Mitotic block in HeLa cells by vinblastine: ultrastructural changes in kinetochore-microtubule attachment and in centrosomes

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1 Journal of Cell Science 104, (1993) Printed in Great Britain The Company of Biologists Limited Mitotic block in HeLa cells by vinblastine: ultrastructural changes in kinetochore-microtubule attachment and in centrosomes Kim Livezey Wendell, Leslie Wilson and Mary Ann Jordan Department of Biological Sciences, University of California Santa Barbara, Santa Barbara, CA 93106, USA SUMMARY Previous work from this laboratory has indicated that very low concentrations of vinblastine block HeLa cells at mitosis in the presence of a full complement of microtubules and without major disruption of spindle organization. In the present study we analyzed the structural organization of mitotic spindle microtubules, chromosomes and centrosomes by electron microscopy after incubating HeLa cells for one cell cycle with 2 nm vinblastine. We found that mitotic block of HeLa cells by vinblastine was associated with alterations of the fine structure of the spindle that were subtle but profound in their apparent consequences. The cell cycle was blocked in a stage that resembled prometaphase or metaphase; chromosomes had not undergone anaphase segregation. Neither the structure of the microtubules nor the structure of the kinetochores was detectably altered by the drug. However, the number of microtubules attached to kinetochores was decreased significantly. In addition, the centrosomes were altered; the normal close association of mother and daughter centriole was lost, numerous membranous vesicles were found in the centrosomal region, and many centrioles exhibited abnormal ultrastructure and had microtubules coursing through their interiors. These findings are consistent with our previous results and indicate that inhibition of the polymerization dynamics of mitotic spindle microtubules and perhaps of centriole microtubules, rather than microtubule depolymerization, is responsible for the mitotic inhibition by vinblastine. Key words: vinblastine, mitosis, kinetochore, centriole, centrosome, HeLa INTRODUCTION Vinblastine is a powerful antitumor drug in widespread use in cancer chemotherapy. The mechanism by which vinblastine effects its unusually potent antiproliferative action is by inhibition of mitosis (Jordan et al., 1991), and often has been ascribed to its capacity to depolymerize mitotic spindle microtubules (e.g. Malawista et al., 1968; Wilson and Bryan, 1974). However, in recent studies we found that low concentrations of vinblastine (0.1-6 nm) blocked progression of mitosis from metaphase to anaphase in HeLa cells without causing net depolymerization of microtubules (Jordan et al., 1991, 1992). Some blocked spindles appeared normal in organization and had apparently normal arrangements of microtubules, but in many cells the block induced by low vinblastine concentrations was associated with subtle alterations in the organization of the spindle microtubules, chromosomes and centrosomes. For example, in such cells astral microtubules were more numerous and prominent than those in control cells while the microtubules of the central spindle became shorter. Centrosomes were fragmented, and some chromosomes were stranded or locked at or near the spindle poles and appeared unable to congress to the otherwise compact metaphase plate. Spindle organization became increasingly distorted with increasing drug concentrations. Spindle microtubules are highly dynamic and the dynamics of some spindle microtubules change as the cell progresses through mitosis (Saxton et al., 1984; Mitchison et al., 1986; Mitchison, 1989; Hamaguchi et al., 1987; Gorbsky et al., 1987; Wadsworth et al., 1989; Hayden et al., 1990; Rieder and Alexander, 1990). For example, the rapid dynamic instability at the plus ends of centrosome-anchored microtubules that occurs during prometaphase appears to be involved in formation of the bipolar metaphase spindle and the attachment of microtubules to the kinetochores (Hayden et al., 1990; Rieder and Alexander, 1990). During metaphase, kinetochore microtubules incorporate tubulin at the kinetochores and undergo a continuous poleward treadmilling or flux of subunits (Hamaguchi et al., 1987; Mitchison, 1989) while during anaphase, the kinetochore microtubules selectively shorten by losing tubulin subunits at the kinetochores. At the same time, interpolar microtubules selectively lengthen by incorporation of tubulin subunits at their plus ends (Gorbsky et al., 1987, Saxton and McIntosh, 1987; Masuda and Cande, 1987). In our previous studies we hypothesized that the defects in spindle organization induced in HeLa cells by low vinblastine concentrations were caused by suppression of microtubule polymerization dynamics rather than by depolymerization of the spindle microtubules (Jordan et al., 1991, 1992). In further support of this idea, we also had

2 262 K. L. Wendell, L. Wilson and M. A. Jordan found that the high affinity binding of vinblastine at the ends of microtubules in vitro caused the kinetic suppression of tubulin exchange at microtubule ends without significant microtubule depolymerization (Wilson et al., 1982; Jordan and Wilson, 1990). More recently, we have found by video microscopy that low concentrations of vinblastine strongly suppress the rates of growing and shortening at plus ends of individual microtubules in vitro and greatly increase the percentage of time that microtubules spend in a state of attenuated activity, neither growing nor shortening detectably (Toso et al., 1993). However, since vinblastine, at high concentrations, can induce the formation of non-microtubular polymers of tubulin (Bensch and Malawista, 1969) and enlarged kinetochores (Krishan, 1968), we questioned whether mitotic block by vinblastine at low concentrations might result from some structural perturbation of a component of the mitotic spindle. Thus, in the present work, we analyzed the ultrastructural effects of 2 nm vinblastine on spindle microtubules, kinetochores and centrosomes of HeLa cells blocked in metaphase in order to elucidate further the antimitotic mechanism of this powerful drug. In the previous experiments, 2 nm vinblastine induced the accumulation of 80% of the cells in metaphase with no decrease in the mass of microtubules (Jordan et al., 1991). Approximately 8% of the cells blocked in metaphase had bipolar spindles with a completely normal arrangement of chromosomes in a compact metaphase plate, and approximately 9% had bipolar spindles with most chromosomes in a compact metaphase plate but with a few chromosomes near the poles of the spindle. Cells remained blocked in metaphase with virtually no transition to anaphase and no significant further degradation of spindle organization for 20 h after the observations reported here (M.A. Jordan, K.L. Wendell, and L. Wilson, unpublished data, see Discussion). Because many of the blocked metaphase spindles were minimally altered as compared with control cells, they offered the opportunity to determine the most sensitive ultrastructural changes in the spindles which accompanied the mitotic block. MATERIALS AND METHODS Cell culture and incubation with vinblastine Human HeLa S3 cells (American Type Culture Collection, Rockville, MD) were grown at 37 C in an atmosphere of 5% CO 2 and 95% air in the absence of antibiotics in Dulbecco s modified Eagle s medium (Sigma Chemical Co., St. Louis, MO) supplemented with 10% fetal bovine serum (Intergin Co., Purchase, NY, or Sigma Chemical Co.) and non-essential amino acids (Sigma Chemical Co.). They were plated in mm Permanox Lux tissue culture dishes (Electron Microscopy Services [EMS], Fort- Washington, PA) at a density of cells/ml so that they would reach approximately 75% confluency on day three, at which time vinblastine (a generous gift of Eli Lilly and Co., Indianapolis, IN) in fresh media or fresh media alone was added. Incubation with vinblastine was at a concentration of 2 nm and for a period of h in all experiments. Immunofluorescence microscopy Cells grown on coverslips were prepared for immunofluorescence microscopy as described previously (Jordan et al., 1991). Centromeres were detected with human anticentromere serum G, a gift from Dr Kevin Sullivan, Scripps Clinic and Research Foundation, La Jolla, CA; this antiserum binds to CENP A and CENP B on western blots (Kevin Sullivan, personal communication). Tubulin was detected with a mouse monoclonal antibody (E7, IG 1, a gift from Dr Michael Klymkowsky, Universty of Colorado, Boulder, CO; Chu and Klymkowsky, 1989), and chromosomes were stained with DAPI (4,6-diamino-2-phenylindole, Sigma Chemical Co.). Second antibodies were from Cappel (West Chester, PA). Photomicrographs were obtained using a Zeiss Photomicroscope III equipped with an epi-fluorescence condenser and a 100 Olympus UVFL oil immersion objective as described previously (Jordan et al., 1991). Electron microscopy Fixation and embedding of the cells in monolayers were carried out directly in culture dishes at room temperature. Cells were fixed for 30 min in 3% glutaraldehyde (EMS) plus 0.5% tannic acid (Ted Pella, Redding, CA) in M sodium phosphate buffered at ph 7.2, followed by three 5 min washes with M sodium phosphate buffer, ph 7.2. Cells were then postfixed in 2% osmium tetroxide in M sodium phosphate buffer, ph 7.2, for 30 min and washed three times with M sodium phosphate buffer. After fixation, cells were dehydrated in ethanol and embedded in LR White resin (EMS) by the method of Erickson et al. (1987) and polymerized by incubation at 52 C for 3-5 days. The culture dishes were pulled away from the hardened resin and small cubes (~4 4 2 mm) of the embedded cell monolayers were cut out and glued to empty plastic blocks so that the bottom of the monolayer was facing up. During mitosis, cells grown in monolayer tend to orient themselves such that the spindle axis is parallel to the surface of the culture dish. Hence, cells were usually sectioned parallel to the main spindle axis. Ribbons of serial sections were cut using a diamond knife (Dupont Instruments, Burbank, CA) on a Porter Blum microtome (Sorvall, Wilmington, DE). Ribbons of serial sections were mounted on Formvar (Ladd, Burlington, VT)-coated tabbed slot grids. Section thickness was approximately 70 nm, as determined by interference colors. Sections were stained with lead citrate and uranyl acetate and examined and photographed with a Philips CM10 electron microscope at 80 kv. Microtubule width measurements Microtubules were measured in three control cells and three cells incubated with vinblastine on micrographs printed at magnifications of >70,000. Measurements were made at regular intervals along the entire microtubule length to obtain an average diameter for each microtubule. The values were averaged to obtain the mean microtubule diameter for each condition. Determination of the number of microtubules per kinetochore Serial sections through spindles were photographed at a magnification of 31,400. The entire metaphase plate of chromosomes and kinetochores was documented in two overlapping micrographs. Every kinetochore encountered was included in the data set, except in cases in which stain precipitate or dirt obscured the kinetochore in any of the sections within the series. All clear microtubules that converged toward and made contact with the kinetochore, as well as microtubules that made contact with the chromatin immediately adjacent to the distinct trilaminar disc of a kinetochore and were part of the same kinetochore fiber, were counted. Centrosome and centriole analysis The distance between centrioles within a pair was estimated in

3 HeLa spindles blocked in metaphase by vinblastine 263 two ways: (1) when centrioles within a pair were found in the same section, the distance between the edge of one centriole and the closest edge of the other in the same section was measured with an ocular micrometer; (2) when centrioles within a pair were in different sections of a series, the number of sections that separated the two centrioles was multiplied by the section thickness. The typical final magnification used for making the measurements was 14,200. For studying centriole ultrastructure and inclusions, only longitudinal or transverse sections through the center of the centriole were used. Sections in which the centrioles began and ended were not used; in this way, only structures that were clearly included in the centrioles were identified. Computer-aided reconstruction of a chromosome located at the spindle pole of a cell blocked in metaphase by vinblastine Micrographs of serial sections through an entire chromosome located at the pole of a cell after incubation with vinblastine (46 sections total) were printed at a final magnification of 21,000. Contours of the chromosome were traced from each micrograph into Swivel 3D Pro (Macromedia Inc., San Francisco, CA) using a digitizing pad (CalComp, Anaheim, CA) on an Apple Macintosh II computer. The software was then used to build a threedimensional model of the chromosome from the traced sections. The resulting 8-bit color images were printed on a Canon color copier at approximately 150 dpi resolution. An animated rotation sequence was generated for the purpose of analyzing the structure (data not shown). The animated sequence was played back using Director 3.0 (Macromedia Inc.), also on a Macintosh c o m p u t e r. RESULTS The overall appearance of control HeLa cells and the comparison with cells incubated with 2 nm vinblastine for 20 h are shown in Figs 1-3. Control cells (Fig. 1A) were predominantly in interphase. Control cells in metaphase had generally spherical spindles consisting primarily of kinetochore and interpolar microtubules (curved arrows, Fig. 2A) extending from the polar region toward the equator of the spindle. Few astral microtubules were present. Spindle poles (Fig. 2A, straight arrows, and Fig. 2B) were well separated from the metaphase plate (approximately 3-4 µm). Spindle poles consisted of two slightly barrel-shaped centrioles in close association with each other, oriented at right angles, and surrounded by dense pericentriolar material. Microtubules were in close proximity to the exterior surface of the centrioles but were excluded from the interior (Fig. 2B). Membranous organelles were excluded from the central spindle and from the centrosomal region. Ribosomes were present throughout the central spindle but were scarce in the region of the centrosome. In contrast, the majority of cells were in metaphase or a metaphase-like stage after incubation with 2 nm vinblastine (Fig. 1B, described further below). Cells incubated with vinblastine were larger than control cells, and the cytoplasm was often considerably less dense than that of control cells. A few dying cells were present (Fig. 1B, arrowheads); however, cell viability is not significantly decreased by incubation for 20 h with 2 nm vinblastine (Jordan et al., 1991). Approximately 20% of the cells blocked in metaphase had bipolar spindles that, in many ways, appeared normal. The blocked, bipolar spindles were generally round and had compact metaphase plates of condensed chromosomes (Fig. 1B, arrows). However, in agreement with our previous light microscopic measurements (Jordan et al., 1992), the spindles were shorter than spindles of control cells (compare Fig. 1. Low magnification electron micrographs of sections of (A) a control culture of HeLa cells grown in monolayer and (B) a similar culture after incubation for 20 h with 2 nm vinblastine. The control cells (A) are predominantly in interphase. Two cells are in metaphase (large arrows) and one is in telophase (small arrow). After incubation with vinblastine (B), most of the cells are arrested in a metaphaselike stage of mitosis. The spindle of one cell appears normal (large arrow); however, images through the entire spindle would be required to discern whether any chromosomes were excluded from the metaphase plate. Four spindles are bipolar but have some chromosomes near the spindle poles (small arrows). Two cells have monopolar spindles (asterisks); by serial sectioning it was determined that they were monopolar rather than bipolar spindles with their spindle axes perpendicular to the substrate. (Most bipolar spindles were found to lie with the spindle axis parallel to the substrate.) Three or four cells appear to be dying (arrowheads). In general, the cytoplasm of cells appears less dense after incubation with vinblastine. Bar, 10 µm.

4 264 K. L. Wendell, L. Wilson and M. A. Jordan Fig. 2. Control cells in metaphase. (A) Section showing both poles of a metaphase spindle (large arrow and open arrow). Microtubules (curved arrows) extend from the centrosomes toward the chromosomes (Ch) on the metaphase plate. Large membranous organelles such as mitochondria and Golgi vesicles are absent from the interior of the spindle. A mother-daughter pair of centrioles is included in the section of the pole on the right (large arrow). An oblique section of one centriole is present at the opposite pole (open arrow). The interpolar distance is 9.3 µm. The distance between the two centrioles of the pair on the right (large arrow) is 56 nm. (B) Centrosomal region of a control cell including a longitudinal section of one centriole. Microtubules run close to the centriole but are absent from its interior. The centriole shows a typical taper at one end and a hollow lumen. The centrosome contains flocculent pericentriolar material (P), but membranous vesicles and ribosomes are absent from the centrosome. Bars, 1 µm (A), 200 nm (B).

5 HeLa spindles blocked in metaphase by vinblastine 265 Fig. 3. Cells blocked in metaphase after incubation with 2 nm vinblastine. (A) Section of a metaphase spindle perpendicular to the plane of the metaphase plate of chromosomes and including both centrosomes (asterisks). The chromatin is condensed into darkly stained chromosomes (Ch) that are aligned along the metaphase plate (large arrows) between the two centrosomes; no chromosomes were outside the central spindle. The distance between the two poles (4.1 µm) is much shorter than in control spindles (compare with Fig. 2A). Microtubules (curved arrows) extend from both centrosomal regions toward the chromosomes. One kinetochore (small arrow) can be identified at this low magnification. One centriole is visible in the centrosome at the right side of the metaphase plate. Mitochondria (M) are found in the periphery of the cell. Many vesicles (v) are found near both spindle poles. (B) Centrosomal region of a cell after incubation with vinblastine. Centrioles (arrows) have lost their normal close orthogonal apposition and are separated by 1 µm distance. Many membranous vesicles (v) are found in the centrosomal region. Microtubules extend toward both the chromosomes (Ch) in the metaphase plate (on the left) and toward one chromosome that is located outside the spindle (on the right). Bars, 1 µm.

6 266 K. L. Wendell, L. Wilson and M. A. Jordan Fig. 3A with Fig. 2A). By immunofluorescence light microscopy, astral microtubules in cells treated with vinblastine appeared more prominent than those in control spindles (Jordan et al., 1991, 1992). Changes in the distribution of microtubules among the subsets of astral, kinetochore and interpolar microtubules were difficult to assess by electron microscopy. However, by electron microscopy it was clear that interpolar microtubules, which were difficult to distinguish by light microscopy, were present in cells incubated with vinblastine (data not shown). The arrangement of chromosomes in some cells blocked in metaphase was indistinguishable from that of control cells in metaphase; that is, all chromosomes were included in a compact metaphase plate (Fig. 1B, large arrow; Fig. 2A). Other bipolar spindles had metaphase plates of chromosomes that were compact and included nearly all chromosomes; however, from one to several chromosomes were located near the spindle poles (Fig. 1B, small arrows). The remaining metaphase-blocked cells had monopolar spindles. Monopolar spindles consisted of ball-shaped arrangements of chromosomes surrounding a single centrosome or two centrosomes that were less than 3 µm apart and starburst-shaped assemblages of microtubules (Fig. 1B, asterisks). The relative proportions of metaphase spindles that were bipolar or monopolar remained approximately the same between 2 h and 39 h after addition of vinblastine to the cell cultures (data not shown). In addition to alterations in chromosome organization and spindle shape, when examined at high magnification cells incubated with vinblastine frequently exhibited numerous small membranous vesicles that were concentrated in the region of the centrosome (inset, Fig. 3A,B; see also Fig. 9A and C, arrows). The vesicles varied in size and shape and appeared to be bounded by a typical single lipid bilayer (Fig. 3B; see also Fig. 9A and C). No accumulation of vesicles occurred in the pericentriolar region (Fig. 2A,B) or in any other region of control cell spindles. Mitochondria and large vesicles invaded the spindle region in many monopolar spindles after incubation with vinblastine. Similar results were obtained in HeLa cells after incubation with high vincristine concentrations (1 µm), which induced total microtubule depolymerization (George et al., 1965). Mitochondria and large vesicles were generally excluded from the spindles of control cells and from bipolar spindles of vinblastine treated cells (note the absence of these organelles in the spindle regions of Figs 2A and 3A). Does incubation with 2 nm vinblastine induce metaphase block or do bipolar spindles with chromosomes at the poles represent cells that have undergone a partial or asynchronous anaphase segregation of chromosomes? Our previous data (Jordan et al., 1991, 1992) indicated that the most sensitive effect of vinblastine involved a block of spindle progression at the metaphase-anaphase boundary. However, a number of chromosomes were situated at the poles in such blocked cells, and it was conceivable that such chromosomes were those that had escaped the block and had segregated to the poles in an anaphase-like manner (Jordan et al., 1992). To characterize the stage of the block more definitively, cells were incubated with vinblastine and prepared for immunofluorescence microscopy using stains for centromeres, microtubules and chromosomes (Fig. 4) (see Materials and Methods). When the chromosomes located near the poles of bipolar spindles were examined, Fig. 4. Light microscopic immunofluorescence of cells blocked in metaphase by incubation with 2 nm vinblastine for one cell cycle. (A) Centromeres and (B) microtubules of a bipolar spindle with two chromosomes located near the spindle pole (outside the compact metaphase plate of chromosomes). (C) Centromeres and (D) chromosomes of a bipolar spindle with one chromosome located near the spindle pole. Arrows in (A) and (C) point to the pairs of centromeres that indicate that chromatids have not undergone premature anaphase segregation. Arrows in (B) point to the long astral microtubules. Arrow in (D) points to the chromosome located near the spindle pole. Bars, 5 µm.

7 HeLa spindles blocked in metaphase by vinblastine 267 microscopy and reconstructed in three dimensions with the aid of a computer (Fig. 5) (see Materials and Methods); it consisted of two chromatids, each with a kinetochore. Thus, vinblastine appears to block the cell cycle at prometaphase or metaphase of mitosis in the absence of significant premature anaphase splitting or segregation. Fig. 5. Computer-aided reconstruction of a chromosome that was located at the pole of a bipolar spindle blocked in metaphase after incubation with 2 nm vinblastine. Two views, rotated approximately 180 from each other, are shown. The chromosome consisted of two chromatids (blue), each with a kinetochore (red). According to the classification scheme of Roos (1973), the chromosome was mono-oriented. The kinetochore nearer to the pole (top image) had microtubules (white) attached (22 in this case), and the kinetochore facing away from the pole was devoid of attached microtubules (bottom image). One centriole of the near pole is shown (green); in the top image it is near the viewer and hence appears large, and in the lower panel it is distant from the viewer and appears small. The chromosome was seriallysectioned and reconstructed from tracings of electron micrographs (see Materials and Methods). we found that nearly all of them contained paired centromeres (Fig. 4A and C), indicating that the chromosomes had not yet undergone separation and segregation. Of 145 such chromosomes located at the spindle poles in 65 cells with otherwise compact metaphase plates, 122 (84%) clearly had 2 centromeres, 19 (13%) were somewhat unclear but appeared to consist of 2 or more centromeres that were close together and formed an elongated fluorescent mass, and only 4 (3%) of the chromosomes exhibited unpaired centromeres. In addition, one chromosome that was located at the pole of a bipolar spindle was sectioned completely for electron Effects of vinblastine on the ultrastructure of microtubules and kinetochores and on the attachment of microtubules to kinetochores Microtubules in vinblastine-treated cells were indistinguishable from those in control cells. They had a mean width of 22.6 ± 0.2 nm (n = 43) as compared with 23.6 ± 0.3 nm (n = 32) in control cells. The frequent occurrence of spindles with imperfect metaphase plates of chromosomes after incubation with nm vinblastine (Jordan et al., 1991, 1992) indicated that vinblastine might induce alterations in the structure of the kinetochores or in the interaction of spindle microtubules with the kinetochores. Examination of 24 kinetochores from control cells and 33 kinetochores from cells incubated with vinblastine showed that, in chromosomes on the metaphase plate, vinblastine appeared to have no effect on the size or ultrastructure of kinetochores (Fig. 6). Like the kinetochores of control cells, the kinetochores of chromosomes in the metaphase plate in metaphase-blocked cells exhibited a characteristic trilaminar structure composed of a thin band of electron dense material separated from the chromatin by a lighter staining band (Jokelainen, 1967; Roos, 1973). (Compare the kinetochore of a control cell in Fig. 6A with kinetochores incubated with vinblastine in Fig. 6B- D.) In control cells, the average length and width of the electron dense band were 203 ± 6 nm and 55 ± 2 nm, respectively (n = 16); and the maximum length and width were 239 nm and 83 nm, respectively. The dimensions of kinetochores on chromosomes located in the metaphase plate in vinblastine blocked cells were essentially the same; they had an average length and width of 192 ± 6 nm and 59 ± 3 nm, respectively (n = 17), and a maximum length and width of 239 nm and 80 nm, respectively. A few kinetochores, however, were larger and more prominent in cells after incubation with vinblastine. These prominent kinetochores were never observed on chromosomes that were included in the metaphase plate, but were found only on the outer surfaces of chromosomes located at the poles of bipolar spindles or in monopolar spindles. The large kinetochores (Fig. 6E) had longer and wider electron dense bands than those of control cells. The large kinetochore shown in Fig. 6E was 478 nm long and 130 nm wide. Enlarged kinetochores in cells blocked in metaphase by vinblastine never had any attached microtubules. Large kinetochore structures have also been noted in cells in prometaphase, and the large kinetochores found in vinblastine-treated cells may be a secondary consequence of the absence of attached microtubules (reviewed by Rieder, 1982; also see Discussion). The majority of the kinetochores in cells incubated with vinblastine exhibited no detectable structural changes with respect to size, shape and density. We then determined whether the number of microtubules attached to the kinetochores of chromosomes that were

8 268 K. L. Wendell, L. Wilson and M. A. Jordan Fig. 6. Kinetochores (arrows) from a control cell (A) and from cells after incubation with 2 nm vinblastine (B-E). There was no detectable effect of vinblastine on the structure of kinetochores that had microtubules attached (compare control in A with B-D). (E) Large kinetochore (width = 130 nm, length = 478 nm) devoid of microtubules and facing away from the pole in a monopolar spindle. This kinetochore was devoid of microtubules in all six of the sections spanning the kinetochore. Kinetochores that had no attached microtubules after incubation with vinblastine appeared larger and had more distinct coronas; they resembled prometaphase kinetochores. The opposite kinetochore faced the central pole and had microtubules attached (not shown). Bar, 0.25 µm. included in the metaphase plate was altered by vinblastine. Using series of sections through kinetochores, the number of microtubules attached per kinetochore was determined on 24 kinetochores from 3 control cells in metaphase and on 33 kinetochores from 4 cells blocked in bipolar metaphase by 2 nm vinblastine (Fig. 7). While there was a broad distribution in the number of attached microtubules both in cells incubated with vinblastine and in control cells, the mean number of microtubules attached to the kinetochores was significantly decreased after incubation with the drug. The mean number of microtubules attached to the kinetochores of control cells was 17.1 ± 0.6, while in cells incubated with vinblastine, the mean number of attached microtubules per kinetochore was reduced to 12.4 ± 0.7 (Fig. 7). This represents a statistically significant 27% decrease in the average number of microtubules per kinetochore, indicating that microtubule capture and/or retention by kinetochores was inhibited by vinblastine. Effects of vinblastine on the association of centrioles within centriole pairs All cells examined in serial sections after incubation with vinblastine (approximately 20 cells) had four centrioles; a pair of centrioles was located at each pole in bipolar spindles and four centrioles were located at the centers of monopolar spindles. The two centrioles of a centriole pair were often situated considerable distances from each other in cells incubated with vinblastine (Fig. 3B) whereas centrioles within the same centrosome in control cells were always close to each other (Fig. 2A). As shown in Fig. 8, the mean distance between the two centrioles of a pair in control cells was 60 ± 7 nm (n = 10), and the centrioles were never separated by a distance of more than 100 nm. The mean separation distance between nearest centrioles increased to 443 ± 66 nm (n = 28) after incubation with vinblastine. Approximately 75% of the centriole pairs were separated by more than 200 nm in vinblastine-incubated cells (twice the maximum separation in control cells); approximately 50% of the centrioles were separated by more than 400 nm and, in some cells, they were separated by more than 1 µm. Thus, the normal close association of mother and daughter centrioles within centrosomes was disrupted by vinblastine. Effects of vinblastine on the structure and integrity of centrioles Centrioles in control cells were cylindrical structures composed of nine triplet blades of microtubules. The interiors of the cylinders were devoid of any structures other than the occasional presence of structures known as the hub and spokes which line the periphery of the centriolar lumen (Gall, 1961; de Harven, 1968) (Figs 2B and 9A). Transverse and longitudinal sections of centrioles from cells incubated with vinblastine are shown in Fig. 9. The centriole

9 HeLa spindles blocked in metaphase by vinblastine 269 Fig. 7. Effect of vinblastine (2 nm) on the number of microtubules attached to a kinetochore. The number of microtubules attached to kinetochores included in the metaphase plate in bipolar spindles after incubation with vinblastine was counted in serial sections of kinetochores (bottom panel) and compared with similar counts for control cells (top panel). The mean number for control cells was 17.1 ± 0.6 whereas the mean number after incubation with vinblastine was 12.4 ± 0.7. The difference is significant at the 99.8% confidence level (Student s t-test). The mean number of microtubules in individual cells was as follows (the number of kinetochores is indicated in parentheses): control; cell 1, 17.9 (8 kinetochores); cell 2, 17.4 (10); cell 3, 15.7 (6); vinblastinetreated; cell 1, 12.1 (21 kinetochores); cell 2, 12.0 (10); cell 3, 23 (1); cell 4, 10 (1). shown in Fig. 9A looks normal while the centrioles shown in B-D are markedly abnormal. In Fig. 9B and C, the centrioles contain extra microtubules of normal diameter (25 nm) coursing through their lumens. In Fig. 9D, the centriole is split open apparently within one of the blades of triplets. Of a total of 18 centrioles examined from control cells, all were intact and lacked extra microtubules. In contrast, approximately one-third of the centrioles of cells blocked in metaphase by vinblastine exhibited abnormal structure. Of a total of 34 centrioles from cells incubated with vinblastine, nine had between one and five central microtubules (five of the nine were from monopolar spindles and four were from bipolar spindles), suggesting that by some mechanism, incubation with vinblastine allowed microtubules to invade the centriolar lumen. The organization of the nine outer triplet microtubules was disrupted in 2 of the 34 centrioles examined. Of four mother-daughter pairs of centrioles examined in which the structural integrity of both members of the pair could be assessed, only one member of the pair was impaired structurally. DISCUSSION We have previously found that incubation of HeLa cells Fig. 8. Effect of incubation with vinblastine on the association between the two centrioles of a centriole pair. The distance between the two nearest centrioles in the same centrosomal region was measured for control cells (top panel) and for cells after incubation with 2 nm vinblastine for 20 h (bottom panel). Measurements were made on single sections or using series of sections as described in Materials and methods. Pairs after incubation with vinblastine include 20 pairs from bipolar spindles and 8 pairs of nearest centrioles from monopolar spindles; they are combined in the graph because there was no difference in centriole separation within a pair between bipolar and monopolar spindles. The mean separation distance for control cells was 60 ± 7 nm in control cells whereas the mean separation distance for cells incubated with vinblastine was 443 ± 66 nm. with low concentrations of vinblastine (0.1-6 nm) blocks cells in mitotic metaphase in the absence of net microtubule depolymerization (Jordan et al., 1991). While some blocked spindles appeared normal in organization, in most cells the metaphase block was associated with subtle alterations in the overall organization of the spindle microtubules, chromosomes and centrosomes (Jordan et al., 1992). In the present work we sought to determine whether there were any detectable ultrastructural alterations of the microtubules, kinetochores or centrosomes of cells blocked in metaphase configuration by vinblastine that might further illuminate the mechanism of mitotic block by the drug. Many of our observations were performed on the bipolar metaphase spindles because they represent a configuration that superficially appears to have the organization necessary to transit from metaphase into anaphase. However, no cells were in anaphase after incubation of cells with 2 nm vinblastine for h (the duration of one cell cycle) (Jordan et al., 1992). In addition, we found virtually no cells in anaphase at any time between 2 h and 48 h after addition

10 270 K. L. Wendell, L. Wilson and M. A. Jordan Fig. 9. Electron micrographs of the ultrastructure of centrioles in cells incubated with 2 nm vinblastine (A-D). (A) Transverse section of a normal looking centriole in a cell incubated with vinblastine. The centriolar cylinder is composed of nine outer triplet microtubules. The interior of the lumen is devoid of any structures except for some material of medium electron density known as the hub, which is arranged symmetrically around the periphery of the lumen. (B) Transverse section of a centriole with what appear to be five central microtubules in the lumen. The average diameter of the five central microtubules was 24.2 nm. (C) Two centrioles of a pair, one of which is in oblique section and the other longitudinal section. The longitudinally-sectioned centriole has at least three microtubules coursing through the lumen. Microtubules numbered 1, 2 and 3 in this panel have average diameters of 24.4, 25.6 and 25.0 nm respectively measured along their entire included lengths. (D) Transverse section of a centriole which is split open within one of the triplets of microtubules. There is one microtubule in the center of the lumen; this may have derived from the broken triplet blade of microtubules. This section is from the mid-region of the centriole as were all sections of centrioles used in this analysis (see Materials and Methods). A and C are from monopolar spindles, B and D are from bipolar spindles; there was no consistent difference in the abnormalities associated with bipolar or monopolar spindles. Bar, 100 nm. of 2 nm vinblastine (one cell was in anaphase out of 5839 cells observed after vinblastine incubation, whereas 70 cells were in anaphase out of 3190 control cells, M.A. Jordan, K.L. Wendell and L. Wilson, unpublished data). In other words, the metaphase/anaphase block is not a transitory block. In addition, the relative proportion of bipolar and monopolar spindles did not change significantly between 2 h and 39 h after addition of 2 nm vinblastine; after 31 h increasing numbers of cells revert to interphase without dividing (M.A. Jordan, K.L. Wendell and L. Wilson, unpublished data). Thus abnormal bipolar spindles do not appear to represent merely a stage in a process of spindle collapse to a more aberrant monopolar configuration in the presence of drug; rather they represent unsuccessful attempts to form anaphase-competent spindles. Monopolar spindles also appear unable to evolve to an anaphase-competent bipolar configuration. Neither the structure of the microtubules nor the struc-

11 HeLa spindles blocked in metaphase by vinblastine 271 ture of kinetochores on chromosomes in the metaphase plate was detectably altered by the drug. However, the number of microtubules attached to kinetochores of chromosomes in the metaphase plate of bipolar spindles was decreased significantly (Fig. 7). In addition, centrosomes of both bipolar and monopolar spindles were altered; the close association between the mother and daughter centrioles was lost, numerous membranous vesicles were found in the centrosomal region and many centrioles exhibited abnormal ultrastructure. As discussed further below, these perturbations of spindle organization by vinblastine could all be caused by suppression of microtubule polymerization dynamics (dynamic instability and treadmilling) rather than by microtubule depolymerization. In addition, the effects of vinblastine on centrosomal structure and organization not only may be involved with inhibition of mitosis by vinblastine, but also may be involved with the ability of vinblastine to kill cells. Effects of vinblastine on kinetochore structure and on the attachment of microtubules to kinetochores Several antimitotic drugs are known to alter kinetochore structure (Brinkley and Stubblefield, 1966; Krishan, 1968; Cassimeris et al., 1990). For example, incubation of Earle s L cell fibroblasts with 11 nm vinblastine induced formation of prominent and large kinetochores (Krishan, 1968), and the kinetochore corona material became increasingly distinct in PtK 1 cells as the microtubules were depleted by high concentrations of nocodazole (Cassimeris et al., 1990). Thus, we were particularly interested in determining whether low concentrations of vinblastine induced alterations in kinetochore fine structure. Incubation with 2 nm vinblastine did not affect the size or structure of kinetochores when the kinetochores had attached microtubules. Some large prominent kinetochores were observed on a few chromosomes, but these had no attached microtubules. Such large kinetochores were situated only on the outwardfacing chromatids of chromosomes at the poles of bipolar spindles or in monopolar spindles, and they resembled kinetochores of control cells during prometaphase prior to microtubule attachment (reviewed by Rieder, 1990). Thus, vinblastine did not detectably affect kinetochore structure in a way that could be related causally to its ability to inhibit mitosis. Vinblastine significantly reduced the attachment of microtubules to kinetochores of chromosomes which were aligned in the metaphase plates of bipolar spindles (Fig. 7). The reduction in the numbers of attached kinetochore microtubules constituted one of the major structural alterations associated with vinblastine-induced metaphase block. There are two potential sources of error in the analysis of microtubule attachment to kinetochores. It is known that the number of kinetochore-attached microtubules varies with the size of the kinetochore, and kinetochore size varies among different chromosomes (Cherry and Johnston, 1987; Cherry et al., 1989; reviewed by Brinkley, 1990; Rieder, 1982). Thus, we had to control for the variation in kinetochore size. For this study, we analyzed between 6 and 21 kinetochores in each cell. Because the average number of attached microtubules per kinetochore per cell agreed closely for the three cells of the control group and for the two cells incubated with vinblastine (Fig. 7), we believe that a sufficient number of kinetochores was sampled to negate the effects of variability in kinetochore s i z e. Another potential source of error was the possible presence of overlapping microtubules or of unclear microtubules. With a section thickness of approximately 70 nm and a microtubule diameter of 24 nm, it is conceivable that one microtubule could have been superimposed upon another. However, such an occurrence should have occurred with equal or higher probability in control cells than in cells incubated with vinblastine which have fewer attached microtubules than the control cells. Only the numbers of clearly distinguishable microtubules were reported in the data of Fig. 7. However, we also tallied separately any structures that were unclear but were possibly microtubules attached to kinetochores. The incidence of these structures was also reduced by 24% in cells incubated with vinblastine as compared with control cells. Thus neither potential source of error appears to affect the result that the number of attached kinetochore microtubules was reduced significantly by incubation with vinblastine. As discussed above, some kinetochores had no attached microtubules after incubation with vinblastine; these kinetochores were located on the outward facing chromatids of chromosomes in monopolar spindles or at the poles of bipolar spindles. In addition, incubation with relatively high concentrations of vinblastine (10 nm) resulted in the absence of microtubules from nearly all kinetochores (data not shown). Thus, inhibition of microtubule attachment to kinetochores appears to be vinblastine concentration dependent. Effects of vinblastine on microtubule dynamics and relationship to inhibition of chromosome congression at metaphase The sensitive effects of vinblastine on the dynamics of tubulin exchange of MAP-rich bovine brain microtubules in vitro is brought about by the binding of small numbers of vinblastine molecules at microtubule ends (Wilson et al., 1982) and occurs in the absence of significant depolymerization (Jordan and Wilson, 1990). For example, vinblastine inhibits the treadmilling (flux) addition of tubulin at the ends of the microtubules half-maximally at 0.15 µm while the polymer mass is reduced less than 3% as compared with controls. In addition, we have recently found by video microscopy that vinblastine markedly suppresses dynamic instability at the (+) ends of MAP-depleted microtubules in vitro. For example, 0.5 µm vinblastine suppresses the rates of growing and shortening by 50-60%, and it shortens the duration of an average growing phase by 33%. In addition, microtubules incubated with 0.5 µm vinblastine spend 2.5 times longer than control microtubules in a pause or attenuated state during which no growing or shortening can be detected (Toso et al., 1993). In prometaphase of mitosis, microtubules originating at the centrosomes are thought to probe repeatedly the cytoplasm by continuous growing and shortening until kinetochore attachment is achieved (Rieder and Alexander, 1990;

12 272 K. L. Wendell, L. Wilson and M. A. Jordan Hayden et al., 1990). If vinblastine suppresses the growing and shortening dynamics of microtubules in cells as it does in vitro, such kinetic stabilization might readily result in the inability of microtubules nucleated at the spindle poles to reach both kinetochores of a chromosome and establish bipolar attachment. As described in the preceding studies and in the present work, addition of very low concentrations of vinblastine (eg., 2 nm) to the growth media of HeLa cell cultures induces significant metaphase arrest. In contrast, kinetic suppression of microtubule dynamics in vitro requires approximately 100-fold higher concentrations of the drug. However, the apparent contradiction in the effective concentration of vinblastine in cells and in vitro can be explained because vinblastine is concentrated in HeLa cells under the conditions examined. For example, after incubation of HeLa cells with 2 nm vinblastine for 20 h, the actual intracellular concentration of vinblastine was found to be 103 ± 3 nm (Jordan et al., 1991), approximately 50 times the concentration in the medium, and well within the concentration range in which microtubule growing and shortening dynamics are suppressed in vitro. Despite the accumulation of vinblastine in cells under the conditions used, the intracellular concentration of vinblastine was substoichiometric to intracellular tubulin (1 vinblastine: 200 tubulin molecules, Jordan et al., 1991). Thus it is reasonable to hypothesize that inhibition of microtubule growing and shortening dynamics by vinblastine in cells may lead to the inability of some chromosomes to congress to the metaphase plate because the microtubules emanating from both spindle poles are prevented from reaching the kinetochores. Similarly, suppression of microtubule growing and shortening dynamics by vinblastine may also be responsible for the reduced number of microtubules attached to kinetochores of chromosomes located in the metaphase plate. An alternative possibility, also consistent with the present data, is that the drug, when bound at the plus ends of the microtubules, might sterically block or weaken the binding of the microtubules to kinetochores, thus causing failure of microtubule attachment to the kinetochores or fragility of the attachment. Murray and Kirschner (1989) have suggested that one of the important control points of the cell cycle involves a signal or switch that prevents the completion of mitosis until the spindle has been correctly assembled. The fact that kinetochores of spindles blocked at the metaphase-anaphase boundary by vinblastine have fewer microtubules than kinetochores of control cells in metaphase suggests that attachment of a full complement of microtubules may be required to initiate the onset of anaphase. Another possibility is that HeLa cells must wait for all chromosomes to become aligned at the metaphase plate before initiating anaphase (Zirkle, 1970) although, in some cells, anaphase can begin while mono-oriented chromosomes are still present (reviewed by Rieder, 1990). We have suggested that progression from metaphase to anaphase may require that microtubules attached to the kinetochores have specific assembly and disassembly dynamics, and failure of progression from metaphase to anaphase in the presence of vinblastine might be due to suppression of such dynamics (Jordan et al., 1992). Effects of vinblastine on centrosome and centriole organization In previous work we found that incubation with vinblastine at concentrations that blocked mitosis but did not cause net microtubule depolymerization induced fragmentation of centrosomal material at the spindle poles (Jordan et al., 1992). Considering the well-established role of the mitotic centrosome in organizing the spindle and in regulating spindle microtubule lengths and dynamics (Snyder and McIntosh, 1975; Telzer and Rosenbaum, 1979; Kuriyama and Borisy, 1981; Verde et al., 1990; Belmont et al., 1990), we examined the structure of mitotic centrosomes after incubation with vinblastine. We found significant and interesting alterations in the components of the centrosomes, in the structures of the centrioles, and in the relationship between mother and daughter centrioles. Like centrosomes of control cells, centrosomes in cells incubated with vinblastine contained many microtubules with normal morphology and flocculent pericentriolar material; the centrosomes excluded ribosomes and most cell organelles. Unlike centrosomes of control metaphase spindles, however, centrosomes of vinblastine-blocked spindles contained accumulations of many small membranebounded vesicles of unknown identity (compare Fig. 2A, B with Figs 3A, B and 9A-D). In normal mitosis, membranous vesicles accumulate in the region of the centrioles during S phase and disappear during prometaphase or metaphase (Robbins et al., 1968; Vorobjev and Chentsov, 1982). The observations reported here suggest that vinblastine may induce blockage of both centrosomes and chromosomes in a stage prior to metaphase. Blockage of the centrosomes in prometaphase might conceivably play a causal role in the mitotic block induced by vinblastine. The results of many studies have suggested that the mitotic centrosome plays a significant role in the regulation of mitosis (Berns et al., 1977; Kuriyama and Borisy, 1981; Bornens and Karsenti, 1984; Vandre et al., 1984; Bailly et al., 1989; Maniotis and Schliwa, 1991). Thus, vinblastine might block mitosis by binding directly to the ends of the microtubules of the centrioles or perhaps by binding to γ-tubulin in the centrosome (Zheng et al., 1991; Stearns et al., 1991; Joshi et al., 1992). Such interactions could perturb the assembly dynamics of centriole microtubules themselves or of the centrosome-anchored microtubules, or perhaps block the interactions of the microtubules with other centrosomal components. To our knowledge there have not been any studies in which the binding of vinblastine to centriolar components has been examined. However, the present data indicate that vinblastine may exert a direct effect on centrioles. Thirty per cent of the centrioles were altered structurally by incubation with vinblastine; many had extraneous microtubules coursing through their lumens and some appeared to be split open either between or within microtubule triplets (Fig. 9A-D). Due to the loss of defining orientation between mother and daughter centrioles after incubation with vinblastine, it was not possible to determine whether the affected centrioles were mothers (formed prior to vinblastine incubation) or daughters (formed in the presence of vinblastine). However, in all four cases in which distortions were observed and both centrioles were clearly vis-