Role of microtubule organization in centrosome migration and mitotic spindle formation in PtK1 cells

Protoplasma (1993) 173:133-143

�9 Springer-Verlag 1993 Printed in Austria

Role of microtubule organization in centrosome migration and mitotic spindle formation in PtK1 cells

Lydia Armstrong** and Judith A. Snyder*

Department of Biological Sciences, University of Denver, Denver, Colorado

Received September 9, 1992 Accepted November 24, 1992

Summary. Quinacrine, an acridine derivative, has previously been shown to disrupt lateral associations between non-kinetochore microtubules (nkMTs) of opposite polarity in PtKI metaphase spindles such that the balance of spindle forces is significantly altered. We extended the analysis of the spatial relationship of spindle microtubules (MTs) in this study by using quinacrine to compare ATP- dependent requirements for early prometaphase centrosome separation and spindle formation. The route used for centrosome migration can take a variety of pathways in PtK1 cells, depending on the location of the centrosomes at the time of nuclear envelope breakdown. Following quinacrine treatment centrosome separation decresased by 1.9 to 14.0 ~tm depending on the pathway utilized. However, birefringence of the centrosomal region increased approximately 50% after quinacrine treatment. Quinacrine-treated mid-prometaphase cells, where chromosome attachment to MTs had occurred, showed a decrease in spindle length of approximately 6.0 ktm with only a slight increase in astral birefringence. Computer- generated reconstructions of quinacrine-treated prometaphase cell~ were used to confirm changes in MT reorganization. Early-prometaphase cells showed more astral MTs (aMTs) of varied length while mid-prometaphase cells showed only a few short aMTs. Late prometaphase cells again showed a large number of aMTs. Our results suggest that: (1) quinacrine treatment affects centrosome separation, (2) recruitment of nkMTs by kinetochores is quinacrine- sensitive, and (3) development of the prometaphase spindle is dependent on quinacrine-sensitive lateral interactions between nkMTs of opposite polarity. These data also suggest that lateral interactions between MTs formed during prometaphase are necessary for centrosome separation and normal spindle formation but not necessarily chromosome motion.

Keywords: Centrosomes; Microtubule; Mitosis; Prometaphase; Quinacrine.

* Correspondence and reprints: Department of Biological Sciences, University of Denver, Denver, CO 80208, U.S.A. ** Present address: Schering-Plough Research Institute, Bloomfield, New Jersey, U.S.A.

Abbreviations: aMT(s) astral microtubule(s); DIC differential interference contrast; MT(s) microtubule(s); kMT(s) kinetochore microtubule(s); NEB nuclear envelope breakdown; nkMT(s) non-kinetochore microtubule(s).

Introduction

In PtK cells, the timing of centrosome migration during prophase relative to other mitotic events such as formation of the asters, chromosome condensation, and nuclear envelope breakdown (NEB) varies considerably (Roos 1973, Aubin etal. 1980). Generally, both centrosomes are motile for approximately 7-10min, although in some instances one centrosome remains stationary while the other continues to migrate (Ratt- ner and Berns 1976). As centrosomes separate, MT initiation and elongation increase at each centrosome to form astral complexes (Kuriyama and Borisy 1981, Snyder and McIntosh 1975). Once interaction between MTs of opposite polarity is established, the distance between centrosomes varies (Rattner and Berns 1976 a, b; Roos 1973; Aubin et al. 1980) until the final positioning of the spindle is defined at metaphase. Congression of chromosomes in PtK~ cells is dependent on attachment of kinetochores to the prometaphase spindle which occurs: (1) well after the centrosomes generate MTs, (2) in an order which is influenced by their proximity to a centrosome, and (3) initially as mono-orientation (monotelic orientation) followed by bipolar association to the developing spindle (Rieder and Borisy 1981). Centrosome separation, however, is not dependent on chromosome movements since cen-

134 Lydia Armstrong and Judith A. Snyder: Role of microtubule organization in centrosome migration in PtK1 cells

trosome separation in PtK1 cells can occur without association of MTs with kinetochores. Congression of chromosomes with concomitant reduction in spindle length during the prometaphase-to- metaphase transition is an apparent reflection of the mitotic motor. This reduction in spindle length during the prometaphase-to-metaphase transition is evidence that forces generated in the mitotic spindle prior to anaphase act to pull the poles together (discussed in Snyder et al. 1985). Ostergren (1951) was the first to postulate that the force(s) which moves chromosomes during prometaphase could be the same force(s) responsible for chromosome movements during anaphase. Anaphase A movements are dependent on the depolymerization of MTs at the kinetochore (Gorbsky et al. 1987, Koshland etal. 1988, Mitchison and Kirschner 1985), while anaphase B pole-pole separation may result from MT-MT sliding (Baskin and Cande 1988; Cande and McDonald 1985, 1986; Spurck and Pickett-Heaps 1987) and/or from the release of compression loaded into the nkMT population of MTs (Armstrong and Snyder 1989; Sny- der etal. 1984, 1985). In either case anaphase B is thought to be mediated by the non-kinetochore class of MTs where MTs generated at each pole overlap in the spindle domain (Oppenheim et al. 1973, Ris 1949). It is probable that vanadate-sensitive, dynein-like linkages found in diatom spindles (Cande 1982), or the quinacrine-sensitive linkages found in PtK1 cells (Arm- strong and Snyder 1989) are capable of producing spindle pole separation during anaphase B. It is most likely that spindle elongation during anaphase is dependent on lateral MT-MT interactions where MTs of opposite polarity are interdigitated (Euteneuer and McIntosh 1981). Although quinacrine treatment is thought to affect numerous ATP-requiring molecules, it has been shown to reduce anaphase B with almost no effect on anaphase A (Armstrong and Snyder 1989). Therefore, in PtK~ cells only the forces which maintain pole-to- pole distance during metaphase and those responsible for pole-to-pole separation during anaphase B were selectively affected by quinacrine treatment. This study suggests that the relative energy requirements for MT- MT interactions which are responsible for centrosome separation, prometaphase spindle shortening, and chromosome congression are differentially sensitive to quinacrine treatment at different stages of mitosis.

Materials and methods

PtK1 cells were grown in monolayer culture in Ham's F-12 medium supplemented with 10% fetal bovine serum (Gibco) in a 37 ~ CO2

incubator. For experimentation cells were subcultured onto 22 Iili1"12 coverslips 48 h prior to use. Coverslips were inverted onto glass microscope slides with coverslip fragments used as spacers and sealed on two edges with valap. Quinacrine [6-chloro-9-{(4-(diethylamino)- 1-methylbutyl)amino}- 2-methyloxyacridine] dihydrochloride (Sigma) was dissolved in Ham's F-12 medium containing 10% fetal bovine serum to final concentrations of 0.5, 1, and 2 ~tM. Quinacrine-containing medium was introduced to cells on the microscope stage by exchanging con- ditioned medium with several volumes of quinacrine-containing medium. Polarization and differential interference contrast (DIC) light micrographs were taken on a Zeiss Photomicroscope II with a 40 x objective (N.A. = 0.85). A ~ 30 Brace Kohler compensator was used for birefringenee measurements. Light micrographs were recorded on Kodak Tri-X film and developed in Diafine. Cells were prepared for electron microscopy by standard techniques as described elsewhere (Snyder etal. 1983). Cells experimentally treated and monitored with light optics were processed for electron microscopy, reidentified, serial sectioned, and examined with a Hi- tachi 7000-3 electron microscope. For computer graphics an IBM PC-based three dimensional reconstruction system was employed (Young et al. 1987). Data were en- tered into a DTK-1000 PC by digitizing MT profiles and contours directly from electron micrographs of serial sections. The reconstruction program aligned the plane information from each section and displayed the final reconstruction on a high resolution color monitor (NEC Multisynch). Object types were differentiated by line width, line color, and color fill. Computer images were directly recorded from the CRT with Kodak Pan-X film.

Results

Controls

The definition of prometaphase, for the purpose of this study, is broadened to be the time between NEB and metaphase, a fairly-well defined period of 29 4- 1.1 min in PtK1 cells (Hamilton and Snyder 1982). Figure 1 is a graph representing two different pathways that PtK1 centrosomes can take during prometaphase prior to establishing the length of the late metaphase spindle. During the first 10-12 min after NEB occurs, the greatest variation in centrosome separation occurred (6- 20 ~tm) and depended on the original location of each centrosomes at NEB. If the centrosomes were located on opposite sides of the nucleus prior to NEB (Fig. 1, path A), then separation steadily decreased as the spindle formed prior to metaphase. Centrosomes which had not separated prior to NEB (< 6 I~m apart Fig. 1, path B) showed an increase in centrosome separation before spindle shortening occurred later in prometaphase. Most of the chromosomes during early prometaphase are mono-oriented and showed poleward motions by oscillations and/or sliding motions until they became amphi-oriented. By mid-prometaphase most chromosomes were amphitelic in orientation, and the pole-pole

Lydia Armstrong and Judith A. Snyder: Role of microtubule organization in centrosome migration in PtK~ cells 135

E ::L v r,. O

D..

g,

O

E: o CO

20

18

16"

t4-

10"

S'

6,

EARLY PSOMETAPRASE

NEB ~ 10

"-~....

j

/ MID- I LATE ] M ] PROMETAPHASE IPROMETAPHASE] I ANAPHASE

t I, I

t~ 2b ~ ao [min]

Fig. 1. Graph showing the various pathways (A, B) in which centrosomes migrate during prometaphase in PtK~ cells. Prometaphase begins with NEB. Early prometaphase, the first t5min after NEB, shows the greatest variation in centrosome migration. Mid-prometaphase is defined as the prometaphase-to-metaphase transition during which the spindle may shorten as much as 4~tm. The period 10min prior to anaphase onset is defined as late prometaphase. Metaphase itself may last only 2-3 rain prior to anaphase onset

Tablel. Quinacrine-induced reduction in centrosome-centrosome distance (~tm) in PtKl cells

2 lXM 1 ~tM 0.5 ~tM

NM 3.0 2.4 2.7 2.0 2.6 1,0 2.9 1.2 3.1 3.2 1.2 2.0 2.6 • 0.6 2.0 4- 0.7

Early prometaphase (< 12 l,m)

Average

Mid-prometaphase (14-16 txm)

NM 7.6 4.8 5.2 8.8 5.6 6.4 6.8 6,9 3.4 5.8 5.7 4.2 5.7 4- 1.3 6.2 Average

Late prometaphase 7.2 7.2 5.6 (12-14~n) 7.2 4.8 7.2

8.8 6.6 5.2 4.3 7.2 5.6 6.0 7.5 6.8 5.2 6.6 6.0

Average 6.5 • 1.5 6.7 4- 0.9 6.1 • 0,7

• 1.5

Duration of quinacrine treatments was 3 rain NM Measurements not made due to loss of birefringence

distance decreased at comparable rates (Fig. 1). During the transition between mid-to-late prometaphase (last- ing approximately 10 min, Fig. 1) the spindle continued to shorten by an average of 2.0 • 0.3 pro. Approxi- mately 25 rain after NEB spindle length reached an average of 13 ~tm, a length which was consistent and independent of the pathways shown in Fig. 1. Table 1 shows the effects of a 3 rain treatment of various concentrations of quinacrine on prometaphase spindle formation. Early prometaphase cells treated with 1 ~tM quinacrine for 3 rain where the centrosome-centrosome distance was less than 12 gm showed an average decrease in spindle length of 2.6 • 0.6 ~ n (n = 6). Early prometaphase cells treated with 0.5 IxM quinacrine re- suited in a decrease of 0.2 pm in centrosome distance if pathway A was utilized. Centrosome movements of early and mid-prometaphase cells treated with 1 and 2 gM quinacrine were not measured due to loss of birefringence. Cells treated with 1 pM quinacrine for 3 rain during mid- and late prometaphase, where spindle lengths measured 14-16 txm and 12-14 Ixm, respectively, resulted in an average decrease in spindle lengths of 5.7 • 1.3 and 6.7 • 0.9 ~ma (n = 6). Treatment of mid-

and late prometaphase cells with 0.5 gM quinacrine for 3 rain resulted in decreased centrosome separations of 6.1 • 0.7 and 6.2 • 1.5pm, respectively (n = 6). Late prometaphase cells (spindle lengths of 12-14pro) treated with 2 taM quinacrine for 3 min resulted in an average reduction in spindle length of 6.5 • 1.5 pm (n = 6).

Light microscopy

Early prometaphase cells treated with 1 gM quinacrine for 3 rain (Fig. 2) showed distinctive effects on MTs as seen by polarization optics. Figure 2 a is a DIC image of an early prometaphase cell 2 min after NEB; chromosomes remained positioned as in prophase. MT ar- rangements, as identified by astral birefringence patterns, were clearly evident (Fig. 2 b). Following a 3 min treatment centrosome-centrosome distance decreased by 4pro and birefringence increased approximately 50% in both the spindle and astral regions (Fig. 2c). Chromosome positions changed slightly but appeared to retain monotelic orientation with respect to the position of the centrosomes.


Fig. 2. Series of polarization and DIC light micrographs showing the effects of 0.5 gM quinacrine treatment for 3 min on an early prometaphase PtK1 cell. a and b Cell prior to treatment. NEB is not complete and chromosomes appear in a circular pattern. The centrosomes (arrows) are 7 gm apart, e and d Same cell after the 3 min quinacrine treatment. Chromosome organization remains in a circular pattern. There was a 4 ~tm decrease in the distance between the centrosomes. Birefringence has increased in the presence of quinacrine. Bar: 5 gill

Fig. 3. Series of polarization and DIC light micrographs showing the effects of 0.5 ~tM quinacrine treatment for 3 min on an early prometaphase cell approximately 5 rain after NEB. a and b Cell prior to treatment. Chromosomes have no specific organization. The centrosomes (arrows) are located at opposite sides of the cell 16 gm apart, e and tl Same cell after treatment with quinacrine. Chromosomes appear more closely associated to each other compared to before treatment but showed no significant changes in position (arrow). Centrosome separation has decreased to 8gin and birefringence has increased. Bar: 5 gm

An early prometaphase cell treated 5 min after NEB with 1 gM quinacrine for 3 min is shown in Fig. 3. Prior to treatment several chromosomes were located near the metaphase plate while other chromosomes were located beyond the region originally defined by the nuclear envelope (Fig. 3 a and b). Changes in the level of birefringence seen in this early prometaphase spindle are significant following quinacrine treatment (compare Fig. 3 b with d). Pole-to-pole length decreased by 50% (8.0 gm ) with an increase in astral birefringence (Fig. 3 d). Also, following this treatment some chromosomes located near the centrosomes moved towards the recta- phase plate, while others (Fig. 3 c) showed no significant change in position. Chromosomes already aligned close to the metaphase plate showed little change in position (Fig. 3 c). Figures 4 and 5 are series of DIC and polarization light micrographs of mid-prometaphase cells treated with

0.5 gM quinacrine for 3 min. Each cell has a different chromosome organization prior to treatment (compare Fig. 4 a with Fig. 5 a), yet spindle shape and birefringence levels were similar (Figs. 4 b and 5 b). Spindle lengths measured 15.4 and 16.8 gm for Fig. 4 a and 5 a respectively, and some astral birefringence was present (Fig. 4b). Following quinacrine treatment the chromosomes in both cells congressed towards the metaphase plate (Figs. 4 c and 5 c). In each case spindle MT structure was affected similarly; spindle lengths and birefringence decreased, and only a slight increase in astral birefringence could be seen (Figs. 4 d and 5 d). Late prometaphase cells treated with 0.5 gM quinacrine for 3 rain shows a distinct change in chromosome position and MT birefringence patterns. Prior to treatment, all chromosomes had congressed to the metaphase plate but were not tightly aligned (Fig. 6 a). The spindle measured 15.2 gm from pole-to-pole and was more birefringent, both within the spindle domain and

Lydia Armstrong and Judith A. Snyder: Role of microtubule organization in centrosome migration in PtK1 cells 137

Fig.4. Series of polarization and DIC light micrographs showing the effects of 0.5 gM quinacrine on a mid-prometaphase cell. a and b Cell prior to treatment; chromosomes appear gathered in the center of the cell. Spindle length measures 15.4~tm, and some astral birefringence is apparent (arrows). e and d Same cell after the 3 rain quinacrine treatment. Chromosomes appear aligned on the metaphase plate and spindle length has decreased to 7.0 gin. Birefringence has decreased by approximately 50%, Bar: 5 gm

Fig. 5, Series of polarization and DIC light micrographs showing the effects of 0.5 gM quinacrine on a different mid-prometaphase cell. a and b Cell prior to treatment, Chromosome organization is different from that seen in Fig.4b. Spindle length measures 16,8 gm before treatment, c and d Same cell after quinacrine treatment; chromosomes have moved towards the center of the cell. Spindle length decreased by 50%. Birefringence within the spindle domain has decreased concomitant with a slight increase in astral birefringence. Bar: 5 ~tm

Fig. 6. Series of polarization and DIC fight micrographs showing the effects of 0.5 gM quinacrine on a Iate prometapha~ cell. a and b CelI prior to treatment; the chromosomes are all located at or near the metaphase plate. The spindle is more birefringent than the spindles shown in Figs. 4 and 5. e and d Same cell after quinacrine treatment. Concomitant with a decrease in spindle length there is a decrease in spindle birefringence and a slight increase of astral birefringence. Bar: 5 I~m

the as t ra l region, t han m i d - p r o m e t a p h a s e spindles

( compare Fig. 6 b wi th Figs. 4 b and 5 b). Af t e r t rea t -

ment , the c h r o m o s o m e o rgan iza t ion was no t signifi-

can t ly affected (Fig. 6 c) while spindle length decreased

by 6.0 gm or 40% and birefr ingence was s imilar ly de-

creased wi thin the spindle d o m a i n bu t sl ightly increased

in the as t ra l reg ion (Fig. 6 d).

Electron microscopy

Ut t ra s t ruc tu ra l analysis o f a cell t rea ted in mid -p ro -

me taphase with 0.5 g M quinacr ine for 3 min conf i rmed

tha t the 2 gm reduc t ion in spindle length occur red with

min ima l M T depo lymer i za t i on (Fig. 7). N u m e r o u s

n k M T s were seen in the as t ra l region and the spindle

138 Lydia Armstrong and Judith A. Snyder: Role of microtubule organization in centrosome migration in PtKI cells

Fig,7, Electron micrograph showing the effects of 0,5 ~tM quinacrine on a mid-prometaphase cell. nkMT fibers are seen within the spindle domain and associated with chromosomes. There are only a few short aMTs around the centrosome. Bar: 0.5 fxm

domain. Kinetochore fibers appeared unaffected with MTs directly inserted into the outer kinetochore lamina. aMTs in these mid-prometaphase spindles were decreased in number compared with aMTs seen in spindles treated in early prometaphase but were increased in number compared with aMTs in untreated spindles. Figure 8 is an electron micrograph of a late prometaphase cell treated with 0.5 gM quinacrine for 3 rain. Spindle length was reduced by 4 gm and there was a conspicuous paucity of nkMTs in the spindle domain. MTs were predominantly organized in bundles which ended in kinetochore regions. Some aMTs were oriented perpendicular to the longitudinal axis of the spindle (Fig. 8). A single chromosome located close to the centriole showed monotelic orientation, yet appeared

to have connections via MT bundles to a kinetochore on an equatorially aligned chromosome. Careful analysis of serial sections showed that kinetochore structure in quinacrine-treated prometaphase PtK1 cells varied considerably. Some kinetochores re- tained a "corona" (Jokelainen 1967) while others had either two or three distinct lamina. Kinetochores containing all three lamina (trilaminate) were usually found near the centrosomes after quinacrine treatment. Chro- mosomes with immature (bilaminate) kinetochores showed no specific pattern of orientation with respect to centrosome location after quinacrine treatment. Ki- netochore structure following quinacrine treatment was similar to that found in cells treated with dinitrophenot (Snyder 1988).

Lydia Armstrong and Judith A. Snyder: Role of microtubule organization in centrosome migration in P t K l cells 139

Fig. 8. Electron micrograph showing the effects of 0.5 gM quinacrine on a late prometaphase cell. Some aMTs extend perpendicular to the longitudinal axis of the spindle (arrow) and are probably responsible for the birefringence pattern seen using polarization optics. A single chromosome located close to the centriole shows monotelic orientation, yet appears to have connections via MT bundles to a kinetochore on an equatorially aligned chromosome (double arrow). The spindle is composed of mostly kMTs and a large number of aMTs. Bar: 0.5 ~tm

Computer reconstructions

Computer-generated reconstructions of early and mid- prometaphase spindles, respectively, that had been treated with 0.5 gM quinacrine for 3 rain are shown in Figs. 9 and 10. Each reconstruction included digitized locations of organelles from a set of 10 planes or sections. Although a prometaphase spindle extends through approximately 15-20 sections, only 10 sections, including both centrosomes as mid-points, were chosen for comparison of MT and chromosome organization. Planes 1-4 show the organization of predominantly nkMTs. Figure 9 b shows sections 1-10 in-

dicating a larger number of aMTs, with few MTs end- ing on kinetochores, consistent with the arrangement of early prometaphase chromosomes. Mid-prometaphase cells treated with 0.5 pm quinacrine for 3 rain show the increased number of MTs associated with kinetochores and a significant reduction in aMTs (Fig. 10; compared with similarly treated mid-prometaphase spindles shown in Figs. 4 d and 5 d). Chromo- some position was indicative of attachment to spindle fibers and alignment of chromosomes toward the equa- torial region (planes 1-4; Fig. 10 a). In Fig. 10 b (planes 1-10) the relative rearrangement of MTs from the astral region to the spindle region is pronounced. Tracking

140 Lydia Armstrong and Judith A. Snyder: Role of microtubule organization in centrosome migration in PtK l cells

Fig. 9. Computer reconstruction of an early prometaphase cell treated with 0.5 gM quinacrine for 3 min. a Four adjacent planes of the reconstruction, b Composite of planes 1-10. These reconstructions clearly demonstrate that after quiuacrine treatment of an early prometaphase cell there are few nkMTs between the centrosomes, and many long aMTs (arrow) around the centrosome

Fig. 10. Computer reconstruction of a mid-prometaphase cell treated with 0.5 IxM quinacrine for 3 rain. a Planes 1-4 of the reconstruction. b Composite of planes 1-10. The increased number of planes demonstrates an increase in kMTs and a reduction of aMTs

Fig. 11. Computer reconstruction of the same late prometaphase cell shown in Fig. 6. a Two adjacent planes, b Composite of four contiguous planes. MT fragments are prevalent in the spindle domain (arrows). There is an increase in all classes of MTs compared to Figs. 9 a and 10 a


of these MTs showed that over 70% were associated with kinetochores. The computer reconstruction of serial sections from the late prometaphase cell in Fig. 6 is shown in Fig. 11. Two planes in the centriole-containing region show a different rearrangement of MTs compared to a similarly treated mid-prometaphase cell (Fig. 11 a). aMTs are evident with MT fragments prevalent in the spindle domain (Fig. 10 a). The reorganization of MTs which results from this treatment is more clearly seen in the composite reconstruction shown in Fig. 11 b. Four sections are reconstructed which show an increase in all classes ofMTs. A comparison of Fig. 11 b with Fig. 10 a gives a distinct impression of an increase in all classes of MTs (aMTs, kMTs, and nkMTs).

Discussion

Prometaphase spindle formation

The function of many mitotic components, including MTs, is thought to be dependent, in part, on their spatial organization. Chromosome movements are un- doubtedly related to changes in number, length, and arrangement of MTs during the mitotic process (Hir- aoka et al. 1990, Kuriyama and Borisy 1981, Mitchison and Kirschner 1984, Oud et al. 1989, Snyder and Mc- lntosh 1975, Steffen and Fuge 1982). Studies of mitotic prophase-to-prometaphase spindle dynamics (Rattner and Berns 1976 b, Roos 1973, Aubin etal. 1980) have shown that the majority of prometaphase centrosomal MTs extend over long distances and can initially interact diagonally between the pole- pole axis being established (Fig. 12). Few aMTs are found in parallel arrangement along the axis of separation in PtK~ cells. Therefore, movement of centrosomes during prometaphase (pathway B) may be unlike that of anaphase when close, parallel, and lateral interactions are thought to be the driving force for uni- directional pole-pole separation. Centrosome separation is likely dependent on both the lateral interactions between the number and length of MTs from each aster as well as the relative proportion of MTs associated with kinetochores. Quinacrine treatment of PtK1 anaphase cells has been previously shown to disrupt anaphase B but not anaphase A chromosome motions. Treatment of prometaphase cells with concentrations of quinacrine less than 2 ~tM tends to increase aMTs with little change in MT number or length. This increase in astral birefringence can occur without centrosome separation, leaving an astral migration force related to the number of MTs,

aMTs \ J ~ !'~rosome

A

Fig. 12. Diagrammatic representation showing centrosome separation. A aMTs extend and may interact diagonally from the centrosomes forming cross-bridges (arrow). B As centrosomes separate, cross-linked nkMTs may move to a parallel position to the axis of separation temporarily while longer aMTs interact with each other

as examined in anaphase (Bajer etal. 1980, Sullivan and Huffaker 1992), an unlikely mechanism for centrosome separation. However, in taxol-treated cells astral birefringence can increase as centrosome separation is inhibited, yet saltatory motions remain (De Bra- bander et al. 1986). The concentrations of quinacrine used in this study were sufficiently low such that ATP-requiring micro- filament function was probably not affected. Early prometaphase cells treated with 10gM cytochalasin B demonstrated centrosome separation. Therefore, mi- crofilaments may not play a decisive role in centrosome migration. As an internal control we have previously shown that only concentrations of quinacrine greater than 12 ~tM affected the rate of contraction of the con- tractile ring during cytokinesis, and concentrations greater then 10 ~tM showed an increase in metaphase cell rounding, suggesting cell adhesion was affected (Armstrong and Snyder 1987, 1989). Cells in mid-prometaphase treated with 2 ~tM quinacrine showed rapid movement (approximately 2 ~tm/ min) of the centrosomes towards the chromosomes compared to similarly treated metaphase cells (0.6 Ixm/


min; Armstrong and Snyder 1987). In untreated mid- prometaphase cells spindle shortening occurred at 0.3 gm/min. Spindle shortening was maximal in quinacrine-treated mid-prometaphase cells, a result which could be associated with the relatively low number of nkMTs in the spindle domain. A considerable proportion of nkMTs were aMTs during early prometaphase and by metaphase the percentage of aMTs and/ or nkMTs decreased concomitant with an increase in kMTs (Church and Lin 1986). Therefore, during mid- prometaphase, nkMTs from the astral regions were mostly recruited by kinetochores instead of forming lateral associations with MTs from the other aster. This could also explain the normal spindle shortening seen during the prometaphase-to-metaphase transition. In the absence of nkMTs, which is thought to provide the counterbalancing force which ultimately limits the plateward centrosome movement at metaphase and de- fines spindle length (for discussion see Snyder etal. 1984, 1985), a rapid decrease in spindle length would be expected after quinacrine treatment during early prometaphase. The number of kinetochore fibers were unaffected by the low concentrations of quinacrine used in this study, data which were enhanced by computer- aided reconstructions. Although, once chromosomes attached to aMTs during mid-prometaphase, quinacrine treatment affected the directed movement of centrosomes towards the metaphase plate. Quinacrine may also modulate MT depolymerization which could ac- count for modifications in chromosome congression. However, in quinacrine-treated cells, where chromosomes are not yet associated to MTs, the computer reconstructions suggest the lengths of aMTs are unaffected by treatment. The results presented in this study indicate that there are energy-requiting MT-MT linkages formed during prometaphase which are necessary for centrosome separation and spindle formation during prometaphase. However, this result does not eliminate the possibility that quinacrine disrupts the normal ATP-requiring process of MT instability which is also relevant to centrosome separation. Centrosome migration away from each other (prior to spindle formation) and the rate of centrosome migration towards each other (during spindle formation) are both highly quinacrine sensitive. These results also suggest that prometaphase consists of three distinct periods: (1) centrosome migration during early prometaphase, (2) recruitment of nkMTs by the kinetochore during mid-prometaphase, and (3) development of the nkMT continuum necessary to con- tinue spindle formation during late prometaphase.

Acknowledgements We would like to thank Sandra McLelland for her expert technical assistance. This research was supported by the National Science Foundation (Grant DCB 8903966 to J.A.S.).

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Role of microtubule organization in centrosome migration and mitotic spindle formation in PtK1 cells

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