 h at room temperature. Following a repeat wash sequence, bound antibodies were detected by incubation with a solution containing I-chloro-lnaphthol and H,O,.
Antibodies were raised in ra bits to a synthetic peptide corresponding to the N-ter inal (antibody designation NEl) or C-terminal regions ( CEI) of the yy isotype of enolase. body was raised in rabbits to y’y e i.solated from bovine brain (antibody designation Each of these antibodies showed strong reactivit brain yy enolase (Fig. 1). When each of these antibodies was reacted with total HeLa mitotic cell nent reactivity was detected at the 4%kDa region of the blot which was comparable to the Gtion of enolase (Fig. 1). However, each of the anti ies, witth the exception of GGf, showed weak reactivity with other mitotic cell proteins, producing a series of nately within the 14- to GO-kDa region o parative analysis, however, suggested
47.7 -
I
3
4
FIG. 2. Immunoblot of anti-enolase antibodies reacted with protein obtained from isolated HeLa interphase cell centrosome preparations. Lane 1 Commassie blue-stained gei, iane 2 reacted with NE1, lane 3 reacted with @El, and lane 4 reacted with GG1. Left arrows, origin (top) and dye front (bottom); right arrow denotes position of enolase.
460
JOHNSTONE,
WAISMAN,
AND
RATTNER
ENOLASE
I
AT THE
CENTROSOME
2
FIG. 4. Immunoblot of the CEl antibody reacted with isolated HeLa spindle protein. Lane 1, Commassie blue-stained gel; lane 2, spindle protein reacted with CEl. Left arrows, origin (top) and dye front (bottom); right arrow denotes position of enolase.
region of reactivity common to all the antibodies was the enolase band (Fig. 1). A 40-kDa band was present in lanes containing mitotic cell protein as well as purified bovine brain enolase. The persistence of this band suggests that it likely represents a breakdown product of enolase. The variation in the intensity of this band is likely due to differences in the epitopes recognized by the different peptide antibodies. To verify that enolase was the only centrosome component identified by each of the antibodies, we isolated centrosomes from interphase HeLa cells and used the proteins from these preparations in immunoblots with each of the enolase antibodies. As shown in Fig. 2, each antibody recognized a single 48-kDa protein in these centrosomal preparations. The failure to detect the putative 40-kDa breakdown product in this blot may be due to the relatively low yield of enolase obtained in these preparations. Each of the antibodies was used in IIF studies in order to obtain cytological information on the distribution of enolase during the cell cycle of HeLa cells. IIF studies using the peptide antibody CEI revealed a diffuse staining pattern throughout the interphase cell. In addition, prominent reactivity was detected at a focus near the nuclear periphery (Fig. 3A). This region was found to correspond to the centrosome in double label experiments using anticentrosomal antibodies (see below). At metaphase, this antibody showed reactivity with the entire spindle including the centrosomal region (Fig. 3B).
OF HeLa
CELLS
461
At the completion of telop ase reactivitgr was seen at the centrosome, along some centrosomal a crotubules, and at the midbody (Fig. 3C). plasmic staining was observed throughout the cytoplasm during cell division in all cells e To determine if the spindle and detected with the CEl antibody wa h a spindle prote epared Western HeLa spindle protein. Figure 4 illu tion to enolase, the major reae directed toward a protein et with molecular weights of 62 and 70 kDa, whi nent of isolated spindles. Since teins were not detected in the Western blots il do not appear to be compo somes. Therefore, cross-reaeti y with the 6%.kDa TOkDa doublet may provide an e nation for the spindle reactivity seen with the CE1 When interphase HeLa cells were i~~~ba~ed with t peptide antibody n tbrougbQut the cytoplasm. In antibody, the signal was localized to the centrosom’es (Fig. 3D). Cytoplasmic and centrosomal reactivity was also detected in metaphase and telophase cells (Figs. 3E and 3F). The intensity of the cytoplasmie signal ~rod~~@d by this antibody often interfered with the detection of centrosoma1 reactivity. Double staining, ‘ng a human antiee trosomal antibody [6] and the N antibody, confirm that the localized region of reac ity produced by t anti-enolase antibody correspo d to the site of the centrosome (Figs 36 and 3H). IIF studies using the GGl antibody raised against bovine brain yy enolase reve attern of staining identical to that observed wi -terminal antibody with a localized site of reactivity only at the centrosome 0th the anti-~e~t~~so~al antibody and antibodies showed reaetiv the anti-enola with isolated interphase centrosomes (Figs 3L and 9. In general, the region of reactivity educed by the autoimmune serum was broader th produced by the anti-enolase antibodies. It is that the autoimmune serum recognizes other d the centromere in addition to tb Preimmune rabbit sera did not cell reactivity (Fig. 3N).
FIG. 3. Mitotic HeLa cells reacted with anti-enolase antibodies. A, interphase; B, metaphase; C, telophase reacted with the CEl antibody. Large arrows denote centrosomal reactivity and small arrows microtubule reactivity (M = midbody). D, interphase; E, metaphase; F, telophase reacted with the NE1 antibody. Arrows denote centrosomal reactivity. G and H, a telophase cell double stained reacted with an anti-centrosoma1 autoimmune serum (G) and the NE1 antibody (H). Arrows indicate that the region stained by the NE1 antibody overlap with the centrosomal region identified by the autoimmune serum. I, interphase; J, metaphase; K, telophase reacted with the GGl antibody. Arrows denote centrosomal reactivity. L and M, double staining of a preparation of isolated interphase HeLa centrosomes reacted with a human anticentrosomal serum (L) and the GGl antibody (M). N, a control panel illustrating an interphase cell reacted with preimmune rabbit sera. Bar, 10 pm.
462
JOHNSTONE,
WAISMAN,
DISCUSSION
Using antibodies to two different regions of bovine yy enolase, as well as an antibody produced against yy enolase isolated from bovine brain, we have shown that in addition to its distribution within the cytosol of cycling cells, a subset of this enzyme is found at the centrosome throughout the cell cycle. Several lines of evidence suggest that the centrosome pattern we have detected reflects the differential distribution of enolase: (i) Three different antibodies, two of which are known to recognize two diverse regions of enolase produce centrosome staining in HeLA cells. (ii) Each of these antibodies recognizes enolase in immunoblots of whole mitotic cell protein. (iii) Each of these antibodies recognizes a single 4% kDa protein in immunoblots of isolated centrosome proteins. (iv) The antibodies show reactivity with isolated centrosome. In addition to the antibodies used in this study, we have also investigated the ability of commercially available antibodies to yy enolase to react with the centrosome of HeLa cells. Of the three antisera tested (DakoPatt, Carpinteria, CA), two produced a pattern of centrosomal reactivity identical to that found with the NE1 and GGl antibodies. The third produced pronounced cytoplasmic reactivity but did not show reactivity specific to the centrosome. It is possible that some anti-enolase antibodies recognize determinants that are masked at the centrosome. One unique feature of the CEl antibody is that it also shows reactivity within the mitotic spindle. We have shown that this is likely due to cross-reactivity with two prominent protein components found in isolated spindle preparations. A GenBank search failed to reveal homology between the peptide used to generate the CEl antibody and the protein sequence of any other known protein. Thus, the cross-reactivity seen with the CEl antibody provides a means for the identification of additional protein(s) that colocalize with the microtubules of the mitotic spindle. It is interesting to note that at late telophase, the CEl antibody not only reacts with the centrosome and intercellular bridge (midbody) but also with the microtubule array associated with the centrosome. During late telophase in these cells, the centrosome migrates from its original poleward position to a position at the midbody side of the reforming nucleus. The microtubule array stained by the CEl antibody extends along this path, suggesting a possible role for the antigen(s) recognized by this antibody in centrosome migration. While our antibodies were raised to either the purified bovine brain yy enolase or the C-terminal or N-terminal sequence of the molecule, we have observed slight cross-reactivity between these antibodies
AND
RATTNER
and CY~!enolase (data not shown). Although our data provide strong support for the presence of yy enolase at the centrosome (for example, the presence of a single band of reactivity at 48 kDa in the blots of isolated centrosomal protein Fig. 2), we cannot rule out the possibility that LYLY enolase could also colocalize to the centrosome. Several recent studies have suggested that glycolytic enzymes may be localized to specific sites within the cell. For example, some glycolytic enzymes have been shown to bind to structural proteins, particularly actin [26] and tubulin [27, 281. The association of glycolytic enzymes with membranous structures has also been observed. For example, electron-immunocytochemical studies have illustrated the localization of yy enolase to the membrane of primary and metastatic cerebral tumors, and also to the glial filaments of glioma cells [14, 291. Knull 1301 and Lin et al. [32] have documented the association of several glycolytic enzymes with synaptic membranes and Gosti et al. [32] used an anti-centrosoma1 serum to identify an epitope of lactate dehydrogenase at the centrosome. The detection of enolase at the centrosome lends further support to the concept that glycolytic enzymes can have a site-specific distribution within the cell. The compartmentalization of glycolytic enzymes implies that there is a requirement for these enzymes in site-specific cellular events. There are several lines of evidence that suggest that the glycolytic enzymes may be required for these events not only for their glycolytic capacity, but also for their ability to perform a structural function [17]. For example, some glycolytic enzymes such as lactate dehydrogenase type M, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, and aldolase have been found to copellet with microtubules [28]. In the case of glyceraldehyde-3-phosphate dehydrogenase, the binding of the enzyme to microtubules shows considerable specificity and is associated with changes in the assembly and disassembly parameters of these cytoskeletal elements [33]. Further, several glycolytic enzymes have been shown to function in vitro in the bundling of actin [17] and microtubules [27,34]. In addition, Masters [35] has postulated that glycolytic enzymes may “plate” the filamentous actin core of the microfilament. Thus, the glycolytic enzymes may play a structural role affecting the organization of cytoskeletal elements. The presence of enolase at the centrosome may be correlated with this role either through a direct interaction with cytoskeletal components or indirectly as a member of an enzyme complex. The apparent inability of enolase to bind to tubulin, for example, would support the latter possibility [28]. In conclusion, we have demonstrated the presence of a portion of cellular enolase at the centrosome throughout the cell cycle of HeLa cells. This subset of enolase may be correlated with a structural and/or glycolytic
ENOLASE
role carried out by glycolytic bule organizing center.
AT THE
CENTROSQME
enzymes at this microtu-
This work is supported by a grant from the Medical Research Council of Canada (D.M.W.) and the National Cancer Institute of Canada (J.B.R.). The authors thank Craig Litwin for help in preparing the centrosomal preparations and Jerry Wang for his helpful comments during the course of this investigation and manuscript preparation. REFERENCES 1. 2. 3.
4. 5. 6. 7. 8. 9. 40. 11. 12. 13. 14.
Rattner, J. B. (1992) in The Centrosome (Kalnins, V., Ed.), Academic Press, San Diego. Brenner, S., Pepper, D., Turner, D., Boyd, A. E., and Brinkley, B. R. (1980) J. Cell Biol. 87, 240a. Moroi, Y., Murata, I., Takeuchi, R., Kamatani, N., Tanimoto, K., and Yokohari, R. (1983) Ciin. Zmmunol. Immunopathol. 29, 381-390. Tuffanelli, D. L., McKeon, F., Kleinsmith, D. M., Burnham, T. K., and Kirschner, M. (1983) Arch. Dermatol. 119, 560-566. Turksen, K., Aubin, J. E., and Kalmus, V. I. (1982) Nature (London) 298, 763-164. Rattner, J. B., Martin, L., Waisman, D. M., Johnstone, S. A., and Fritzler, M. J. (1991) J. Immunol. 146, 2341-2344. Rider, C. C., and Taylor, 6. B. (1974) Biochim. Biophys. Acta 365, 285-300. Fletcher, L., Rider, C. C., and Taylor, C. B. (1976) Biochim. Biophys. Acta 452, 245-252. Marangos, P. J., Zomzely-Neurath, C., and York, C. (1976) Bio&em. Biophys. Res. Commun. 68, 1309-1316. Marangos, P. J., and Schmechel, D. G. (1987) Annu. Reu. Neurosci. 10, 269-295. Cras, P., Martin, J. J., and Gheuens, H. J. (1988) Acta Neuropathol. 75,377-384. Haimoto, II., Takahashi, Y., Koshikawa, T., Nagura, H., and Kato, K. (1985) Lab. dnuest. 82, 257-263. Piihlman, S., Esscher, T., and Mitsson, K. (1986) Lab. Inuest. 54,554-560. Vinores, S. A., Bonnin, J. M., Rubinstein, L. J., and Marangos, P. J. (1984) Acta Pathol. Lab. Med. 108,536-540.
Received March 18, 1992 Revised version received June 15, 1992
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Eigenbrodt, E., Fister, P., Rubsamen, EMBO J. 2, 1565-1570.
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Takei, N., Kondo, J., Nagaike, K. Kohsaka, S. (1991) J. Neurochem. Clarke, F. M., Morton, D. J., Step (1985) in Cell Motility: Mechanism and Regulation (10th Yamada Conference) (Ishikawa, H., Hatano, S., and Sato, II., Eds.), pp. 235-250, Tokyo University Press, Tekyo.
17.
., and Feiis, R. R. (1983)
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Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY.
19.
Green, N., Alexander, H., Olson, A., Alexander, S., Shinnick, T. M., Sutcliffe, J. C., and Lerner, R. A. (1982) Cell 28,477-487. Tokuda, M., Khanna, N. C., and Waisman, D. M. (1987) &f&hods Enzymol. l39,68-79.
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Perlman, R., Bottaro, D. P., White, (1989) J. Biol. Chem. 15,8946-8950.
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Mitch&on, 232-245.
(London)
312,
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Towbin, II., Staehelin, T., and Gordon, J. (1979) Pro, Acad. Sci. USA 76,4350-4354. Laemmli, U. K. (1970) Nature (London) 227, 680-685.
Natl.
24. 25. 26. 27.
T., and Kirschner,
. F.. and Kahn,
M. (1984) Nature
C. R.
Kuroiyama, R., Keryer, G., and Borisy, 6. G. (1984) j. Cell Sci. 66, 265-275. Bronstein, W. W., and Knull, H. R. (‘,981) Can. j. Rio&em. 5 494-499. Huitorel, P., and Pantaioni, D. (1985) UI. BioE, Chem. 150, 265269.
28.
Walsh, J. L., Keith, T. J., and Knull, Biophys. Acta 999? 64-70.
H. R. (1989) Biochim.
29.
Vinores,
ubinstein,
30.
Knull,
31.
Lin, L., Hall, C., Leung, T., Mahadevan, L., and Whatley, S. (1983) J. New-o&em. 41, 1177-1182. Gosti, F., Marty, M-C., Courvalin; J. C., Maunoury, R., and Bornens, M. (1987) Proc. NatZ. Acad. Sci. USA
32. 33. 34. 35.
S. A., Herman,
M. M., and
L. J. (1986)
H. R. (1980) J. BioZ. Chem. 28%, 6439-6444.
Durrieu, C., Bernier-Valentin, F., and Rousset, B. (1987) Mol. CeZZ.Biochem. 74,55-65. Kumagai, II., and Sakai, I-I. (1983) J. Biocizem. 93, 1259-1269. ,222s-225s. Masters, C. J. (1984) J. Cell BioZ.
CELL
RESEARCH
202,4X3-463
(19%)
Enolase Is Present at the Centrosome of HeLa Cells SHARONA. JOHNSTONE,DAVIDMORTONWAISMAN,ANDJ. Department
of Medical Biochemistry,
*Department
of Anatomy,
Antibodies raised against the C-terminus and N-terminus region of yy enolase, as well as a polyclonal antibody raised against bovine brain yy enolase, were used to study the distribution of this glycolytic enzyme during the cell cycle in HeLa cells. Enolase was found to be present throughout the cytoplasm of both interphase and dividing cells. In addition, a portion of cellular enolase was detected at the centrosome throughout the cell cycle. The capacity of glycolytic enzymes to play a structural as well as a glycolytic role suggests that the presence of enolase at the centrosome may be correlated with the organization of both the interphase cytoskeleton and the mitotic spindle. o 1992 Academic PWSS, IIIC.
INTRODUCTION The centrosome of animal cells functions as the site for the organization of both the interphase cytoskeleton and the spindle of dividing cells. Ultrastructurally, the centrosome in most vertebrate cells has two distinct domains, one containing the centrioles and the other the pericentriolar material. It is this material that appears to serve as the major site for the organization of cytoskeletal elements and is composed of a complex set of proteins [l]. A survey of known centrosomal proteins indicates that they fall into two groups: those associated with the centrosome throughout the cell cycle (e.g., tubulin) and those that associate with the centrosome only during the period of cell division (e.g., ~34”~“~). One role of the centrosome may be to act as a focal point for the concentration of many ubiquitous cellular proteins, since many centrosomal proteins are also found throughout the cytoplasm [l]. The sera of patients with systemic rheumatic disease contain autoantibodies to many cellular components including the centrosome [2-41. These autoantibodies react with the centrosome throughout the cell cycle and with the basal bodies of some cell types 151. Recently, it has been demonstrated that autoantibodies responsible for centrosome reactivity in some patients react with
1 To whom dressed.
correspondence
0014.4827/92 $5.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
and reprint
requests
should
University
of Calgary, Calgary, Alberta,
Canada T2N 4Nl
determinants present on enolase and specifically the yy isotype [6]. Enolase (2-phospho-D-glycerate hydrolase) is a glycolytic enzyme that is present as three homodimerit isozymes in eukaryotes [7-lo]. A muscle-specific form, /3& has been demonstrated, as has a nonneuronal isozyme, OXX.This form is also present in a variety of tissues including glial cells of adult neuronal tissue. The third isozyme of enolase, yy, is also referred to as the neuronal-specific form of the enzyme [9], although several reports have indicated that the yy isotype is present in nonneuronal tissue [6, 11-151. This isotype has also been shown to serve as a neuronal survival factor [16]. Although the glycolytic pathway is well defined at the biochemical level, the dynamic and spatial organization of glycolysis in living cells has not been well characterized. While it has been generally assumed that these enzymes are free “floating,” several recent studies have suggested that they may also be localized to specific domains within the cell. Clarke et al. [17] have presented the concept that glycolytic enzymes are capable of dual functions, playing both a structural and catalytic role. Structurally, some glycolytic enzymes have been shown to function in vitro in the bundling of actin and tubulin. When these proteins bind their substrate, their ability to participate in the bundling of actin or tubulin is attenuated. In this study we have investigated the relationship between the distribution of enolase and the centrosome. Using a series of specific antibodies, we show that a subset of enolase localizes to the centrosome throughout the cell cycle. Since both a structural and catalytic role have been ascribed to the glycolytic enzymes [17], our study suggests that the presence of enolase at the centrosome may be related to the role of the centrosome as a microtubule organizing center.
EXPERIMENTAL PROCEDURES Antibody production. Antibody production was based on the procedures described in Harlow and Lane [B]. Peptides corresponding to the N-terminal (SZ-SVVEQEKLDNLMC-93) and C-terminal (321-VTNPKRIERAVEEC-333) regions of yy enolase were chemically synthesized, purified by reverse-phase high-performance liquid chromatography, and coupled to keyhole limpet hemocyanin via an additional cysteine residue attached to the C-terminus of each pep-
be ad-
458 Inc. reserved.
B. RATTNER*'~
ENOLASE AT THE CENTROSOME OF HeLa CELLS
123456789 FIG. 1. Immunoblot of anti-enolase antibodies reacted against 2 pg of purified yy enolase (lanes 1, 4, 7) or total HeLa mitotic cell protein (lanes 2,3, 5, 6,8, 9). Antibodies used in these reactions were Northern (lanes i, Z), CEl (lanes 4, 5), GGl (lanes 7, S), and preimmune sera form NE1 rabbits (lane 3), CEl rabbits (lane 6), and GGl rabbits (lane 9). Antibody dilutions were l:lOO, 1:250, and l:lOOO, respectively. Left arrows, origin (top) and dye front (bottom); right arrow denotes the position of enolase.
tide [19]. New Zealand white rabbits were injected with 250 pg of antigen in Freund’s incomplete adjuvant. Rabbits were bled 10 days subsequent to boosting. Antibodies to yy enolase were generated using yy enolase purified from frozen bovine brain as described by Tokuda et al. (201. Antisera were purified by affinity chromatography on Sepharoseimmobilized antigen. One milligram of peptide or protein was coupled to 1 ml of A&Gel 10 (Bio-Rad) in 10 mM Mops, pH 7.4, according to the manufacturer’s instructions. Chromatography was carried out according to the procedure of Perlman et al. [21], using the pellet obtained after ammonium sulfate fractionation (50% saturation) of whole sera. Tissue culture and indirect immunofluorescence. Monolayer cultures of HeLa cells (American Type Tissue Collection, Bethesda, Maryland) were grown in Joklik’s suspension medium supplemented with 10% fetal calf serum. Forty-eight hours prior to use, cells were seeded onto coverslips. To prepare cells for indirect immunofluorescence (IIF), cells were washed in PBS and fixed in 100% methanol at -20°C for 10 min and air dried. We also prepared cells using different fixation protocols including paraformaldehyde and methanol-acetone. These procedures had no effect on the ability to detect enolase antibody reactivity at the eentrosome. Some fixation procedures (paraformaldehyde) resulted in a reduced cytoplasmic signal, facilitating the visualization of centrosome reactivity. Methanol-fixed cells were rehydrated in PBS and incubated for 1 h in primary antibody at 37°C. After three washes in PBS, the coverslips were incubated with fluorescein-conjugated anti-rabbit IgG + IgM + Ig (H + L) (Dimension Labs) at a dilution of 1:ZO. After 1 h at 37’C the coverslips were washed three times in PBS and mounted in 90% glycerol containing paraphenylenediamine and observed using a Nikon Optophot fluorescent microscope. Images were recorded on Ilford HP-5 film. Double-label experiments were preformed using HeLa cells grown on coverlips or centrosomal preparations prepared as described below and deposited on coverslips according to the procedure of Mitchison and Kirschner [22]. After fixation (3.5% paraformaldehyde), the preparations were incubated in a mixture of a human autoimmune serum reactive with the centrosome [6] and the appropriate anti-enolase antibody (see text). Tie samples were processed as described above using the following secondary antibodies: TRIT-conjugated anti-hu-
459
man IgG + IgM + IgA (H + L) and a FITC anti-rabbit IgG + IgM + IgA (H + L) (Dimension Labs). Western blotting. Immunoblotting was performed on nitrocellulose strips containing proteins separated by 12% SDS-polyacrylamide gel electrophoresis according to the procedures of Tobwin et al. [23] and Laemmli 1241. The proteins used for these blots included mitotie cell protein from cells obtained by selective mitotic detachment from monolayer cultures of HeLa cells, mterpbase centrosomal proteins prepared by the procedure of Mitcbison and Kirschner [22], and spindle proteins isolated according to the procedure of Kuriyama et al. [25]. Antibody binding to nitrocellulose strips was detected using peroxidase-conjugated anti-rabbit IgG + I& t IgM (II + L) at the manufacturer’s recommended dilution (1:fOOO) for 2 h at room temperature. Following a repeat wash sequence, bound antibodies were detected by incubation with a solution containing I-chloro-lnaphthol and H,O,.
Antibodies were raised in ra bits to a synthetic peptide corresponding to the N-ter inal (antibody designation NEl) or C-terminal regions ( CEI) of the yy isotype of enolase. body was raised in rabbits to y’y e i.solated from bovine brain (antibody designation Each of these antibodies showed strong reactivit brain yy enolase (Fig. 1). When each of these antibodies was reacted with total HeLa mitotic cell nent reactivity was detected at the 4%kDa region of the blot which was comparable to the Gtion of enolase (Fig. 1). However, each of the anti ies, witth the exception of GGf, showed weak reactivity with other mitotic cell proteins, producing a series of nately within the 14- to GO-kDa region o parative analysis, however, suggested
47.7 -
I
3
4
FIG. 2. Immunoblot of anti-enolase antibodies reacted with protein obtained from isolated HeLa interphase cell centrosome preparations. Lane 1 Commassie blue-stained gei, iane 2 reacted with NE1, lane 3 reacted with @El, and lane 4 reacted with GG1. Left arrows, origin (top) and dye front (bottom); right arrow denotes position of enolase.
460
JOHNSTONE,
WAISMAN,
AND
RATTNER
ENOLASE
I
AT THE
CENTROSOME
2
FIG. 4. Immunoblot of the CEl antibody reacted with isolated HeLa spindle protein. Lane 1, Commassie blue-stained gel; lane 2, spindle protein reacted with CEl. Left arrows, origin (top) and dye front (bottom); right arrow denotes position of enolase.
region of reactivity common to all the antibodies was the enolase band (Fig. 1). A 40-kDa band was present in lanes containing mitotic cell protein as well as purified bovine brain enolase. The persistence of this band suggests that it likely represents a breakdown product of enolase. The variation in the intensity of this band is likely due to differences in the epitopes recognized by the different peptide antibodies. To verify that enolase was the only centrosome component identified by each of the antibodies, we isolated centrosomes from interphase HeLa cells and used the proteins from these preparations in immunoblots with each of the enolase antibodies. As shown in Fig. 2, each antibody recognized a single 48-kDa protein in these centrosomal preparations. The failure to detect the putative 40-kDa breakdown product in this blot may be due to the relatively low yield of enolase obtained in these preparations. Each of the antibodies was used in IIF studies in order to obtain cytological information on the distribution of enolase during the cell cycle of HeLa cells. IIF studies using the peptide antibody CEI revealed a diffuse staining pattern throughout the interphase cell. In addition, prominent reactivity was detected at a focus near the nuclear periphery (Fig. 3A). This region was found to correspond to the centrosome in double label experiments using anticentrosomal antibodies (see below). At metaphase, this antibody showed reactivity with the entire spindle including the centrosomal region (Fig. 3B).
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At the completion of telop ase reactivitgr was seen at the centrosome, along some centrosomal a crotubules, and at the midbody (Fig. 3C). plasmic staining was observed throughout the cytoplasm during cell division in all cells e To determine if the spindle and detected with the CEl antibody wa h a spindle prote epared Western HeLa spindle protein. Figure 4 illu tion to enolase, the major reae directed toward a protein et with molecular weights of 62 and 70 kDa, whi nent of isolated spindles. Since teins were not detected in the Western blots il do not appear to be compo somes. Therefore, cross-reaeti y with the 6%.kDa TOkDa doublet may provide an e nation for the spindle reactivity seen with the CE1 When interphase HeLa cells were i~~~ba~ed with t peptide antibody n tbrougbQut the cytoplasm. In antibody, the signal was localized to the centrosom’es (Fig. 3D). Cytoplasmic and centrosomal reactivity was also detected in metaphase and telophase cells (Figs. 3E and 3F). The intensity of the cytoplasmie signal ~rod~~@d by this antibody often interfered with the detection of centrosoma1 reactivity. Double staining, ‘ng a human antiee trosomal antibody [6] and the N antibody, confirm that the localized region of reac ity produced by t anti-enolase antibody correspo d to the site of the centrosome (Figs 36 and 3H). IIF studies using the GGl antibody raised against bovine brain yy enolase reve attern of staining identical to that observed wi -terminal antibody with a localized site of reactivity only at the centrosome 0th the anti-~e~t~~so~al antibody and antibodies showed reaetiv the anti-enola with isolated interphase centrosomes (Figs 3L and 9. In general, the region of reactivity educed by the autoimmune serum was broader th produced by the anti-enolase antibodies. It is that the autoimmune serum recognizes other d the centromere in addition to tb Preimmune rabbit sera did not cell reactivity (Fig. 3N).
FIG. 3. Mitotic HeLa cells reacted with anti-enolase antibodies. A, interphase; B, metaphase; C, telophase reacted with the CEl antibody. Large arrows denote centrosomal reactivity and small arrows microtubule reactivity (M = midbody). D, interphase; E, metaphase; F, telophase reacted with the NE1 antibody. Arrows denote centrosomal reactivity. G and H, a telophase cell double stained reacted with an anti-centrosoma1 autoimmune serum (G) and the NE1 antibody (H). Arrows indicate that the region stained by the NE1 antibody overlap with the centrosomal region identified by the autoimmune serum. I, interphase; J, metaphase; K, telophase reacted with the GGl antibody. Arrows denote centrosomal reactivity. L and M, double staining of a preparation of isolated interphase HeLa centrosomes reacted with a human anticentrosomal serum (L) and the GGl antibody (M). N, a control panel illustrating an interphase cell reacted with preimmune rabbit sera. Bar, 10 pm.
462
JOHNSTONE,
WAISMAN,
DISCUSSION
Using antibodies to two different regions of bovine yy enolase, as well as an antibody produced against yy enolase isolated from bovine brain, we have shown that in addition to its distribution within the cytosol of cycling cells, a subset of this enzyme is found at the centrosome throughout the cell cycle. Several lines of evidence suggest that the centrosome pattern we have detected reflects the differential distribution of enolase: (i) Three different antibodies, two of which are known to recognize two diverse regions of enolase produce centrosome staining in HeLA cells. (ii) Each of these antibodies recognizes enolase in immunoblots of whole mitotic cell protein. (iii) Each of these antibodies recognizes a single 4% kDa protein in immunoblots of isolated centrosome proteins. (iv) The antibodies show reactivity with isolated centrosome. In addition to the antibodies used in this study, we have also investigated the ability of commercially available antibodies to yy enolase to react with the centrosome of HeLa cells. Of the three antisera tested (DakoPatt, Carpinteria, CA), two produced a pattern of centrosomal reactivity identical to that found with the NE1 and GGl antibodies. The third produced pronounced cytoplasmic reactivity but did not show reactivity specific to the centrosome. It is possible that some anti-enolase antibodies recognize determinants that are masked at the centrosome. One unique feature of the CEl antibody is that it also shows reactivity within the mitotic spindle. We have shown that this is likely due to cross-reactivity with two prominent protein components found in isolated spindle preparations. A GenBank search failed to reveal homology between the peptide used to generate the CEl antibody and the protein sequence of any other known protein. Thus, the cross-reactivity seen with the CEl antibody provides a means for the identification of additional protein(s) that colocalize with the microtubules of the mitotic spindle. It is interesting to note that at late telophase, the CEl antibody not only reacts with the centrosome and intercellular bridge (midbody) but also with the microtubule array associated with the centrosome. During late telophase in these cells, the centrosome migrates from its original poleward position to a position at the midbody side of the reforming nucleus. The microtubule array stained by the CEl antibody extends along this path, suggesting a possible role for the antigen(s) recognized by this antibody in centrosome migration. While our antibodies were raised to either the purified bovine brain yy enolase or the C-terminal or N-terminal sequence of the molecule, we have observed slight cross-reactivity between these antibodies
AND
RATTNER
and CY~!enolase (data not shown). Although our data provide strong support for the presence of yy enolase at the centrosome (for example, the presence of a single band of reactivity at 48 kDa in the blots of isolated centrosomal protein Fig. 2), we cannot rule out the possibility that LYLY enolase could also colocalize to the centrosome. Several recent studies have suggested that glycolytic enzymes may be localized to specific sites within the cell. For example, some glycolytic enzymes have been shown to bind to structural proteins, particularly actin [26] and tubulin [27, 281. The association of glycolytic enzymes with membranous structures has also been observed. For example, electron-immunocytochemical studies have illustrated the localization of yy enolase to the membrane of primary and metastatic cerebral tumors, and also to the glial filaments of glioma cells [14, 291. Knull 1301 and Lin et al. [32] have documented the association of several glycolytic enzymes with synaptic membranes and Gosti et al. [32] used an anti-centrosoma1 serum to identify an epitope of lactate dehydrogenase at the centrosome. The detection of enolase at the centrosome lends further support to the concept that glycolytic enzymes can have a site-specific distribution within the cell. The compartmentalization of glycolytic enzymes implies that there is a requirement for these enzymes in site-specific cellular events. There are several lines of evidence that suggest that the glycolytic enzymes may be required for these events not only for their glycolytic capacity, but also for their ability to perform a structural function [17]. For example, some glycolytic enzymes such as lactate dehydrogenase type M, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, and aldolase have been found to copellet with microtubules [28]. In the case of glyceraldehyde-3-phosphate dehydrogenase, the binding of the enzyme to microtubules shows considerable specificity and is associated with changes in the assembly and disassembly parameters of these cytoskeletal elements [33]. Further, several glycolytic enzymes have been shown to function in vitro in the bundling of actin [17] and microtubules [27,34]. In addition, Masters [35] has postulated that glycolytic enzymes may “plate” the filamentous actin core of the microfilament. Thus, the glycolytic enzymes may play a structural role affecting the organization of cytoskeletal elements. The presence of enolase at the centrosome may be correlated with this role either through a direct interaction with cytoskeletal components or indirectly as a member of an enzyme complex. The apparent inability of enolase to bind to tubulin, for example, would support the latter possibility [28]. In conclusion, we have demonstrated the presence of a portion of cellular enolase at the centrosome throughout the cell cycle of HeLa cells. This subset of enolase may be correlated with a structural and/or glycolytic
ENOLASE
role carried out by glycolytic bule organizing center.
AT THE
CENTROSQME
enzymes at this microtu-
This work is supported by a grant from the Medical Research Council of Canada (D.M.W.) and the National Cancer Institute of Canada (J.B.R.). The authors thank Craig Litwin for help in preparing the centrosomal preparations and Jerry Wang for his helpful comments during the course of this investigation and manuscript preparation. REFERENCES 1. 2. 3.
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