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Human anticentromere antibodies: Distribution, characterization of antigens, and effect on microtubule organization
Cell, Vol . 35, 33 1 -339, November 1983, Copyright m 1983 by MIT
0092-8674/83/110331-09 $02 .00/0
Human Anticentromere Antibodies: Distribution, Characterization of Antigens, and Effect on Microtubule Organization John V. Cox,* Eric A. Schenk,t and J. B . Olmsted* *Department of Biology tDepartment of Pathology University of Rochester Rochester, New York 14627
Summary Properties of human anticentromere .autoantibodies were analyzed . In intact cells or isolated cell fractions, these sera stain the centromeres of mitotic chromosomes and discrete speckles (prekinetochores) in nuclei. Staining is also retained in matrix preparations from nuclei or chromosomes . Immunoprecipitation or immunoblotting demonstrates protein antigens of 14, 20, 23, and 34 kd in HeLa nuclei and chromosomes; immunoprecipitates of nuclei also contain a protein of 15 .5 kd . Matrix preparations contain only the 20, 23, and 34 kd species . Absorption of the anticentromere serum with any one of the four nuclear antigens immobilized on nitrocellulose is sufficient to eliminate centromere staining . Using a lysed cell model for microtubule nucleation, anticentromere sera are shown to inhibit specifically the organization of microtubules at the kinetochore . Introduction Light and electron microscopic studies have shown that the kinetochore of chromosomes is involved in the attachment of spindle fiber microtubules to chromosomes during mitosis . However, the nature of this interaction is still very poorly understood, partly because of lack of information on the biochemical composition of the kinetochore . The recent discovery that sera from patients having the CREST variant of scleroderma stain the centromere region of mitotic chromosomes and discrete speckles within interphase nuclei (Fritzler and Kinsella, 1980 ; Moroi et al ., 1980 ; Tan et al ., 1980) has made it possible to study the organization of this region in more detail . Immunoelectron microscopic studies (Brenner et al ., 1981) have established that one anticentromere serum stains the inner and outer plates of the trilaminar kinetochore of mitotic chromosomes . This and another study (Moroi et al ., 1981) have shown that these sera stain diffuse foci within the nucleus of interphase cells, and have also indicated that the location of these regions relative to the nuclear envelope may vary among cell types . Synchrony experiments have demonstrated that the interphase speckles (prekinetochores) double during late G2 of the cell cycle, and these then segregate with daughter chromosomes during anaphase (Brenner et al ., 1981) . Studies in which enzymatic digestion has been used to determine which treatments abolish immunofluorescent staining patterns have suggested that at least some of the antigenic
determinants with which these antibodies react are protein in nature (Moroi et al ., 1980). In the studies described in this report, we present the first evidence that antigens recognized by an anticentromere serum are a discrete set of proteins . Some of these proteins are also components of nuclear and chromosomal matrix preparations . In addition, we demonstrate that these anticentromere sera can selectively block microtubule organization at one of the two mitotic microtubule-organizing centers, the kinetochore . Results Distribution of Anticentromere Staining Of 50 ANA (antinuclear antibody) antisera we screened, five were identified as anticentromere by immunofluorescent staining of PtK 1 and HeLa cells and isolated HeLa chromosomes . The distribution of the antigens recognized by two of these sera, cen-1 and cen-2, at various stages of the PtK, cell cycle is illustrated in Figure 1 . The anticentromere fluorescence is localized to a pair of spots per mitotic chromosome at prophase (Figures 1 b and l e), while only a single spot per chromosome is found at anaphase (Figures 1 c and 1f) . These antigens were also found in isolated Hela chromosomes (Figures 2a and 2b), where the staining can be more precisely localized to the region of the primary constriction . Double immunofluorescent staining of a metaphase PtK, cell with cen-1 and tubulin antibodies indicates that spindle fibers terminate at the region of the chromosome stained by cen-1 (Figures 1 g and 1 h) . In addition to the localization of these antigens at the primary constriction of mitotic chromosomes, they were maintained as discrete speckles (prekinetochores; Brenner et al ., 1981), in interphase nuclei of PtK 1 cells (Figures 1 a and 1 d) and in isolated Hela nuclei (Figures 2c and 2d) . All mammalian cell lines thus far examined (Hela, 3T3, BHK, L, neuroblastoma, and Indian muntjac cells) show the same pattern of nuclear fluorescence when stained with cen-1 and cen-2 sera as that described for the marsupial PtK, cells . Effect of Anticentromere Sera on Microtubule Organization A lysed cell model was developed to examine the effect of the anticentromere sera on microtubule organization . PtK 1 cells grown on coverslips were gently lysed in buffer containing 2 mM CaCl 2 to break down preexisting microtubules . Free Ca' was then chelated with EGTA and the cells were incubated with phosphocellulose-purified porcine brain tubulin . By employing purified brain tubulin at a concentration of 1 mg/ml, it was possible to examine the addition of tubulin subunits onto cellular nucleation sites, such as centrosomes and kinetochores, in the absence of any significant self-assembly of tubulin into microtubules . After incubation with tubulin, the cells were fixed and the microtubule distribution assayed by immunofluorescent staining with tubulin antibodies . Initiation of microtubules
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Figure 1. Cell-Cycle
Distribution
of Anti-Centromere
Staining
PtK, cells were stained with ten-I (a-c,g) or ten-2 (d-f) at various stages of the cell cycle. (a) interphase (d) interphase (860x); (e) prophase (1075X); (f) anaphase (1100X). (g and h) double immunofluorescent 1 (g) and tubulin (h) antibodies (1300X).
from the centrosome region was seen in 15% of interphase cells as compared to greater than 90% of mitotic cells, In neither case were there significant numbers of microtubules assembled randomly in the cytoplasm. Approximately 50% of the mitotic cells showed organization of microtubules from the centromere region of several chro-
(600X); (b) prophase (1220X); (c) anaphase. (950x); staining of a metaphase RK, cell stained with cen-
mosomes. As can be seen in Figures 3a and 3b, double immunofluorescence staining with tubulin and ten-I antibodies served to identify the region of the chromosomes from which microtubules initiated. To determine whether the anticentromere sera would interfere with microtubule formation on chromosomes,
Human Anticentromere 333
Antrbodres
Frgure 2. lmmunofluorescent Nuclei
Stainrng of isolated HeLa Chromosomes
and
HeLa chromosomes and nucler were isolated as described in Experimental Procedures and stained with ten-I. (a and b) phase and fluorescence micrographs of isolated HeLa chromosomes (1675x). (c and d) phase and fluorescence mrcrographs of an Isolated HeLa nucleus (1960X).
lysed cells were incubated with IgG from ten-1 or ten-2 for 30 min at room temperature prior to incubation with tubulin. After incubation with tubulin and fixation, cells preincubated with ten-1 or ten2 were stained with tubulin antibodies and the appropriate fluorochrome-conjugated second antibodies, By scoring only those cells that were positive for anticentromere fluorescence, one could be assured that any effect on microtubule initiation at the kinetochore region was due to the presence of the antibody at this site prior to incubation with tubulin. As shown in Figures 3c and 3d, initiation of microtubules from chromosomal kinetochores was inhibited by ten-I ; similar results were obtained with ten-2 serum. Only 10% of the cells preincubated with either centromere antiserum showed microtubule initiation at the kinetochore, as compared to 50% of the cells preincubated with normal human sera (Figures 3a and 3b) or buffer. The anticentromere sera had no effect on the initiation of microtubules from mitotic or interphase centrosomes. In addition, when human sera containing noncentromeric ANA were employed in this assay, there was no effect upon initiation of microtubules from centrosomes or the kinetochore region. These results indicate that both ten-1 and ten-2 sera inhibit the organization of microtubules at the kinetochore in this lysed cell system.
Distribution of ten-2 Antigens in Matrix Preparations To determine the subnuclear distribution of the antigens, a number of fractionation procedures were investigated. Using previously published methods for the preparation of nuclear matrix fractions (Adolph et al., 1977; Capco et al., 1982) we established that matrices prepared from HeLa cells metabolically labeled with 3H-thymidine, 3H-uridine, or 35S-methionine contained 2.5% of the DNA, 0.2% of the
RNA, and 15%-20% of the protein of the intact cell. When nuclear matrices prepared from PtK, cells by either Procedure 1 (Figure 4a) or Procedure 2 (data not shown) were stained with ten-2 serum, a pattern similar to that observed for intact interphase cells (Figure Id) was obtained. In addition, the number of spots/nucleus in matrix preparations of either isolated HeLa nuclei (average of 43/nucleus for 14 cells) or PtK, cells (average of 12,5/nucleus for 12 cells) was the same as that seen in intact cells and, as had been found previously (Moroi et al., 1980; Brenner et al., 1981) corresponded closely to the normal chromosome number for the respective cell lines. Analyses were also performed using mitotic cells to determine whether the antigens were retained in extracted chromosomes. However, following extraction, chromosomes were no longer visible by phase microscopy, and it was difficult to ascertain whether punctate fluorescent staining with ten-2 was due to specific centromere labeling or background debris. A modified procedure was therefore developed, in which spindle microtubules were stabilized by the inclusion of 5 PM taxol in all extraction buffers, and were used as markers for the location of centromeres. As shown in Figures 4b and 4c, double immunofluorescence staining of mitotic matrices shows spots stained by ten-2 in the region of termini of taxol-stabilized spindle fibers. All of these results indicate that at least some of the antigens recognized by ten-2 are tightly bound components of both interphase and mitotic matrices.
Biochemical Identification of Antigens Further characterization of the antigens recognized by cen2 was carried out by immunoprecipitation of various fractions derived from HeLa cells labeled with 35S-methionine. As shown in Figure 5a, immunoprecipitates from labeled nuclei (lane 3) and chromosomes (lane 6) contain proteins of 14, 20, 23, and 34 kd, although the 34 kd protein is only faintly visible in the precipitate from chromosomes. A protein of 15.5 kd is also seen in the nuclear immunoprecipitate. The proteins of higher molecular weight are also in immunoprecipitates obtained using normal human sera (Figure 5a, lane 4) or human sera containing noncentromerit ANA (data not shown), and are nonspecifically bound proteins. lmmunoprecipitates of matrix preparations (Figure 5b) from interphase (lane 2) and mitotic cells (lane 4) also contain proteins of 20, 23, and 34 kd. However, the 14 and 15.5 kd proteins are almost entirely absent. lmmunoprecipitates of matrices prepared from isolated nuclei (lane 6) and isolated chromosomes (lane 8) show the same proteins as seen in matrices from intact cells; the 34 kd protein is again present in reduced amounts in matrices prepared from isolated chromosomes. Two-dimensional gel analysis of immunoprecipitates from 35S-labeled HeLa nuclei shows that the proteins range in isoelectric point from 5.2 to 5.9, and that the 20 and 23 kd proteins exist in at least one modified state (Figure 5~). As shown in Figure 5d, an immunoprecipitate obtained from 3’P-labeled nuclei shows that the 14, 15.5, 20, and 23 kd proteins are all phosphorylated.
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Figure 3. Lysed Cell Model Double immunofluorescent staining of RK, cells that have been lysed under the conditions described in the text and incubated tubulin. (a and b) Control cell was lysed and incubated with phosphocellulose-purified tubulin and then fixed and stained antrbodies. Note that individual foci of tubulin nucleation correspond to the region of centromere staining (arrows, 925x). (c and with ten-1 prior to incubation with phosphocellulose-purified tubulin. Note the lack of nucleation from centromeres while polar (1540X).
Figure 4. Interphase
with phosphocellulose-purified with ten-I (a) and tubulin (b) d) Cells that were preincubated nucleation appears unaffected
and Mitotic Matrices
(a) Nuclear matrices from PtK, cells were prepared according to Procedure 1 described in Experimental Procedures and stained with ten-2. Note speckles that appear identical to those seen in nuclei of intact cells (Figure Id) (1320x). (b and c) Double immunofluorescent staining of a PtK, cell that has been prepared in the same manner as Figure 4a except for the inclusion of 5 WM taxol in all steps of the protocol. The cell was then stained with ten2 (b) and tubulin (c) antibodies. As can be seen in Figure 4c. a subset of spindle microtubules is stabilized and the anticentromere fluorescence is located in the region of the termini of stabilized fibers. The hash mark in the upper right hand corner of Figures 4b and 4c provides a reference point for comparing the double immunofluorescent pair (1050X).
lmmunoprecipitates obtained from 35S-iabeled nuclei of various cell types, including L, PtK,, neuroblastoma, 3T3, and Indian muntjac cells, also contain the 14, 15.5, 20, and 23 kd proteins (Figure 6) and indicate that these proteins are conserved in other mammalian cell lines.
However, the 34 kd protein found in HeLa cells is not precipitated from these cell lines. The antigens reacting with ten-2 were also examined by immunoblotting. As shown in Figure 7, proteins in HeLa nuclear fractions of 14, 20, 23, and 34 kd proteins are
Human Anticentromere 33.5
Antrbodres
Figure 6. lmmunoprecrprtates
from Nucler of Varrous Cell Types
Nuclei were isolated from cell lines labeled wrth “S-methronrne, and proteins rmmunoprecipitated wrth ten-2: lndran muntjac (lane 1); L (lane 2); PtK, (lane 3); neuroblastoma (lane 4); 3T3 (lane 5).
recognized by the ten-2 serum; this is the same pattern as seen by immunoprecipitation. However, the 15.5 kd protein seen by immunoprecipitation is rarely seen on immunoblots. Additional proteins of 70 and 90 kd were also labeled on the immunoblots. lmmunoblots using normal human sera or sera from patients with noncentromeric ANA showed no reaction with nuclear proteins from HeLa, or they generated a pattern distinct from that obtained with ten-2. To determine whether the proteins that react with the ten-2 serum by immunoblotting and immunoprecipitation corresponded to the antigens localized at the kinetochore region of chromosomes and the prekinetochores of interphase nuclei, absorption of the ten-2 serum was carried out using proteins immobilized on nitrocellulose. Proteins from isolated HeLa nuclei were separated on a preparative SDS slab gel and transferred to nitrocellulose paper. A vertical strip of this blot was probed with ten-2, generating a pattern like that shown in Figure 7, and used for orientation The regions in the remainder of the blot corresponding to the individual proteins of 14, 20, 23, 34, and 90 kd were then cut out and incubated with the ten-2 serum prior to staining F’tK, cells. A 2 mm band at -150 kd, where no antiserum reaction was seen, was used as a control for
Figure 5. lmmunoprecrpitates
from HeLa Cell Fractions
Autoradrograms of tmmunoprecipitates of fractions from HeLa cells labeled with %methionine (a-c) or zP-orthophosphate (d). (a and b) One-dimensronal SDS gels of total %-labeled proterns and rmmunopreciprtates obtarned using ten-2 are shown for nuclei (5a, lanes 2-3), chromosomes @a, lanes 5-6) matrix from interphase cells (5b. lanes l-2) matrix from mrtotic cells (5b. lanes 3-4). matrix from isolated nuclei (5b, lanes 5-6) and matrix
from isolated chromosomes (5b, lanes 7-8). An immunoprecrpitate from Isolated nuclei using a normal human serum (5a, lane 4) illustrates the nonspecrfically bound proteins observed with the human sera tested. 5a. lane 1: “C-molecular weight markers: myosin, 200 kd; phosphorylase 6, 92.5 kd; BSA. 68 kd; ovalbumin, 43 kd; a-chymotrypsrnogen. 25.7 kd: plactoglobukn, 18.4 kd; cytochrome-c. 12.3 kd. Dashes indicate positron of antigens referred to in the text. (c) Two-dimensronal NEPHGE gel of proteins rmmunoprecrpitated wrth ten-2 from %labeled nucler. Note multiple rsoelectnc pornts for the 20 and 23 kd proterns. Bars at the top of the gel Indicate pH gradient in units of 1 .O. (d) One-drmensional SDS gel of proteins rmmunoprecipitated wrth ten-2 from %labeled nucler.
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Figure 7. Western
Blot of Isolated HeLa Nuclei Probed with cen9
Proteins from Isolated HeLa nuclei were run on a 1O%-25% polyacrylamide gradient gel, electrophoretically transferred to nitrocellulose paper, and probed with ten-2. As described in the text, specific bands (lines) or a control region of the blot (bracket) were used for absorption of ten-2 prior to immunofluorescent staining. The right-hand panel indicates the regions that absorbed (+) or had no effect on (-) centromere staining.
nonspecific binding of the antibody to nitrocellulose. As listed in Figure 7, the proteins of 14, 20, 23, and 34 kd eliminated the centromere staining pattern in isolated chromosomes or intact cells, whereas the protein at 90 kd and the control strip had no effect on the staining pattern.
Discussion Human autoantibodies have been useful probes for studying the molecular composition (Douvas et al., 1979; Lerner and Steitz, 1979; Hendrick et al., 1981; Lerner and Steitz, 1981; Lerner et al., 1981b; Hardin et al., 1982; Matter et al., 1982) and possible function (Lerner et al., 1980; Yang et al., 1981) of various small nuclear and cytoplasmic RNPs. In many instances, the antigenic determinants recognized by these sera are complex. However, autoantibodies can provide a means of investigating an organelle that cannot otherwise be isolated or assayed for function. Thus studies with human anticentromere antibodies have demonstrated that antigens reacting with these sera remain as discrete structures during interphase and duplicate in late G2 (Brenner et al., 1981). We have used these sera in a lysed cell system to determine whether the antibodies react with sites at the kinetochore that are important in the organization of microtubules. The protocols described previously (Pepper and Brinkely, 1979) were modified slightly in order to eliminate formation of random microtubules. Under the conditions employed, 15% of interphase centrosomes were competent to initiate microtubule assembly. This is consistent with previous results that indicate no (Telzer and Rosenbaum, 1979) or low (Snyder and McIntosh, 1975; Kuriyama and Borisy, 1981) initiating activity for interphase centro-
somes. However, nucleation of microtubules occurred at approximately 90% of mitotic centrosomes, in agreement with previous studies (Snyder and McIntosh, 1975; Telzer and Rosenbaum, 1979; Kuriyama and Borisy, 1981) that indicate that centrosome-nucleating activity increases at the onset of mitosis. The kinetochore region of chromosomes initiated microtubule assembly in 50% of the mitotic cells that were scored. Gould and Borisy (1978) have shown that 55% of isolated CHO chromosomes initiated microtubule assembly from kinetochores when concentrations of tubulin similar to those used in our study were employed. By using double immunofluorescence analyses with the lysed cell model, it was possible to determine if ten-I or ten-2 sera have any significant effect on microtubule initiation. Preincubation of lysed cells with either serum prior to incubation with tubulin specifically inhibited microtubule initiation in the region of chromosomal kinetochores, but had no effect on the initiation activity of mitotic or interphase centrosomes. EM analyses (Pepper and Brinkley, 1979) demonstrated that tubulin antibodies were capable of blocking the assembly of microtubules from both centrosomes and kinetochores in a lysed cell model. However, this is the first report of an antiserum that selectively inhibits microtubule assembly from only one mitotic organizing site, the kinetochore. Although the mechanism remains to be elucidated, this inhibition could be due to one or more of the following events: the antibodies may react directly with proteins involved in microtubule initiation at the kinetochore; the binding of the antibody may steritally block the accessibility of tubulin to the kinetochore, while not binding directly to structures mediating tubulin function; or the binding of the antibody may alter the conformation of the kinetochore such that microtubule initiation is inhibited. The immunoprecipitation and immunoblotting data indicated that the ten-2 antiserum reacts with at least four proteins (14, 20, 23, and 34 kd) in isolated chromosomes and nuclei. Absorption of the serum with any of these four molecular weight species immobilized on nitrocellulose caused loss of staining, suggesting that the antibody recognizes determinants shared by the four proteins. The complexity of the antigens recognized by ten-2 is not unlike that observed for human autoantibodies associated with systemic lupus erythematosus. The Sm antigen has been shown to be made up of five snRNAs, each of which is complexed with seven proteins; however, the antigenic determinant is known to be localized on only one of these proteins (Lerner et al., 1981a). The interaction of Ul RNA with the same seven proteins mentioned above is thought to comprise the RNP antigenic determinant (Lerner and Steitz, 1981). Both the Sm and RNP antigens are highly conserved and abundant in most mammalian cells (Hendrick et al., 1981). Like these snRNP antigens, the 14, 15.5, 20, and 23 kd proteins recognized by ten-2 are conserved in the mammalian cell lines we examined. Previous cytochemical analyses have shown that an RNP
Human Anticentromere Antibodies 337
component is present at the kinetochore of chromosomes (Rieder, 1979) . The possibility that the proteins recognized by cen-2 comprise a portion of an RNP or interact with specific regions of centromeric heterochromatin remains to be analyzed . Two-dimensional gel analysis has shown that the proteins recognized by cen-2 have an acidic isoelectric point and are thus distinct from the bulk of protein associated with chromatin . An acidic protein of approximately 38 kd has been identified in Drosophila (Will and Bautz, 1981) and an antiserum against this protein has been shown to stain the chromocenter of salivary gland chromosomes, as well as metaphase chromosomes from Hela cells . This protein may be homologous to the 34 kd protein that is recognized by cen-2 . We have also shown that the 20, 23, and 34 kd proteins are components of matrices from interphase and mitotic Hela cells ; these antigens comprise only a minor fraction of the proteins found in these preparations . Although the bulk of the DNA, RNA, and protein have been extracted from these matrices, the staining pattern obtained with cen-2 is the same as that seen in intact cells, suggesting that both kinetochores and prekinetochores are very stable structures . Our through-focus analyses of the distribution of prekinetochores in nuclei and nuclear matrices from PtK, and HeLa cells, as well as the EM observations of Brenner et al . (1981), indicate a nuclear location for these proteins distinct from the nuclear pore-lamina complex . We have shown that preparations of mitotic cells extracted in a manner similar to that used in obtaining chromosomal scaffolds (Adolph et al ., 1977) exhibit the same distribution of these proteins as seen in intact cells . The finding that the localization of anticentromere antigens is unchanged in mitotic matrices suggests that the matrix or scaffold may be important in maintaining the organization of this region in mitotic cells . A number of biochemical studies have suggested that the matrix serves as a nuclear substructure important in the organization, replication, and transcription of interphase chromatin (Berezney and Coffey, 1974 ; Long et al ., 1979 ; Berezney and Buchholtz, 1981 ; Van Eekelen and Van Venrooij, 1981 ; Robinson et al ., 1982) . The manner in which proteins recognized by centromere antisera are related to others defined as biochemical components of matrices is as yet undetermined . However, our findings do suggest that cen-2 antigens are involved in the organization of chromosomes throughout the cell cycle . With the exception of the 15 .5 kd protein, the composition of the antigens recognized by cen-2 are the same in both isolated HeLa nuclei and chromosomes . However, the region with which these proteins are associated undergoes major morphological rearrangement in changing from the diffuse foci of interphase nuclei to the trilaminar kinetochores of mitotic chromosomes . Concomitant with this transition, the kinetochore also becomes competent to interact with microtubules of the mitotic spindle . It will be interesting to determine whether the phosphorylation of
the proteins recognized by cen-2 is involved in the reorganization of chromosomes at the kinetochore and the ability of this site to interact with microtubules during mitosis . Experimental Procedures Cell Culture Monolayer cultures of Hela S3, PtK,, neuroblastoma, L, 3T3, BHK, and Indian muntjac cells were grown at 37°C in F12 with 10% fetal calf serum . Cultures were labeled with 35S-methionine, 3H-thymidine, 3H-uridine or 32 Porthophosphate at approximately 10 µCi/ml for 36 hr in order to obtain labeled cell fractions . Antisera Human sera were obtained from the Department of Pathology, University of Rochester. Sera that had been scored as ANA (antinuclear antibody)positive on frozen sections of tissue were examined further by immunofluorescence microscopy of cultured cells . The antisera used in this report, cen-1 and cen-2, represented individual aliquots of nonpooled serum obtained from two different patients diagnosed as having the CREST variant of scleroderma. Additional normal human IgG was the gift of Dr . George Abraham, Department of Immunology and Microbiology, University of Rochester . IgG fractions were prepared from the sera by taking the void volume fraction from an Affigel Blue DEAE column (BioRad) . Tubulin antisera were prepared and characterized as described previously (Van De Water et al ., 1982) . Chromosome Isolation Chromosomes were isolated from HeLa S3 cells using slight modifications of previously published methods (Stubblefield and Wray, 1971 ; Spandidos and Siminovitch, 1977) . Mitotic cells were accumulated by incubating cultures in 60 ng/ml nocodazole overnight, and were harvested by sedimentation at 450 xg for 5 min. All subsequent steps were performed at 04°C unless otherwise noted . The resulting cell pellet was resuspended in 75 mM KCI and incubated for 5 min . The cells were centrifuged at 450 xg for 3 min and the pellet was resuspended in HCP buffer (1 M hexylene glycol, 0 .5 mM CaCl2 , 0 .1 mM PIPES, pH 6 .94) . The cells were again pelleted and resuspended in HCP + 0 .5% NP-40 and incubated at room temperature for 10 min . The cells were then sheared four times with a 22 gauge needle and the lysate centrifuged at 50 xg for 2 min . In some experiments, chromosomes from the suspension were allowed to settle onto poly-L-lysine-coated coverslips for immunofluorescence microscopy . Alternatively, the supernatant was centrifuged at 800 xg for 10 min . The resulting chromosomal pellet was resuspended in 10 mM Tris-HCI, 1 mM MgCl2 (pH 7 .4) containing 50 ag/ml DNAase I and incubated on ice for 1 hr for immunochemical assays . Nuclei Isolation Nuclei were isolated from the various cell types using the procedure of Warner (1979) . The final nuclear pellet was resuspended in 10 mM TrisHCI, 1 mM MgCI2 (pH 7.4) . These nuclei were allowed to settle onto polyL-lysine-coated coverslips for immunofluorescence microscopy or digested with 50 µg/ml DNAase I for 1 hr on ice for immunochemical assays . Nuclear and Chromosomal Matrix Isolation Procedure 1 : Matrices were prepared from interphase and mitotic HeLa and PtK, cells according to the procedure of Capco et al . (1982). The cells were washed in phosphate-buffered saline (PBS) and pelleted by centrifugation at 450 xg for 5 min . The pellet was resuspended in CSK buffer (10 mM PIPES, pH 6 .8, 250 mM (NH4)2SO4 , 300 mM sucrose, 3 mM MgCl2 , 1 .2 mM PMSF, 0.5% Triton X-100), incubated on ice for 3 min, and washed once in digestion buffer (10 mM PIPES, pH 6 .8, 50 mM NaCl, 300 mM sucrose, 3 MM MgCl 2, 1 .2 mM PMSF, 0 .5% Triton X-100) . The pellet was then resuspended in digestion buffer containing 200 µg/ml DNAase I and 30 µg/ml RNAase A and incubated at RT for 20 min. This material was then pelleted at 450 xg for 5 min, resuspended in CSK, and incubated for 5 min at RT. The final pellet was then washed in 10 mM Tris-HCI (pH 7 .4) and prepared for immunoprecipitation .
Cell 3 38
Procedure 2 : Interphase and mitotic cells were washed in PM buffer (0 .1 M PIPES, 0.5 mM MgC1 2, pH 6 .94), lysed in PM with 0 .25% Triton X-100, and then incubated in PM containing 200,ug/ml DNAase I and 30,ug/ml RNAase A for 20 min at RT . The cells were pelleted and resuspended in 10 mM Tris-HCI (pH 9 .0), 2 .0 M NaCl, 10 mM EDTA, 0 .1% NP-40 (Adolph et al ., 1977) and incubated on ice for 10 min . The suspension was centrifuged at 450 xg for 5 min and the pellet prepared for immunoprecipitation . Matrices prepared for immunofluorescence microscopy were treated as described in Procedure 1 or 2 above, except cells remained attached to coverslips throughout the protocols . For determination of the percent DNA, RNA, or protein remaining in matrix preparations, cold TCA precipitates from labeled cell fractions were counted . Lysed Cell Models A modification of the procedure of Pepper and Brinkley (1979) was developed to examine the nucleation of microtubules in lysed cells . PtK 1 cells were grown on coverslips and incubated with 60 ng/ml nocodazole for 8 hr to accumulate mitotic cells . Cells were refed with fresh medium without nocodazole and incubated at 37°C for 20 min . Cells were then placed at 4°C, rinsed with PMG (0.1 M PIPES, 0 .5 mM MgC12, 0 .5 mM GTP) and then lysed for 2 min in PMG containing 0 .09% Triton X-100 and 2 .0 mM CaCl2 . Cells were washed twice in PMG with 2 mM CaCI2 (2 min/wash), and then incubated in PMG with 1 mM EGTA for 30 sec . Cells were rinsed in PMG, returned to 37°C, and incubated for 15-20 min with phosphocellulosepurified porcine brain tubulin (1 mg/ml) (Sloboda et al ., 1976) in PM with 2 mM GTP. In some experiments, lysed cells were incubated with anticentromere or normal human sera prior to incubation with purified tubulin (see Results) . Cells were then fixed in 3 .7% formaldehyde in PMG for 15 min and processed for immunofluorescence microscopy . Cells were scored positive for kinetochore microtubule initiation if two chromosomes exhibited microtubule formation ; the majority of cells showed initiation from 4-6 chromosomes/cell . Cells initiating microtubules from a single kinetochore were rare, and inclusion of these data did not alter the results for control or experimental samples . However, due to the presence of tripolar cells in the population, and the possible ambiguity in scoring initiation from a single kinetochore, these cells were routinely scored as negative . Gel Electrophoresis Cell fractions were analyzed on one- or two-dimensional gels . Nonequilibrium isoelectric focusing gels (NEPHGE) were prepared according to O'Farrell et al . (1977), as modified by Waring et al. (1978); 2 .0% of pH 211 ampholytes were used . Separation of proteins on SDS gels (Laemmli, 1970) was carried out using 10%-25% polyacrylamide gradient gels . Immunological Procedures Immunoprecipltatlon Cells were labeled with 10 µCi/ml °S-methionine for 36 hr. Various cell fractions were isolated as described above and solubilized by sonication in DOC buffer (0 .1 M Tris-HCI, pH 8 .3, 0 .1% SDS, 0 .5% NP-40,0.5% DOC, 2 mM EDTA, and 0 .25% gelatin) . Samples were then incubated with 1015 µl of IgG (1 mg/ml) per 500 µl of solution for 2 hr at 37°C. Immune complexes were precipitated with protein A-Sepharose 4B beads (15 µl packed beads/10 µg IgG) . The beads were washed five times with DOC buffer (2 ml/wash) and once in 0 .1 M Tris-HCI, pH 8.3 . Immune complexes were released from the beads by boiling in SDS sample buffer (Laemmli, 1970) and run on 1- or 2-D gels . After fixing in glacial acetic acid for 30 min, gels were impregnated with 20% PPO in glacial acetic acid for 1 hr, rinsed in H 2O for 1 hr, dried, and autoradiographed. Immunoblotting Electrophoretic transfer and immunoblotting on nitrocellulose paper was performed according to the procedure of Towbin et al . (1979). Blots were incubated with cen-2 for 6-8 hr at RT, washed, and incubated with affinitypurified GAH-IgG coupled with peroxidase for 2 hr at RT . Following washing, antibody binding was detected by reacting peroxidase with the following mixture: 20 ml of 3 mg/mI 4-chloro-l-naphthol in MeOH ; 36 µl of 50% H20 2; 100 ml of 10 mM Tris-HCI, pH 7 .4 . lmmunoabsorption The protein from -107 isolated HeLa nuclei was separated on a 10%25% preparative SDS gel and transferred to a 9 X 9 cm sheet of nitrocellulose as described above . Vertical strips (5 mm) from either end of the
blot were probed with cen-2 and the pattern that was generated was used for orientation . A 2 mm band was cut out from the regions corresponding to the proteins of 14, 20, 23, 34, and 90 kd, plus a control strip at -150 kd . Each strip was cut into eight equal pieces, and incubated with 300 µl of a 1/50 dilution of IgG from cen-2 (1 mg/ml) for 4 hr on ice . Immunofluorescence Microscopy Cells grown on coverslips in 35 mM petri dishes or isolated HeLa chromosomes and nuclei attached to poly-L-lysine-coated coverslips were fixed in 3 .7% formaldehyde in PBS for 15 min followed by -20°C acetone for 30 sec . Alternatively, samples were fixed in 0 .2% glutaraldehyde in PBS for 15 min, followed by washing in 1 % Triton X-100 in PBS for 30 min and five changes (3 min each) of 1 mg/ml sodium borohydride in 95% EtOH. Samples were then incubated with one of the human antisera or tubulin antisera for 30 min at 37°C . Samples were rinsed in PBS and incubated with fluorochrome-conjugated GAH-IgG or GAR-IgG for 30 min at 37°C . The samples were rinsed and mounted on glass slides in 50% glycerol . For double immunofluorescence, samples were simultaneously incubated with both primary antibodies . The samples were then rinsed and incubated sequentially with GAH-IgG-rhodamine with 1% normal rabbit serum, followed by GAR-IgG-fluorescein with 1 % normal human serum . Photographs were taken with a Zeiss microscope using Tri-X film (Kodak) . Acknowledgments We thank Drs . R . Angerer, M . Gorovsky, and L . Parysek for critical reading of the manuscript. Taxol was a generous gift from the Natural Products Division, National Cancer Institute . This work was supported by grants from the National Institutes of Health and ACS to J . B. 0. The costs of publication of this article were defrayed in part by the payment of page charges . This article must therefore be hereby marked "advertisement" in accordance with 18 U .S.C . Section 1734 solely to indicate this fact. Received June 17, 1983 ; revised July 29, 1983 References Adolph, K . W ., Cheng, S . M ., Paulson, J . R ., and Laemmli, U . K. (1977). Isolation of a protein scaffold from mitotic HeLa cell chromosomes . Proc. Natl . Acad . Sci . USA 74, 4937-4941 . Berezney, R ., and Buchholtz, L. A . (1981) . Dynamic association of replicating DNA fragments with the nuclear matrix of regenerating liver . Exp . Cell Res . 132, 1-13 . Berezney, R ., and Coffey, D . S . (1974) . Identification of a nuclear protein matrix . Biochem . Biophys . Res . Commun . 60, 1410-1417 . Brenner, S ., Pepper, D ., Berns, M. W., Tan, E., and Brinkley, B . R . (1981) . Kinetochore structure, duplication, and distribution in mammalian cells : analysis by human autoantibodies from scleroderma patients . J . Cell Biol. 91,95-102 . Capco, D . G ., Wan, K . M ., and Penman, S . (1982). The nuclear matrix: three-dimensional architecture and protein composition . Cell 29, 847-858. Douvas, A. S ., Stumph, W . E., Reyes, P ., and Tan, E . M . (1979) . Isolation and characterization of nuclear ribonucleoprotein complexes using human antinuclear ribonucleoprotein antibodies . J. Biol . Chem . 254, 3608-3616 . Fritzler, M . J., and Kinsella, T . D. (1980) . The CREST syndrome: a distinct serologic entity with anticentromere antibodies . Am . J . Med . 69, 520-526. Gould, R . R., and Borisy, G. G. (1978) . Quantitative initiation of microtubule assembly by chromosomes from chinese hamster ovary cells . Exp. Cell Res . 113, 369-374 . Hardin, J . A., Rahn, D. R ., Shen, C ., Lerner, M . R., Wolin, S. L., Rosa, M . D ., and Steitz, J. A. (1982) . Antibodies from patients with connective tissue diseases bind specific subsets of cellular RNA-protein particles . J . Clin . Invest. 70, 141-147. Hendrick, J. P ., Wolin, S . L ., Rinke, J ., Lerner, M . R., and Steitz, J. A . (1981). Ro scRNP are a subclass of La RNP : further characterization of the Ro and La small ribonucleoproteins from uninfected mammalian cells . Mol . Cell . Biol . 1, 1138-1149. Kuriyama, R ., and Borisy, G . G . (1981) . Microtubule-nucleating activity in chinese hamster ovary cells is independent of the centriole cycle but coupled to the mitotic cycle. J. Cell Biol . 91, 822-826 .
Human Anticentromere Antibodies 339
Laemmli, U. K . (1970) . Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature 227, 680-685 . Lerner, M. R ., and Steitz, J . A . (1979) . Antibodies to small nuclear RNAs complexed with proteins are produced by patients with systemic lupus erythematosus . Proc . Nat . Acad . Sci . USA 76, 5494-5499 . Lerner, M. R ., and Steitz, J. A. (1981). Snurps and scyrps . Cell 25, 298300. Lerner, M. R., Boyle, J. A ., Mount, S . M., Wolin, S . L., and Steitz, J. A . (1980) . Are snRNPs involved in splicing? Nature 283, 220-224 . Lerner, M . R ., Boyle, J . A ., Hardin, J . A ., and Steitz, J . A. (1981 a) . Two novel classes of small ribonucleoproteins detected by antibodies associated with lupus erythematosus . Science 211, 400-402. Lerner, E . A ., Lerner, M . R ., Janeway, C. A ., and Steitz, J . A . (1981b). Monoclonal antibodies to nucleic acid-containing cellular constituents : probes for molecular biology and autoimmune disease. Proc. Natl . Acad . Sci. 78, 2737-2741 . Long, B. C ., Huang, C .-Y ., and Pogo, A, O . (1979) . Isolation and characterization of the nuclear matrix in Friend erythroleukemia cells : chromatin and hnRNA interactions with the nuclear matrix . Cell 18, 1079-1090 . Matter, L., Schopfer, K., Wilhelm, J . A., Nyffenegger, R . F ., Parisot, R . F., and DeRobertis, E . M . (1982) . Molecular characterization of ribonucleoprotein antigens bound by antinuclear antibodies . Arth . Rheum . 25, 12781283 . Moroi, Y., Peebles, C ., Fritzler, M ., Steigerwald, J ., Tan, E. M . (1980) . Autoantibody to centromere (kinetochore) in scleroderma sera . Proc . Nat . Acad . Sci . USA 77, 1627-1631 . Moroi, Y ., Hartmen, A ., Nakane, P ., Tan, E . (1981). Distribution of kinetochore (centromere) antigen in mammalian cell nuclei . J . Cell Biol . 90, 254259. O'Farrell, P . Z ., Goodman, H . M ., and O'Farrell, P . H . (1977) . High resolution two-dimensional electrophoresis of basic as well as acidic proteins . Cell 12,1133-1142 . Pepper, D. A., and Brinkley, B . R . (1979) . Microtubule initiation at kinetochores and centrosomes in lysed mitotic cells . Inhibition of site-specific nucleation by tubulin antibody . J . Cell Biol . 82, 585-591 . Rieder, C . L . (1979). Ribonucleoprotein staining of centrioles and kinetochores in newt lung cell spindles . J . Cell Biol. 80, 1-9 . Robinson, S . I ., Nelkin, B. D ., and Vogelstein, B . (1982) . The ovalbumin gene is associated with the nuclear matrix of chicken oviduct cells . Cell 28, 99-1065 . Sloboda, R . D., Dentler, W . L ., and Rosenbaum, J . L . (1976) . Microtubule associated proteins and the stimulation of tubulin assembly in vitro . Biochemistry 15, 4497-4505 . Snyder, J. A ., and McIntosh, J . R. (1975) . Initiation and growth of microtubules from mitotic centers in lysed mammalian cells . J . Cell Biol . 67, 744760 . Spandidos, D. A ., and Siminovitch, L. (1977) . Transfer of anchorage independence by isolated metaphase chromosomes in hamster cells . Cell 12,675-682, Stubblefield, E ., and Wray, W . (1971) . Architecture of the Chinese hamster metaphase chromosome. Chromosoma 32, 262-294 . Tan, E . M ., Rodnan, G . P ., Garcia, I ., Moroi, Y ., Fritzler, M ., and Peebles, C . (1980). Diversity of antinuclear antibodies in progressive systemic sclerosis. Anti-centromere antibody and its relationship to CREST syndrome . Arth . Rheum . 23, 617-625. Telzer, B . R ., and Rosenbaum, J. L. (1979) . Cell cycle-dependent, in vitro assembly of microtubules onto the pericentriolar material of HeLa cells . J . Cell Biol . 81, 484-497 . Towbin, H ., Staehelin, T ., and Gordon, J . (1979) . Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets : procedure and some applications . Proc . Nat . Acad . Sci . USA 76, 4350-4354 . Van De Water, L., Guttman, S. D ., Gorovsky, M . A ., and Olmsted, J . B . (1982). Production of antisera and radioimmunoassays for tubulin . Meth . Cell Biol . 24A, 79-96. Van Eekelen, C ., and Van Venrooij, W . (1981) . hnRNA and its attachment to a nuclear protein matrix . J. Cell Biol . 88, 554-563 .
Waring, G . L ., Allis, C . D ., and Mahowald, A . P. (1978) . Isolation of polar granules and the identification of polar granule-specific protein . Dev . Biol . 66,197-206. Warner, J . R . (1979) . Distribution of newly formed ribosomal proteins in HeLa cell fractions. J. Cell Biol . 80, 767-772 . Will, H ., and Bautz, E . K. (1981) . Immunological identification of a chromocenter associated protein in polytene chromosomes of Drosophila . Exp. Cell Res. 125, 401-410 . Yang, V. W ., Lerner, M . R ., Steitz, J . A ., and Flint, S . J. (1981). A small nuclear ribonucleoprotein is required for splicing of adenoviral early RNA sequences. Proc. Nat. Acad. Sci . USA 78, 1371-1375 .
0092-8674/83/110331-09 $02 .00/0
Human Anticentromere Antibodies: Distribution, Characterization of Antigens, and Effect on Microtubule Organization John V. Cox,* Eric A. Schenk,t and J. B . Olmsted* *Department of Biology tDepartment of Pathology University of Rochester Rochester, New York 14627
Summary Properties of human anticentromere .autoantibodies were analyzed . In intact cells or isolated cell fractions, these sera stain the centromeres of mitotic chromosomes and discrete speckles (prekinetochores) in nuclei. Staining is also retained in matrix preparations from nuclei or chromosomes . Immunoprecipitation or immunoblotting demonstrates protein antigens of 14, 20, 23, and 34 kd in HeLa nuclei and chromosomes; immunoprecipitates of nuclei also contain a protein of 15 .5 kd . Matrix preparations contain only the 20, 23, and 34 kd species . Absorption of the anticentromere serum with any one of the four nuclear antigens immobilized on nitrocellulose is sufficient to eliminate centromere staining . Using a lysed cell model for microtubule nucleation, anticentromere sera are shown to inhibit specifically the organization of microtubules at the kinetochore . Introduction Light and electron microscopic studies have shown that the kinetochore of chromosomes is involved in the attachment of spindle fiber microtubules to chromosomes during mitosis . However, the nature of this interaction is still very poorly understood, partly because of lack of information on the biochemical composition of the kinetochore . The recent discovery that sera from patients having the CREST variant of scleroderma stain the centromere region of mitotic chromosomes and discrete speckles within interphase nuclei (Fritzler and Kinsella, 1980 ; Moroi et al ., 1980 ; Tan et al ., 1980) has made it possible to study the organization of this region in more detail . Immunoelectron microscopic studies (Brenner et al ., 1981) have established that one anticentromere serum stains the inner and outer plates of the trilaminar kinetochore of mitotic chromosomes . This and another study (Moroi et al ., 1981) have shown that these sera stain diffuse foci within the nucleus of interphase cells, and have also indicated that the location of these regions relative to the nuclear envelope may vary among cell types . Synchrony experiments have demonstrated that the interphase speckles (prekinetochores) double during late G2 of the cell cycle, and these then segregate with daughter chromosomes during anaphase (Brenner et al ., 1981) . Studies in which enzymatic digestion has been used to determine which treatments abolish immunofluorescent staining patterns have suggested that at least some of the antigenic
determinants with which these antibodies react are protein in nature (Moroi et al ., 1980). In the studies described in this report, we present the first evidence that antigens recognized by an anticentromere serum are a discrete set of proteins . Some of these proteins are also components of nuclear and chromosomal matrix preparations . In addition, we demonstrate that these anticentromere sera can selectively block microtubule organization at one of the two mitotic microtubule-organizing centers, the kinetochore . Results Distribution of Anticentromere Staining Of 50 ANA (antinuclear antibody) antisera we screened, five were identified as anticentromere by immunofluorescent staining of PtK 1 and HeLa cells and isolated HeLa chromosomes . The distribution of the antigens recognized by two of these sera, cen-1 and cen-2, at various stages of the PtK, cell cycle is illustrated in Figure 1 . The anticentromere fluorescence is localized to a pair of spots per mitotic chromosome at prophase (Figures 1 b and l e), while only a single spot per chromosome is found at anaphase (Figures 1 c and 1f) . These antigens were also found in isolated Hela chromosomes (Figures 2a and 2b), where the staining can be more precisely localized to the region of the primary constriction . Double immunofluorescent staining of a metaphase PtK, cell with cen-1 and tubulin antibodies indicates that spindle fibers terminate at the region of the chromosome stained by cen-1 (Figures 1 g and 1 h) . In addition to the localization of these antigens at the primary constriction of mitotic chromosomes, they were maintained as discrete speckles (prekinetochores; Brenner et al ., 1981), in interphase nuclei of PtK 1 cells (Figures 1 a and 1 d) and in isolated Hela nuclei (Figures 2c and 2d) . All mammalian cell lines thus far examined (Hela, 3T3, BHK, L, neuroblastoma, and Indian muntjac cells) show the same pattern of nuclear fluorescence when stained with cen-1 and cen-2 sera as that described for the marsupial PtK, cells . Effect of Anticentromere Sera on Microtubule Organization A lysed cell model was developed to examine the effect of the anticentromere sera on microtubule organization . PtK 1 cells grown on coverslips were gently lysed in buffer containing 2 mM CaCl 2 to break down preexisting microtubules . Free Ca' was then chelated with EGTA and the cells were incubated with phosphocellulose-purified porcine brain tubulin . By employing purified brain tubulin at a concentration of 1 mg/ml, it was possible to examine the addition of tubulin subunits onto cellular nucleation sites, such as centrosomes and kinetochores, in the absence of any significant self-assembly of tubulin into microtubules . After incubation with tubulin, the cells were fixed and the microtubule distribution assayed by immunofluorescent staining with tubulin antibodies . Initiation of microtubules
Cell 332
Figure 1. Cell-Cycle
Distribution
of Anti-Centromere
Staining
PtK, cells were stained with ten-I (a-c,g) or ten-2 (d-f) at various stages of the cell cycle. (a) interphase (d) interphase (860x); (e) prophase (1075X); (f) anaphase (1100X). (g and h) double immunofluorescent 1 (g) and tubulin (h) antibodies (1300X).
from the centrosome region was seen in 15% of interphase cells as compared to greater than 90% of mitotic cells, In neither case were there significant numbers of microtubules assembled randomly in the cytoplasm. Approximately 50% of the mitotic cells showed organization of microtubules from the centromere region of several chro-
(600X); (b) prophase (1220X); (c) anaphase. (950x); staining of a metaphase RK, cell stained with cen-
mosomes. As can be seen in Figures 3a and 3b, double immunofluorescence staining with tubulin and ten-I antibodies served to identify the region of the chromosomes from which microtubules initiated. To determine whether the anticentromere sera would interfere with microtubule formation on chromosomes,
Human Anticentromere 333
Antrbodres
Frgure 2. lmmunofluorescent Nuclei
Stainrng of isolated HeLa Chromosomes
and
HeLa chromosomes and nucler were isolated as described in Experimental Procedures and stained with ten-I. (a and b) phase and fluorescence micrographs of isolated HeLa chromosomes (1675x). (c and d) phase and fluorescence mrcrographs of an Isolated HeLa nucleus (1960X).
lysed cells were incubated with IgG from ten-1 or ten-2 for 30 min at room temperature prior to incubation with tubulin. After incubation with tubulin and fixation, cells preincubated with ten-1 or ten2 were stained with tubulin antibodies and the appropriate fluorochrome-conjugated second antibodies, By scoring only those cells that were positive for anticentromere fluorescence, one could be assured that any effect on microtubule initiation at the kinetochore region was due to the presence of the antibody at this site prior to incubation with tubulin. As shown in Figures 3c and 3d, initiation of microtubules from chromosomal kinetochores was inhibited by ten-I ; similar results were obtained with ten-2 serum. Only 10% of the cells preincubated with either centromere antiserum showed microtubule initiation at the kinetochore, as compared to 50% of the cells preincubated with normal human sera (Figures 3a and 3b) or buffer. The anticentromere sera had no effect on the initiation of microtubules from mitotic or interphase centrosomes. In addition, when human sera containing noncentromeric ANA were employed in this assay, there was no effect upon initiation of microtubules from centrosomes or the kinetochore region. These results indicate that both ten-1 and ten-2 sera inhibit the organization of microtubules at the kinetochore in this lysed cell system.
Distribution of ten-2 Antigens in Matrix Preparations To determine the subnuclear distribution of the antigens, a number of fractionation procedures were investigated. Using previously published methods for the preparation of nuclear matrix fractions (Adolph et al., 1977; Capco et al., 1982) we established that matrices prepared from HeLa cells metabolically labeled with 3H-thymidine, 3H-uridine, or 35S-methionine contained 2.5% of the DNA, 0.2% of the
RNA, and 15%-20% of the protein of the intact cell. When nuclear matrices prepared from PtK, cells by either Procedure 1 (Figure 4a) or Procedure 2 (data not shown) were stained with ten-2 serum, a pattern similar to that observed for intact interphase cells (Figure Id) was obtained. In addition, the number of spots/nucleus in matrix preparations of either isolated HeLa nuclei (average of 43/nucleus for 14 cells) or PtK, cells (average of 12,5/nucleus for 12 cells) was the same as that seen in intact cells and, as had been found previously (Moroi et al., 1980; Brenner et al., 1981) corresponded closely to the normal chromosome number for the respective cell lines. Analyses were also performed using mitotic cells to determine whether the antigens were retained in extracted chromosomes. However, following extraction, chromosomes were no longer visible by phase microscopy, and it was difficult to ascertain whether punctate fluorescent staining with ten-2 was due to specific centromere labeling or background debris. A modified procedure was therefore developed, in which spindle microtubules were stabilized by the inclusion of 5 PM taxol in all extraction buffers, and were used as markers for the location of centromeres. As shown in Figures 4b and 4c, double immunofluorescence staining of mitotic matrices shows spots stained by ten-2 in the region of termini of taxol-stabilized spindle fibers. All of these results indicate that at least some of the antigens recognized by ten-2 are tightly bound components of both interphase and mitotic matrices.
Biochemical Identification of Antigens Further characterization of the antigens recognized by cen2 was carried out by immunoprecipitation of various fractions derived from HeLa cells labeled with 35S-methionine. As shown in Figure 5a, immunoprecipitates from labeled nuclei (lane 3) and chromosomes (lane 6) contain proteins of 14, 20, 23, and 34 kd, although the 34 kd protein is only faintly visible in the precipitate from chromosomes. A protein of 15.5 kd is also seen in the nuclear immunoprecipitate. The proteins of higher molecular weight are also in immunoprecipitates obtained using normal human sera (Figure 5a, lane 4) or human sera containing noncentromerit ANA (data not shown), and are nonspecifically bound proteins. lmmunoprecipitates of matrix preparations (Figure 5b) from interphase (lane 2) and mitotic cells (lane 4) also contain proteins of 20, 23, and 34 kd. However, the 14 and 15.5 kd proteins are almost entirely absent. lmmunoprecipitates of matrices prepared from isolated nuclei (lane 6) and isolated chromosomes (lane 8) show the same proteins as seen in matrices from intact cells; the 34 kd protein is again present in reduced amounts in matrices prepared from isolated chromosomes. Two-dimensional gel analysis of immunoprecipitates from 35S-labeled HeLa nuclei shows that the proteins range in isoelectric point from 5.2 to 5.9, and that the 20 and 23 kd proteins exist in at least one modified state (Figure 5~). As shown in Figure 5d, an immunoprecipitate obtained from 3’P-labeled nuclei shows that the 14, 15.5, 20, and 23 kd proteins are all phosphorylated.
Cell 334
Figure 3. Lysed Cell Model Double immunofluorescent staining of RK, cells that have been lysed under the conditions described in the text and incubated tubulin. (a and b) Control cell was lysed and incubated with phosphocellulose-purified tubulin and then fixed and stained antrbodies. Note that individual foci of tubulin nucleation correspond to the region of centromere staining (arrows, 925x). (c and with ten-1 prior to incubation with phosphocellulose-purified tubulin. Note the lack of nucleation from centromeres while polar (1540X).
Figure 4. Interphase
with phosphocellulose-purified with ten-I (a) and tubulin (b) d) Cells that were preincubated nucleation appears unaffected
and Mitotic Matrices
(a) Nuclear matrices from PtK, cells were prepared according to Procedure 1 described in Experimental Procedures and stained with ten-2. Note speckles that appear identical to those seen in nuclei of intact cells (Figure Id) (1320x). (b and c) Double immunofluorescent staining of a PtK, cell that has been prepared in the same manner as Figure 4a except for the inclusion of 5 WM taxol in all steps of the protocol. The cell was then stained with ten2 (b) and tubulin (c) antibodies. As can be seen in Figure 4c. a subset of spindle microtubules is stabilized and the anticentromere fluorescence is located in the region of the termini of stabilized fibers. The hash mark in the upper right hand corner of Figures 4b and 4c provides a reference point for comparing the double immunofluorescent pair (1050X).
lmmunoprecipitates obtained from 35S-iabeled nuclei of various cell types, including L, PtK,, neuroblastoma, 3T3, and Indian muntjac cells, also contain the 14, 15.5, 20, and 23 kd proteins (Figure 6) and indicate that these proteins are conserved in other mammalian cell lines.
However, the 34 kd protein found in HeLa cells is not precipitated from these cell lines. The antigens reacting with ten-2 were also examined by immunoblotting. As shown in Figure 7, proteins in HeLa nuclear fractions of 14, 20, 23, and 34 kd proteins are
Human Anticentromere 33.5
Antrbodres
Figure 6. lmmunoprecrprtates
from Nucler of Varrous Cell Types
Nuclei were isolated from cell lines labeled wrth “S-methronrne, and proteins rmmunoprecipitated wrth ten-2: lndran muntjac (lane 1); L (lane 2); PtK, (lane 3); neuroblastoma (lane 4); 3T3 (lane 5).
recognized by the ten-2 serum; this is the same pattern as seen by immunoprecipitation. However, the 15.5 kd protein seen by immunoprecipitation is rarely seen on immunoblots. Additional proteins of 70 and 90 kd were also labeled on the immunoblots. lmmunoblots using normal human sera or sera from patients with noncentromeric ANA showed no reaction with nuclear proteins from HeLa, or they generated a pattern distinct from that obtained with ten-2. To determine whether the proteins that react with the ten-2 serum by immunoblotting and immunoprecipitation corresponded to the antigens localized at the kinetochore region of chromosomes and the prekinetochores of interphase nuclei, absorption of the ten-2 serum was carried out using proteins immobilized on nitrocellulose. Proteins from isolated HeLa nuclei were separated on a preparative SDS slab gel and transferred to nitrocellulose paper. A vertical strip of this blot was probed with ten-2, generating a pattern like that shown in Figure 7, and used for orientation The regions in the remainder of the blot corresponding to the individual proteins of 14, 20, 23, 34, and 90 kd were then cut out and incubated with the ten-2 serum prior to staining F’tK, cells. A 2 mm band at -150 kd, where no antiserum reaction was seen, was used as a control for
Figure 5. lmmunoprecrpitates
from HeLa Cell Fractions
Autoradrograms of tmmunoprecipitates of fractions from HeLa cells labeled with %methionine (a-c) or zP-orthophosphate (d). (a and b) One-dimensronal SDS gels of total %-labeled proterns and rmmunopreciprtates obtarned using ten-2 are shown for nuclei (5a, lanes 2-3), chromosomes @a, lanes 5-6) matrix from interphase cells (5b. lanes l-2) matrix from mrtotic cells (5b. lanes 3-4). matrix from isolated nuclei (5b, lanes 5-6) and matrix
from isolated chromosomes (5b, lanes 7-8). An immunoprecrpitate from Isolated nuclei using a normal human serum (5a, lane 4) illustrates the nonspecrfically bound proteins observed with the human sera tested. 5a. lane 1: “C-molecular weight markers: myosin, 200 kd; phosphorylase 6, 92.5 kd; BSA. 68 kd; ovalbumin, 43 kd; a-chymotrypsrnogen. 25.7 kd: plactoglobukn, 18.4 kd; cytochrome-c. 12.3 kd. Dashes indicate positron of antigens referred to in the text. (c) Two-dimensronal NEPHGE gel of proteins rmmunoprecrpitated wrth ten-2 from %labeled nucler. Note multiple rsoelectnc pornts for the 20 and 23 kd proterns. Bars at the top of the gel Indicate pH gradient in units of 1 .O. (d) One-drmensional SDS gel of proteins rmmunoprecipitated wrth ten-2 from %labeled nucler.
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Figure 7. Western
Blot of Isolated HeLa Nuclei Probed with cen9
Proteins from Isolated HeLa nuclei were run on a 1O%-25% polyacrylamide gradient gel, electrophoretically transferred to nitrocellulose paper, and probed with ten-2. As described in the text, specific bands (lines) or a control region of the blot (bracket) were used for absorption of ten-2 prior to immunofluorescent staining. The right-hand panel indicates the regions that absorbed (+) or had no effect on (-) centromere staining.
nonspecific binding of the antibody to nitrocellulose. As listed in Figure 7, the proteins of 14, 20, 23, and 34 kd eliminated the centromere staining pattern in isolated chromosomes or intact cells, whereas the protein at 90 kd and the control strip had no effect on the staining pattern.
Discussion Human autoantibodies have been useful probes for studying the molecular composition (Douvas et al., 1979; Lerner and Steitz, 1979; Hendrick et al., 1981; Lerner and Steitz, 1981; Lerner et al., 1981b; Hardin et al., 1982; Matter et al., 1982) and possible function (Lerner et al., 1980; Yang et al., 1981) of various small nuclear and cytoplasmic RNPs. In many instances, the antigenic determinants recognized by these sera are complex. However, autoantibodies can provide a means of investigating an organelle that cannot otherwise be isolated or assayed for function. Thus studies with human anticentromere antibodies have demonstrated that antigens reacting with these sera remain as discrete structures during interphase and duplicate in late G2 (Brenner et al., 1981). We have used these sera in a lysed cell system to determine whether the antibodies react with sites at the kinetochore that are important in the organization of microtubules. The protocols described previously (Pepper and Brinkely, 1979) were modified slightly in order to eliminate formation of random microtubules. Under the conditions employed, 15% of interphase centrosomes were competent to initiate microtubule assembly. This is consistent with previous results that indicate no (Telzer and Rosenbaum, 1979) or low (Snyder and McIntosh, 1975; Kuriyama and Borisy, 1981) initiating activity for interphase centro-
somes. However, nucleation of microtubules occurred at approximately 90% of mitotic centrosomes, in agreement with previous studies (Snyder and McIntosh, 1975; Telzer and Rosenbaum, 1979; Kuriyama and Borisy, 1981) that indicate that centrosome-nucleating activity increases at the onset of mitosis. The kinetochore region of chromosomes initiated microtubule assembly in 50% of the mitotic cells that were scored. Gould and Borisy (1978) have shown that 55% of isolated CHO chromosomes initiated microtubule assembly from kinetochores when concentrations of tubulin similar to those used in our study were employed. By using double immunofluorescence analyses with the lysed cell model, it was possible to determine if ten-I or ten-2 sera have any significant effect on microtubule initiation. Preincubation of lysed cells with either serum prior to incubation with tubulin specifically inhibited microtubule initiation in the region of chromosomal kinetochores, but had no effect on the initiation activity of mitotic or interphase centrosomes. EM analyses (Pepper and Brinkley, 1979) demonstrated that tubulin antibodies were capable of blocking the assembly of microtubules from both centrosomes and kinetochores in a lysed cell model. However, this is the first report of an antiserum that selectively inhibits microtubule assembly from only one mitotic organizing site, the kinetochore. Although the mechanism remains to be elucidated, this inhibition could be due to one or more of the following events: the antibodies may react directly with proteins involved in microtubule initiation at the kinetochore; the binding of the antibody may steritally block the accessibility of tubulin to the kinetochore, while not binding directly to structures mediating tubulin function; or the binding of the antibody may alter the conformation of the kinetochore such that microtubule initiation is inhibited. The immunoprecipitation and immunoblotting data indicated that the ten-2 antiserum reacts with at least four proteins (14, 20, 23, and 34 kd) in isolated chromosomes and nuclei. Absorption of the serum with any of these four molecular weight species immobilized on nitrocellulose caused loss of staining, suggesting that the antibody recognizes determinants shared by the four proteins. The complexity of the antigens recognized by ten-2 is not unlike that observed for human autoantibodies associated with systemic lupus erythematosus. The Sm antigen has been shown to be made up of five snRNAs, each of which is complexed with seven proteins; however, the antigenic determinant is known to be localized on only one of these proteins (Lerner et al., 1981a). The interaction of Ul RNA with the same seven proteins mentioned above is thought to comprise the RNP antigenic determinant (Lerner and Steitz, 1981). Both the Sm and RNP antigens are highly conserved and abundant in most mammalian cells (Hendrick et al., 1981). Like these snRNP antigens, the 14, 15.5, 20, and 23 kd proteins recognized by ten-2 are conserved in the mammalian cell lines we examined. Previous cytochemical analyses have shown that an RNP
Human Anticentromere Antibodies 337
component is present at the kinetochore of chromosomes (Rieder, 1979) . The possibility that the proteins recognized by cen-2 comprise a portion of an RNP or interact with specific regions of centromeric heterochromatin remains to be analyzed . Two-dimensional gel analysis has shown that the proteins recognized by cen-2 have an acidic isoelectric point and are thus distinct from the bulk of protein associated with chromatin . An acidic protein of approximately 38 kd has been identified in Drosophila (Will and Bautz, 1981) and an antiserum against this protein has been shown to stain the chromocenter of salivary gland chromosomes, as well as metaphase chromosomes from Hela cells . This protein may be homologous to the 34 kd protein that is recognized by cen-2 . We have also shown that the 20, 23, and 34 kd proteins are components of matrices from interphase and mitotic Hela cells ; these antigens comprise only a minor fraction of the proteins found in these preparations . Although the bulk of the DNA, RNA, and protein have been extracted from these matrices, the staining pattern obtained with cen-2 is the same as that seen in intact cells, suggesting that both kinetochores and prekinetochores are very stable structures . Our through-focus analyses of the distribution of prekinetochores in nuclei and nuclear matrices from PtK, and HeLa cells, as well as the EM observations of Brenner et al . (1981), indicate a nuclear location for these proteins distinct from the nuclear pore-lamina complex . We have shown that preparations of mitotic cells extracted in a manner similar to that used in obtaining chromosomal scaffolds (Adolph et al ., 1977) exhibit the same distribution of these proteins as seen in intact cells . The finding that the localization of anticentromere antigens is unchanged in mitotic matrices suggests that the matrix or scaffold may be important in maintaining the organization of this region in mitotic cells . A number of biochemical studies have suggested that the matrix serves as a nuclear substructure important in the organization, replication, and transcription of interphase chromatin (Berezney and Coffey, 1974 ; Long et al ., 1979 ; Berezney and Buchholtz, 1981 ; Van Eekelen and Van Venrooij, 1981 ; Robinson et al ., 1982) . The manner in which proteins recognized by centromere antisera are related to others defined as biochemical components of matrices is as yet undetermined . However, our findings do suggest that cen-2 antigens are involved in the organization of chromosomes throughout the cell cycle . With the exception of the 15 .5 kd protein, the composition of the antigens recognized by cen-2 are the same in both isolated HeLa nuclei and chromosomes . However, the region with which these proteins are associated undergoes major morphological rearrangement in changing from the diffuse foci of interphase nuclei to the trilaminar kinetochores of mitotic chromosomes . Concomitant with this transition, the kinetochore also becomes competent to interact with microtubules of the mitotic spindle . It will be interesting to determine whether the phosphorylation of
the proteins recognized by cen-2 is involved in the reorganization of chromosomes at the kinetochore and the ability of this site to interact with microtubules during mitosis . Experimental Procedures Cell Culture Monolayer cultures of Hela S3, PtK,, neuroblastoma, L, 3T3, BHK, and Indian muntjac cells were grown at 37°C in F12 with 10% fetal calf serum . Cultures were labeled with 35S-methionine, 3H-thymidine, 3H-uridine or 32 Porthophosphate at approximately 10 µCi/ml for 36 hr in order to obtain labeled cell fractions . Antisera Human sera were obtained from the Department of Pathology, University of Rochester. Sera that had been scored as ANA (antinuclear antibody)positive on frozen sections of tissue were examined further by immunofluorescence microscopy of cultured cells . The antisera used in this report, cen-1 and cen-2, represented individual aliquots of nonpooled serum obtained from two different patients diagnosed as having the CREST variant of scleroderma. Additional normal human IgG was the gift of Dr . George Abraham, Department of Immunology and Microbiology, University of Rochester . IgG fractions were prepared from the sera by taking the void volume fraction from an Affigel Blue DEAE column (BioRad) . Tubulin antisera were prepared and characterized as described previously (Van De Water et al ., 1982) . Chromosome Isolation Chromosomes were isolated from HeLa S3 cells using slight modifications of previously published methods (Stubblefield and Wray, 1971 ; Spandidos and Siminovitch, 1977) . Mitotic cells were accumulated by incubating cultures in 60 ng/ml nocodazole overnight, and were harvested by sedimentation at 450 xg for 5 min. All subsequent steps were performed at 04°C unless otherwise noted . The resulting cell pellet was resuspended in 75 mM KCI and incubated for 5 min . The cells were centrifuged at 450 xg for 3 min and the pellet was resuspended in HCP buffer (1 M hexylene glycol, 0 .5 mM CaCl2 , 0 .1 mM PIPES, pH 6 .94) . The cells were again pelleted and resuspended in HCP + 0 .5% NP-40 and incubated at room temperature for 10 min . The cells were then sheared four times with a 22 gauge needle and the lysate centrifuged at 50 xg for 2 min . In some experiments, chromosomes from the suspension were allowed to settle onto poly-L-lysine-coated coverslips for immunofluorescence microscopy . Alternatively, the supernatant was centrifuged at 800 xg for 10 min . The resulting chromosomal pellet was resuspended in 10 mM Tris-HCI, 1 mM MgCl2 (pH 7 .4) containing 50 ag/ml DNAase I and incubated on ice for 1 hr for immunochemical assays . Nuclei Isolation Nuclei were isolated from the various cell types using the procedure of Warner (1979) . The final nuclear pellet was resuspended in 10 mM TrisHCI, 1 mM MgCI2 (pH 7.4) . These nuclei were allowed to settle onto polyL-lysine-coated coverslips for immunofluorescence microscopy or digested with 50 µg/ml DNAase I for 1 hr on ice for immunochemical assays . Nuclear and Chromosomal Matrix Isolation Procedure 1 : Matrices were prepared from interphase and mitotic HeLa and PtK, cells according to the procedure of Capco et al . (1982). The cells were washed in phosphate-buffered saline (PBS) and pelleted by centrifugation at 450 xg for 5 min . The pellet was resuspended in CSK buffer (10 mM PIPES, pH 6 .8, 250 mM (NH4)2SO4 , 300 mM sucrose, 3 mM MgCl2 , 1 .2 mM PMSF, 0.5% Triton X-100), incubated on ice for 3 min, and washed once in digestion buffer (10 mM PIPES, pH 6 .8, 50 mM NaCl, 300 mM sucrose, 3 MM MgCl 2, 1 .2 mM PMSF, 0 .5% Triton X-100) . The pellet was then resuspended in digestion buffer containing 200 µg/ml DNAase I and 30 µg/ml RNAase A and incubated at RT for 20 min. This material was then pelleted at 450 xg for 5 min, resuspended in CSK, and incubated for 5 min at RT. The final pellet was then washed in 10 mM Tris-HCI (pH 7 .4) and prepared for immunoprecipitation .
Cell 3 38
Procedure 2 : Interphase and mitotic cells were washed in PM buffer (0 .1 M PIPES, 0.5 mM MgC1 2, pH 6 .94), lysed in PM with 0 .25% Triton X-100, and then incubated in PM containing 200,ug/ml DNAase I and 30,ug/ml RNAase A for 20 min at RT . The cells were pelleted and resuspended in 10 mM Tris-HCI (pH 9 .0), 2 .0 M NaCl, 10 mM EDTA, 0 .1% NP-40 (Adolph et al ., 1977) and incubated on ice for 10 min . The suspension was centrifuged at 450 xg for 5 min and the pellet prepared for immunoprecipitation . Matrices prepared for immunofluorescence microscopy were treated as described in Procedure 1 or 2 above, except cells remained attached to coverslips throughout the protocols . For determination of the percent DNA, RNA, or protein remaining in matrix preparations, cold TCA precipitates from labeled cell fractions were counted . Lysed Cell Models A modification of the procedure of Pepper and Brinkley (1979) was developed to examine the nucleation of microtubules in lysed cells . PtK 1 cells were grown on coverslips and incubated with 60 ng/ml nocodazole for 8 hr to accumulate mitotic cells . Cells were refed with fresh medium without nocodazole and incubated at 37°C for 20 min . Cells were then placed at 4°C, rinsed with PMG (0.1 M PIPES, 0 .5 mM MgC12, 0 .5 mM GTP) and then lysed for 2 min in PMG containing 0 .09% Triton X-100 and 2 .0 mM CaCl2 . Cells were washed twice in PMG with 2 mM CaCI2 (2 min/wash), and then incubated in PMG with 1 mM EGTA for 30 sec . Cells were rinsed in PMG, returned to 37°C, and incubated for 15-20 min with phosphocellulosepurified porcine brain tubulin (1 mg/ml) (Sloboda et al ., 1976) in PM with 2 mM GTP. In some experiments, lysed cells were incubated with anticentromere or normal human sera prior to incubation with purified tubulin (see Results) . Cells were then fixed in 3 .7% formaldehyde in PMG for 15 min and processed for immunofluorescence microscopy . Cells were scored positive for kinetochore microtubule initiation if two chromosomes exhibited microtubule formation ; the majority of cells showed initiation from 4-6 chromosomes/cell . Cells initiating microtubules from a single kinetochore were rare, and inclusion of these data did not alter the results for control or experimental samples . However, due to the presence of tripolar cells in the population, and the possible ambiguity in scoring initiation from a single kinetochore, these cells were routinely scored as negative . Gel Electrophoresis Cell fractions were analyzed on one- or two-dimensional gels . Nonequilibrium isoelectric focusing gels (NEPHGE) were prepared according to O'Farrell et al . (1977), as modified by Waring et al. (1978); 2 .0% of pH 211 ampholytes were used . Separation of proteins on SDS gels (Laemmli, 1970) was carried out using 10%-25% polyacrylamide gradient gels . Immunological Procedures Immunoprecipltatlon Cells were labeled with 10 µCi/ml °S-methionine for 36 hr. Various cell fractions were isolated as described above and solubilized by sonication in DOC buffer (0 .1 M Tris-HCI, pH 8 .3, 0 .1% SDS, 0 .5% NP-40,0.5% DOC, 2 mM EDTA, and 0 .25% gelatin) . Samples were then incubated with 1015 µl of IgG (1 mg/ml) per 500 µl of solution for 2 hr at 37°C. Immune complexes were precipitated with protein A-Sepharose 4B beads (15 µl packed beads/10 µg IgG) . The beads were washed five times with DOC buffer (2 ml/wash) and once in 0 .1 M Tris-HCI, pH 8.3 . Immune complexes were released from the beads by boiling in SDS sample buffer (Laemmli, 1970) and run on 1- or 2-D gels . After fixing in glacial acetic acid for 30 min, gels were impregnated with 20% PPO in glacial acetic acid for 1 hr, rinsed in H 2O for 1 hr, dried, and autoradiographed. Immunoblotting Electrophoretic transfer and immunoblotting on nitrocellulose paper was performed according to the procedure of Towbin et al . (1979). Blots were incubated with cen-2 for 6-8 hr at RT, washed, and incubated with affinitypurified GAH-IgG coupled with peroxidase for 2 hr at RT . Following washing, antibody binding was detected by reacting peroxidase with the following mixture: 20 ml of 3 mg/mI 4-chloro-l-naphthol in MeOH ; 36 µl of 50% H20 2; 100 ml of 10 mM Tris-HCI, pH 7 .4 . lmmunoabsorption The protein from -107 isolated HeLa nuclei was separated on a 10%25% preparative SDS gel and transferred to a 9 X 9 cm sheet of nitrocellulose as described above . Vertical strips (5 mm) from either end of the
blot were probed with cen-2 and the pattern that was generated was used for orientation . A 2 mm band was cut out from the regions corresponding to the proteins of 14, 20, 23, 34, and 90 kd, plus a control strip at -150 kd . Each strip was cut into eight equal pieces, and incubated with 300 µl of a 1/50 dilution of IgG from cen-2 (1 mg/ml) for 4 hr on ice . Immunofluorescence Microscopy Cells grown on coverslips in 35 mM petri dishes or isolated HeLa chromosomes and nuclei attached to poly-L-lysine-coated coverslips were fixed in 3 .7% formaldehyde in PBS for 15 min followed by -20°C acetone for 30 sec . Alternatively, samples were fixed in 0 .2% glutaraldehyde in PBS for 15 min, followed by washing in 1 % Triton X-100 in PBS for 30 min and five changes (3 min each) of 1 mg/ml sodium borohydride in 95% EtOH. Samples were then incubated with one of the human antisera or tubulin antisera for 30 min at 37°C . Samples were rinsed in PBS and incubated with fluorochrome-conjugated GAH-IgG or GAR-IgG for 30 min at 37°C . The samples were rinsed and mounted on glass slides in 50% glycerol . For double immunofluorescence, samples were simultaneously incubated with both primary antibodies . The samples were then rinsed and incubated sequentially with GAH-IgG-rhodamine with 1% normal rabbit serum, followed by GAR-IgG-fluorescein with 1 % normal human serum . Photographs were taken with a Zeiss microscope using Tri-X film (Kodak) . Acknowledgments We thank Drs . R . Angerer, M . Gorovsky, and L . Parysek for critical reading of the manuscript. Taxol was a generous gift from the Natural Products Division, National Cancer Institute . This work was supported by grants from the National Institutes of Health and ACS to J . B. 0. The costs of publication of this article were defrayed in part by the payment of page charges . This article must therefore be hereby marked "advertisement" in accordance with 18 U .S.C . Section 1734 solely to indicate this fact. Received June 17, 1983 ; revised July 29, 1983 References Adolph, K . W ., Cheng, S . M ., Paulson, J . R ., and Laemmli, U . K. (1977). Isolation of a protein scaffold from mitotic HeLa cell chromosomes . Proc. Natl . Acad . Sci . USA 74, 4937-4941 . Berezney, R ., and Buchholtz, L. A . (1981) . Dynamic association of replicating DNA fragments with the nuclear matrix of regenerating liver . Exp . Cell Res . 132, 1-13 . Berezney, R ., and Coffey, D . S . (1974) . Identification of a nuclear protein matrix . Biochem . Biophys . Res . Commun . 60, 1410-1417 . Brenner, S ., Pepper, D ., Berns, M. W., Tan, E., and Brinkley, B . R . (1981) . Kinetochore structure, duplication, and distribution in mammalian cells : analysis by human autoantibodies from scleroderma patients . J . Cell Biol. 91,95-102 . Capco, D . G ., Wan, K . M ., and Penman, S . (1982). The nuclear matrix: three-dimensional architecture and protein composition . Cell 29, 847-858. Douvas, A. S ., Stumph, W . E., Reyes, P ., and Tan, E . M . (1979) . Isolation and characterization of nuclear ribonucleoprotein complexes using human antinuclear ribonucleoprotein antibodies . J. Biol . Chem . 254, 3608-3616 . Fritzler, M . J., and Kinsella, T . D. (1980) . The CREST syndrome: a distinct serologic entity with anticentromere antibodies . Am . J . Med . 69, 520-526. Gould, R . R., and Borisy, G. G. (1978) . Quantitative initiation of microtubule assembly by chromosomes from chinese hamster ovary cells . Exp. Cell Res . 113, 369-374 . Hardin, J . A., Rahn, D. R ., Shen, C ., Lerner, M . R., Wolin, S. L., Rosa, M . D ., and Steitz, J. A. (1982) . Antibodies from patients with connective tissue diseases bind specific subsets of cellular RNA-protein particles . J . Clin . Invest. 70, 141-147. Hendrick, J. P ., Wolin, S . L ., Rinke, J ., Lerner, M . R., and Steitz, J. A . (1981). Ro scRNP are a subclass of La RNP : further characterization of the Ro and La small ribonucleoproteins from uninfected mammalian cells . Mol . Cell . Biol . 1, 1138-1149. Kuriyama, R ., and Borisy, G . G . (1981) . Microtubule-nucleating activity in chinese hamster ovary cells is independent of the centriole cycle but coupled to the mitotic cycle. J. Cell Biol . 91, 822-826 .
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