Quinacrine-induced changes in mitotic PtK1 spindle microtubule organization

Cell Motility and the Cytoskeleton 7:lO-19 (1987)

Quinacrine-Induced Changes in Mitotic PtK, Spindle Microtubule Organization

Lydia Armstrong and Judith Armstrong Snyder

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

Quinacrine, an acridine derivative which competitively binds to ATP binding sites, has been used to study the role of ATP requiring molecules in microtubule organization in mitotic PtK, cells. Brief treatments of metaphase cells with concentrations of quinacrine ranging from 2 to 10 p M decreased spindle length and birefringence in a concentration-dependent manner. With either increasing quinacrine concentrations or duration of treatment, metaphase cells demonstrated a specific reorganization of spindle microtubules. Both polarization and electron microscopy showed a substantial loss of non-kinetochore spindle microtubules with an increase in astral microtubules: this was particularly evident in the region adjacent to the spindle domain. Addition of millimolar concentrations of dinitrophenol to quinacrine-containing medium did not potentiate the response of metaphase cells to quinacrine treatment. Time-lapse video analysis demonstrated that the astral microtubules are the result of reorganization of spindle microtubules. These data suggest that functional ATP binding sites are required to maintain stable interactions between microtubule5 and that these interactions are responsible for maintaining the bowed configuration of non-kinetochore spindle microtubules which are under compression at metaphase.

Key words: mitosis, mitotic apparatus

INTRODUCTION

Energy requirements for spindle organizatior, and chromosome motions during mitosis have recently come under investigation in an effort to understand the nature of the bioenergetics which control the various “motors” required for mitotic events. Some studies have employed metabolic poisons to ascertain how changes in cellular ATP levels affect mitotic function in mammalian cells [Spurck et al, 1986a,b] while others have employed re- agents which specifically block ATPase activity [Cande and Wolniak, 1978; Cande, 19821. Vanadate, a potent inhibitor of the flagellar microtubule ATPase dynein, has been shown to block chromosome motion during anaphase in permeabilized cell models. This treatment also induced changes in spindle shape, suggesting that a dynein-like molecule is necessary to maintain spindle microtubule organization at metaphase [Cande and Wolniak, 19781. However, as was cogently pointed out by Hepler and Palevitz [ 19861, permeabilized PtKl cells incubated in millimolar concentrations of ATP only approached

0 1987 Alan R. Liss, Inc.

30 % of normal anaphase chromosome movements; furthermore, inhibitor treatments showed wide variation in chromosome motion over a fraction of the distance chromosomes normally travel. Depletion of cellular ATP levels with millimolar concentrations of dinitrophenoli deoxyglucose also produced changes in PtKl spindle microtubule organization, prominently displayed as a reduction in metaphase spindle length [Spurck et al, 1986al; furthermore this treatment blocked both chromosome-to- pole motion and pole-pole separation in similarly treated anaphase cells.

Each approach has provided information concern- ing the relative energy requirements for chromosome

Received May 20, 1986; accepted August 18, 1986.

Address reprint requests to Dr. Judith A. Snyder, Department of Biological Sciences, University of Denver, College Park, Denver, CO 80208-0178.

a

C

b

d

f Fig. 1. Series of polarization and differential interference contrast show the same cell 2 min after 2 pM quinacrine treatment. Spindle light micrographs showing the effect of 2 p M quinacrine on a meta- length and birefringence are reduced. Polarization optics show an phase PtK, cell. a,d are polarization and differential interference increase in astral birefringence.r and f show the same cell 5 min after contrast light micrographs (respectively) of a metaphase cell prior to quinacrine treatment. Arrows point to regions showing pronounced treatment. The spindle region is clearly dcfined by the birefringence changes in birefringence patterns. f shows increased chromosome pattern in a, and the arrangement of the chromosomcs at the meta- condensation with treatment and a decrcasc in spindle length. Bar is phase plate and the zone of organelle exclusion is seen in d. b and e 10 p m .

motion in PtKl cells, particularly the events during anaphase [Cande, 19821; yet neither approach has directly focused on energy requirements or the molecules necessary to maintain the shape of the metaphase spindle while under compression [LaFountain, 1972; Leslie and Pick- ett-Heaps, 19831. There is also little data which examine

how the relative numbers and organization of spindle microtubules affect the mitotic process [for review Mc- Intosh, 19851. The existence of electron-dense filaments linking adjacent spindle microtubules has previously been documented in spindles from a wide range of organisms [Bajer, 1973; McIntosh, 1974; InouC and Ritter, 1975;

a b

C d

e f Fig. 2. Series of polarization and differential interference contrast light micrographs showing the effect of 5 pM quinacrine on a metaphase PtK, cell. a and d show the cell prior to treatment. b and e are the same cell 5 min after treatment. Spindle length and spindle birefringence are reduced by approximately 25%. c and f show the same cell 10 min after treatment. Increases in astral birefringence and reduction in spindle length are prominent. Bar is 10 pm.

McDonald et al, 19791. These cross-bridges have been ciently strong interactions with adjacent microtubules of implicated as having a dynein-like role in the sliding of opposite polarity (from opposite spindle poles [Haimo microtubules past one another, particularly during the and Telzer, 1981; Euteneuer and McIntosh, 1981 I) such later events of anaphase in both metazoan cells [Cande that a stable continuum is formed between two spindle and Wolniak, 19781 and the diatoms [Cande and Mc- poles via these interactions [McDonald et al, 1978; Sny- Donald, 19851. A second function of this, or perhaps der et al, 1984; Snyder et al, 19861. In metazoan cells another group, of molecular bridges is to form suffi- this continuum is thought to bend in response to spindle

Quinacrine Effects on Spindle Organization 13

For immunofluorescence, PtK, cells were grown as monolayer culture on 22-mm2 coverslips. Following quinacrine treatment they were fixed in anhydrous meth- anol at -20°C for 10 min, then rehydrated in calcium- free phosphate-buffered saline (PBS). The coverslips were then incubated in a 1:20 dilution of a monoclonal anti-tubulin antibody at 37°C for 45 min. The properties of the anti-alpha tubulin antibody have previously been described [Mullins et al, 19861. Following three washes in PBS, the coverslips were incubated in a biotinylated anti-mouse conjugate (Vector), diluted 150 in PBS for 45 min at 37°C. Following three more washes in PBS, the coverslips were incubated in fluorescein-conjugated avidin (Vector), diluted 150 for 30 min at 37°C. Cover- slips were then washed three times in PBS; once in distilled water; and mounted in 10% Gelvatol, 30% glyc- erol, and 4 % n-propyl gallate in PBS. Micrographs were taken on a Zeiss IM 35 microscope with Tri-X film and developed in Kodak HC-110.

Cells were prepared for electron microscopy by standard techniques and as described elsewhere [Snyder et al, 19831, then examined with a Hitachi HU-I IC electron microscope.

shortening during the prometaphase to metaphase transition [Snyder et al, 19861, thus “holding” the compression observed in the metaphase spindle [LaFountain, 1972; Leslie and Pickett-Heaps, 19831.

In this paper we employ quinacrine, an acridine derivative, which has been shown to compete selectively for ATP binding sites with different affinities [Neraj et al, 19831. Quinacrine also has been shown to competitively inhibit 50% of F1 mitochondria1 ATPase activity in vitro at half-millimolar concentrations [Laikind and Allison, 19831. We report here the effects of micromolar quinacrine concentrations on metaphase spindle microtubule organization and spindle functions in both PtK, cells and newt lung cells.

MATERIALS AND METHODS

PtK, cells were grown in monolayer culture in Ham’s F-12 medium supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY) in a 37°C C 0 2 incubator. For experimentation cells were subcultured onto 25-mm round coverslips 48 h prior to use. Coverslips were inverted onto glass microscope slides with coverslip fragments used as spacers, and waxed on two edges with valap. Quinacrine hydrochloride [6-chloro-9-{ (4-(dieth- ylamino) - 1 -methy lbuty 1)amino ] -2-methy oxyacridine] di- hydrochloride (Sigma, St. Louis, MO) was dissolved directly into Ham’s F-12 medium containing 10% fetal bovine serum to final concentrations of 2, 5 , and 10 pM. Quinacrine-containing medium was introduced to cells on the microscope stage by exchanging conditioned medium with several volumes of quinacrine-containing medium.

Sheets of lung mucocilary epithelium were initially removed aseptically from Turicha grunulosa lungs. Lung tissue was minced into approximately 1 mm3 fragments at room temperature into medium as previously described by Hard and Rieder [ 19831. Fragments were placed onto the surface of carbon-coated 22-mm2 coverslips in 35 x 10-mm Corning tissue culture dishes. These explants were incubated at 22°C in a 5% C02 incubator for 8-12 days. For experimentation mitotic cells were identified with an inverted microscope then prepared for light microscopy as described above. A Zeiss Photomicroscope I1 was used to record the effects of quinacrine treatment on newt lung metaphase spindles.

Polarization and differential interference contrast (DIC) micrographs were taken on a Zeiss Photomicro- scope I1 with a 4 0 x objective (N.A. = 0.85). A M30 Brace-Kohler compensator was used to measure birefringence. Light micrographs were recorded on Kodak Tri- X film and developed in Diafine. Video analysis was done with a Dage NC 67M camera and recorded on a Gyyr time lapse recorder.

RESULTS Light Microscopy

Quinacrine was applied to PtKl cells in late metaphase in concentrations ranging from 2 to 10 p M for up

10 ‘

I 2 3 4 a IY IN)

Fig. 3. Graph showing the changes in metaphase spindle length with 2 (A) , 5 ( O ) , and 10 1 M (0) quinacrine treatment. Each point represents the average of at least ten cells. Increasing quinacrine concentrations increase both the rate and extent of spindle shortening as measured by pole-pole distances.

14 Armstrong and Snyder

to 15 min. Figure 1 is a series of polarization and DIC nounced increase in birefringence in the astral region, micrographs showing the results of 2 pM quinacrine concentrated in the areas perpendicular to the spindle treatment on a metaphase cell for 5 min. Figure la,d axis. As with the 2 pM quinacrine treatment, the birefrin- shows the same metaphase cell prior to treatment with 2 gence pattern suggests there are fewer astral microtu- pM quinacrine. Following 2 min of treatment the spindle bules between the centrosome and the cell periphery shows a reduction of approximately I5 % in birefringence parallel with the spindle axis. Longer treatments show a (Fig. lb), and the length of the spindle shortens from an more pronounced spindle shortening and almost complete average of 14 pm to 10.6 pm (n = 10). There is an loss of spindle birefringence (data not shown). increase in astral birefringence in the centrosomal region Treatment of metaphase cells with 10 pM quina- parallel to the spindle axis and perpendicular to this axis, crine show very rapid changes in spindle morphology. rather than uniformly around the centrosome. Rotation After a 2-min treatment both spindle length (Fig. 3) and of the specimen through a 45 O angle confirms preferen- birefringence are reduced by approximately 40%. As tial orientation of astral microtubules close to the spindle seen with lower concentrations of quinacrine treatment, domain as judged by birefringence patterns. Astral bire- the number of astral microtubules increases, particularly fringence is comparatively less in the region parallel to evident in the centrosomal region perpendicular to the the spindle axis between the centrosome and the cell spindle axis (Fig. 8a). Figure 8b is the differential inter- periphery. Changes in the birefringence pattern are more ference contrast image of the same cell in Figure 8a prominent after 5 min of quinacrine treatment (Fig. Ic). showing the reduced zone of exclusion and the condensed Arrows point to a region which shows patterns of bire- chromosomes. After 5 rnin of 10 pM quinacrine treat- fringence outside the original spindle domain. Figure Id ment the spindle shortens to 45% of its original spindle shows after 5 min of treatment that the chromosomes are length, which is also typical of the 5 pM treatment for 15 more condensed and tightly arranged at the metaphase rnin (Fig. 8a,b). Treatments longer than 5 min resulted plate and the region of organelle exclusion is similar to in loss of spindle birefringence as measured with polari- that occupied by the birefringent spindle microtubules. zation optics. Quinacrine treatment induces changes in spindle shape Rates of spindle shortening for each quinacrine such that the spindle takes on a more oval appearance. treatment are compared in Figure 3. The maximum rates The spindle's axial ratio (spindle length to spindle width) of spindle shortening in metaphase cells treated with 2, 5 changes from 1.8:1 to 1.4:l following 5 rnin of 2 pM and 10 pM quinacrine are 0.6, 1.1, and 1.6 pm/min, treatment. Spindle length decreases from 11 pm or 22% respectively (n 2 10). Rates of spindle shortening are of the original length (n 2 lo) following a 10-min treat- roughly equivalent to both loss of spindle birefringence ment. Microtubules remain focused at the spindle poles, and the percentage change in spindle length in the various but the overall organization of microtubules in the quinacrine treatments. mid-region of the spindle takes on a more oval shape Immunofluorescence analysis of anti-tubulin label- (Fig. 1c). ing has also been used to confirm the polarization optical

Treatment of metaphase cells with 5 pm quinacrine images of spindle microtubule organization in quina- causes similar but more rapid changes in spindle morphology compared to the 2 pM quinacrine treatment. After 2 rnin of treatment changes in spindle morphology and spindle length are similar to that of a metaphase cell treated for 5 min with 2 pM quinacrine. However, after 5 min of 5 pM treatment the spindle length decreases to 63% of its original length (compare Fig. 2a with 2b) and the spindle region loses approximately 25% of its birefringence and the axial ratio changes from 2.2: 1 to 1.1: 1. An increase in birefringence in the astral region is prominent (Fig. 2b). Figure 2e shows that the chromosomes remain tightly aligned on the metaphase plate and appear a more condensed after quinacrine treatment. The overall appearance of the cell is more rounded, probably in Fig. 4. Immunofluorescence analysis of tubulin containing structures response to changes in organization of other cytoskeletal in untreated PtK, metaphase spindle (4a) and metaphase cells treated elements. ~i~~~~ 21 shows the effects of 5 p~ quinacrine with 2 pM quinacrine for 5 min. a shows a densely staining spindle

region with a uniform arrangement of astral microtubules. b shows a

to approximately half that of the Pretreatment level, and the astral region, particularly near the spindle domain. Bar 10 is Pm. the spindle length is reduced by 36%. There is a pro-

b

for lo min On morphology' Birefringence drops loss of anti-tubulin staining in the spindle region and an increase in


crine-treated PtK, metaphase cells. Figure 4a shows the dle nonlunetochore microtubules almost exclusively, anti-tubulin antibody localization of metaphase spindle though some kinetochores take on the “ball and cup” microtubules in an untreated metaphase cell. The major configuration which is more typical of prometaphase concentration of staining occurs in the spindle domain, cells [Roos, 19731. Ten micromolar quinacrine treatment and astral assays of microtubules are arranged in a fairly for 5 min dissolves most non-kinetochore microtubules uniform array around each spindle pole. Quinacrine in the original spindle domain and with prolonged treat- treatment induces cell rounding, and for this reason it ment kinetochore structure is affected (Fig. 8c). The was necessary to choose an optical section which dem- trilaminar structure is present but not organized as dis- onstrated changes in spindle microtubule organization, cretely as in untreated cells. Kinetochore microtubules particularly in the astral region. Figure 4b shows the are reduced in number, and those which remain attached effect of 2 pM quinacrine treatment on a metaphase PtKl to the kinetochore no longer form a parallel arrangement. cell for 5 min. There is an increase in astral microtubules

DISCUSSION and a reduction in staining within the spindle domain. The increase in astral microtubules gives the impression of an increase in spindle length; however pole-pole dis- Quinacrine, which binds at ATP requiring sites, tances measure 7.5 pm in Figure 4a and 7.8 pm in 4b. shows specific and concentration-dependent effects on

Metaphase PtK, cells were also treated with deox- spindle microtubule organization when applied to meta- yglucose containing Ham’s F-12 salts with l mM dinitro- phase spindles for short durations and at low concentra- phenol (DNP) in addition to either 2 or 5 pM quinacrine. tions. The magnitude of changes are concentration Cellular depletion of ATP levels showed no potentiation dependent with all treatments showing varying degrees of the quinacrine response as analyzed by polarization of a) loss of spindle birefringence, b) reduction in the (Fig. 5 ) and electron optics, number of non-kinetochore microtubules in the spindle

We have also tested the effects of quinacrine on domain and increases in astral microtubules, and c) re- spindle microtubule organization in newt lung metaphase duction in spindle length with an increase in spindle cells. Figure 6 is a series of polarization light micro- width. Quinacrine not only induces changes in spindle graphs which show the effects of 5 pM quinacrine treat- shape in a manner similar to vanadate treatment [Cande ment on metaphase newt spindles following 5 rnin of and Wolniak, 19781, but also causes the metaphase spin- treatment. Figure 6a,b shows positive and negative bire-

tance measures 4 4 pm) and asters characteristic of this cell type. Following 5 min of treatment (Fig. 6c,d) the spindle shortens by 10 pm and there is some loss of birefringence in the spindle domain and a large increase in astral microtubules (arrows).

Because timing of mitotic stages in PtKl cells is highly consistent [Hamilton and Snyder, 19821, it is pos- sible to select cells that are within 5 min of entering anaphase before quinacrine is applied. In metaphase cells treated with 2 to 10 pm quinacrine for 10 rnin or longer, the cells never anaphased. This may be related to the fact that the effects of quinacrine are not readily reversible within the period of the mitotic cycle.

Electron Microscopy

fringence patterns and the large spindle (pole-pole dis- a b

Electron microscopy of a PtK, cell treated with 10 pm quinacrine for 2 min shows the reorganization of spindle microtubules (Fig. 7). Most of the microtubules c remaining in the spindle domain contribute to kinetochore fibers. Arrows point to microtubules in the astral

d

region, located perpendicular to the spindle axis, which probably account for the birefringence patterns Seen in Fig. 5. Polarization light micrographs showing the effect of 1 mM

dinitrophenol and 2 pM quinacrine on spindle microtubule organiza-

and associated microtubules varies with quinacrine treat- the cell prior to treatment. c and d show a decrease in spindle length ment. Two micromolar quinacrine treatment affects spin- and an increase in birefringence in the astral region. Bar is 10 pm.

light polarization Optics. The Of the kinetochore tion following 10 min. a and b are polarization optical pairs showing

16 Armstrong and Snyder

a b ure 9A is a diagrammatic representation of the metaphase spindle showing normal astral microtubules (aMTs), the kinetochore microtubule continuum (kMTs), and the microtubules of the non-kinetochore microtubule continuum (nkMTs). The region of overlap of non-kinetochore microtubules from each spindle pole is further defined by bridges. This model clearly oversimplifies the classes of microtubules which exist in a typical metazoan spindle: for a recent and more detailed description see Pickett- Heaps et a1 [1986]. Quinacrine may selectively block normal function of an ATP requiring molecule which acts to link non-kinetochore microtubules from each half spindle together. As the stability of the linkages are disrupted by quinacrine action (Fig. 9B), the interactions between microtubules weaken. Those microtubules in the spindle periphery which show the most curvature are the first to break their interactions. At some point the natural rigidity of the microtubule is greater than the forces linking it to another microtubule, allowing some microtubules to splay out of the spindle domain. One candidate for this molecule is the vanadate-sensitive ATPase pro-

C d Fig. 6. Polarization light micrographs of a metaphase newt lung spindle prior to and following 2 p M quinacrine treatment for 5 min. a and b show microtubule organization in an untreated cell. c and 6d are polarization optical images of the same cell treated with 2 pm quinacrine for 5 min. The spindle has shortened 10 pm and there is a large increase in astral birefringence outside the spindle domain (arrows). Bar is 20 pm.

dle to lose birefringence and shorten. Similar changes in spindle length have been documented in cells treated with pressure [Salmon, 1975; reviewed in InouC, 19811 and by tubulin subunit dilution in lysis buffers [Cande et al, 19741. In metaphase or anaphase PtK, cells treated with the microtubule poison nocodazole, at a concentration sufficient to depolymerize both classes of spindle microtubules, the spindle poles move to each side of the metaphase plate [Cande et al, 1974; Snyder et al, 19831. The less stable non-kinetochore microtubules depolymerize first, followed by the kinetochore microtubules; these events presumably allow the poles to move plateward.

These results indicate that non-kinetochore spindle microtubules are responsible for holding the poles apart and defining pole-pole distance at metaphase. Changes in pole-to-metaphase plate distance are usually symmetric in each half-spindle, suggesting a linkage exists, probably mediated by microtubules between both spindle poles. The stability of the non-kinetochore microtubule continuum must be dependent on the relative strength of interactions between microtubules from each half spindle and Fig. 7. Electron micrograph showing the effects of 10 p M quinacrine

treatment for 2 min on a metaphase cell. There is an increase in astral the intrinsic flexibility of microtubules , One hypothesis microtubules (arrows) perpendicular to the spindle domain and a

which could account for quinacrine-induced changes in decrease in non-kinetochore microtubules within the spindle region. mitotic spindle organization is outlined in Figure 9. Fig- Chromosome-to-pole distance is also reduced. Bar is 1 prn.

a


b

C Fig. 8. Effects of 10 pM quinacrine treatment on spindle and kinetochore structure. a and b are polarization and differential interference contrast micrographs, respectively, showing the effects of a 5- rnin, 10 p M quinacrine treatment on spindle morphology. Loss of spindle birefringence and increase in astral microtubules is evident. Bar is 10 prn. c is an electron micrograph of the kinetochore region of a cell treated similarly to that in a and b. The kinetochore trilarninar structure is present but not as discretely organized as in untreated cells. The number of microtubules inserted into the kinetochore is less than seen in control cells and, though attached, do not form a parallel bundle. Bar is 0.25 Fin.

tein identified by Hollenbeck et a1 [ 19841 which has been shown to cross-link microtubules in vitro.

Quindcrine-induced loss of the integrity of the non- kinetochore microtubule continuum reduces the amount of energy which is predicted to be trapped in this class of spindle microtubules by metaphase [see Snyder et al, 1986 for details]. However, the “motor” responsible for reduction in the length of the spindle during the prometaphase-to-metaphase transition remains active at meta-

phase. Micromolar concentrations of quinacrine pref- erentially affect the less stable non-kinetochore microtubule population residing in the spindle domain, thus allowing expression of a mitotic “motor” as the movement of the spindle poles towards the metaphase plate (Fig. 9B).

The fact that addition of DNP to quinacrine-containing medium has little effect on quinacrine action may be significant. DNP treatment apparently does not induce

18

A

Armstrong and Snyder

B

crotubules of opposite polarity. As such they tend to link microtubules together and to act as “brakes” on the motor of mitosis, which is thought to reside in a second continuum formed between the spindle poles, kinetochore microtubules, and the sister chromatids [Snyder et al, 19861. Secondly, spindle microtubule bridges allow compression to be loaded into the metaphase spindle in the form of microtubule curvature which is later used during anaphase to separate the spindle poles.

ACKNOWLEDGMENTS

Fig. 9. Diagrammatic representation of the effect of quinacrine 0 1 1

spindle microtubule organization of a metaphase cell. A: Astral microtubules (aMTs) are located at each spindle pole. Those microtubules ending on chromosomes and forming a continuum via the sister chromatids are designated kMTs. Non-kinetochore microtubules (nkMTs) from each spindle pole are shown to overlap and be linked by bridges. B: Following brief quinacrine treatment some nkMTs in the periphery of the spindle lose the ability to maintain strong lateral interactions. The curvature of these microtubules is lost as the tubules splay out of the spindle domain, forming an increase in astral microtubules. As this class of microtubules is reduced, forces residing in the kMT continuum are expressed as spindle shortening.

We thank Sandra McLelland for her expert techni- - cal assistance with electron microscopy and Timothy

rigor as predicted from muscle and flagellar models; instead it suggests stable interactions between microtubules of opposite polarity exist by metaphase when cellular ATP concentrations are not depleted. This does not preclude analogy with the flagellar model; there may be at least two classes of molecular bridges in the metaphase spindle-one with ATPase activity with active sliding microtubule-microtubule interactions (ie, flagellar dynein), and a second, more stable set, which restricts sliding of microtubules past one another (ie, nexin and radial spokes in the flagellum [for review see Gibbons, 19811).

Quinacrine-induced changes in spindle microtubule organization may include alterations in internal calcium concentrations. Clearly, calcium is an important messen- ger for triggering sister chromatid separation [Izant, 1983; Hepler, 1985; Wolniak and Barth, 19861, yet calcium ionophore treatment has not been shown to affect prometaphase chromosome movement [Fuller and Brink- ley, 19761 or anaphase chromosome motions [Spurck et al, 19861. Therefore, it seems unlikely that micromolar concentrations of quinacrine, which also do not stop anaphase motion significantly, affect internal calcium concentrations.

In summary, low concentrations of quinacrine apparently interfere with ATP requiring linkages between spindle microtubules, particularly non-kinetochore mi-

Spurck for his assistance with newt cell culture. This work was supported by the American Cancer Society (CD-173) , the National Science Foundation (PCM 8409118), and the Milheim Foundation (84-26) to J.A.S.

REFERENCES

Bajer, A.S. (1973): Interaction of microtubules and the mechanism of chromosome movement (Zipper hypothesis). Cytobios 8: 139- 160.

Cande, W.Z., Snyder, J.A., Smith, D., Summers, K., and McIntosh, J.R. (1974): A functional mitotic spindle prepared from mammalian cells in culture. Proc. Natl. Acad. Sci. USA 71:1559- 1563.

Cande, W.Z., and Wolniak, S.M. (1978): Chromosome movement in lysed mitotic cells is inhibited by vanadate. J. Cell Biol.

Cande, W.Z. (1982): Nucleotide requirements for anaphase chromosome movements in permeabilized mitotic cells: Anaphase A but not anaphase B requires ATP. Cell 28: 15-22.

Cande, W.Z., and McDonald, K.L. (1985): In vitro reactivation of anaphase spindle elongation using isolated diatom spindles. Nature 3 16: 168- 170.

Gibbons, I.R. (1981): Cilia and flagella of eukaryotes. J . Cell Bid.

Fuller, M., and Brinkley, B.R. (1976): Structure and control of assembly of cytoplasmic microtubules in normal and trans- formed cells. J . Supramol. Struct. 5:497-514.

Haimo, L.T., and Telzer, B.R. (1981): Dynein-microtubule interactions. ATP sensitive dynein binding and the structural polarity of mitotic microtubules. Cold Spring Habor Quant. Biol. Symp.

Hamilton, B.T., and Snyder, J.A. (1982): Rapid completion of mitosis and cytokinesis in PtK-1 cells following release from nocodazole arrest. Eur. J. Cell Biol. 28:190-195.

Hard, R., and Rieder, C. (1983): Mucilary transport in newt lungs: The ultrastructure of the ciliary apparatus in isolated epithelial sheets and in functional triton-extracted models. Tissue Cell

Hepler, P.K. (1985): Calcium restriction prolongs metaphase in divid- ing Tradescantia stamen hair cells. J. Cell Biol. 100:1363- 1368.

Hepler, P.K., and Palevitz, B.A. (1986): Metabolic inhibitors block anaphase A in vivo. J . Cell Biol. 102:1995-2005.

Hollenbeck, P.J., Suprynowicz, F., and Cande, W.Z. (1984): Cyto- plasmic dynein-like ATPase cross-links microtubules in an ATP sensitive manner. J. Cell Biol. 99:1251-1258.

79~573-580.

91: 107s-123s.

46~207-2 18.

151227-243.

Quinacrine Effects on Spindle Organization

acrine mustard. Biochim. Biophys. Acta 764: 105-113. Pickett-Heaps, J.D., Tippet, D.H., Cohn, S.A., and Spurck, T.P.

(1986): Microtubule dynamics in the spindle. Thcoretical as- pects of assemblyldis embly reactions in vivo. J. Theor. Bid. 118: 153-169.

Ris, H. (1949): The anaphase movement of chromosomes in the spermatocytes of the grasshopper. Biol. Bull. 96:90-106.

Roos, U.-P. (1973): Light and electron microscopy of rat kangaroo cells in mitosis. 11. Kinetochore structure and function. Chro- mosoma (Berl.) 41: 195-220.

Salmon, E.D. ( 1975): Pressure induced depolymerization of spindle microtubulcs. J. Cell Biol. 66: 114-127.

Snyder, J.A., Golub, R.J., and Berg, S.P. (1984): Sucrose-induced spindle elongation in mitotic PtK-I cells. Eur. I. Cell Biol.

Snyder, J.A., Golub, R.J., and Berg, S.P. (1986): Role of non- kinetochore microtubules in spindle elongation in mitotic PtK- 1 cells. Eur. J. Cell Biol. 39:373-379.

Snyder, J.A., Vogt, S. I . , and McLelland, S.M. (1983): Nocodazole selectively reduces anaphase B in PtK-1 cells. Cell Motility

Spurck, T.E., Pickett-Heaps, J.D., and Klymkowsky, M.W. (1986): Metabolic inhibitors and mitosis: I. Effects of dinitrophenoll deoxyglucose and nocodazole on the live spindle. Protoplasm

Spurck, T.E., Pickett-Heaps, J.D., and Klymkowsky, M.W. (1986): Metabolic inhibitors and mitosis: 11. Effects of dinitrophenoll deoxyglucose and nocodazole on the microtubule cytoskeleton. Protoplasma 13 1 :60-74.

Wolniak, S.M., and Barth, K.M. (1986): Nifedipinc reversibily ar- rests mitosis in stamen hair cells of Tradescantia. Eur. J. Cell Biol. 39:273-217.

19

35 :62-69.

3179-91.

131~47-59.

Inoue, S. , and Ritter, H., Jr. (1975): Dynamics of mitotic spindle organization and function. In Inoue, S. and Stephens, R. (eds.): “Moleculcs and Cell Movement.” New York: Raven Press,

InouC, S. (1981): Cell division and the mitotic spindle. J. Cell Bid. 91:131~-147s.

Izant, J.G. (1983): The role of calcium ions during mitosis. Chromo- soma (Berl.) 88: 1-10.

LaFountain, J.R., Jr. (1972): Spindle shape changes as an indicator of force production in crane-fly spermatocytes. J. Cell Sci.

Laikind, P.K., and Allison, W.S. (1983): Quinacrine mustard inacti- vates the bovine heart mitochondria1 F-l ATPase with the modification of the B-subunit. J. Biol. Chem. 11700-11704.

Leslie, R.J., and Pickett-Heaps, J.D. (1983): Ultraviolet microbeam irradiations of mitotic diatoms: Investigations of spindle elongation. J. Cell Biol. 96:548-661.

McIntosh, J.R. (1974): Bridges between microtubules. J. Cell Biol.

McIntosh, J.R. (1985): Spindle structure and the mechanisms of chromosome movement. In Dellarco, V.L., Voyter, P.E., and Hollaender, A. (eds.): “Ancuploidy.” New York: Plenum Press, pp. 197-228.

McDonald, K.L., Edwards, M.K., and McIntosh, J.R. (1979): Cross- sectional structure of the central mitotic spindle of Diutoma vulgare: Evidence for specific interactions between anti-parallel microtubules. I . Cell Biol. 83:443-461.

Mullins, J.M., Wolfe, K.M., and Snyder, J.A. (1986): Immunofluo- rescence analysis of sucrose-induced changes in spindle morphology. Eur. J. Cell Biol. 39:333-340.

Neraj, H., and Kaira, V.K. (1983): Inactivation of the site of labeling of F-1 ATPase from Mycobacterium pheli by dicyclohexylcar- bodiimide, 7-chloro4-nitrobenzo-2-oxa- 1,3-diazole and quin-

pp. 3-30.

10x79-93.

6 1 : 166- 187.

Quinacrine-induced changes in mitotic PtK1 spindle microtubule organization

Documents

Transcript of Quinacrine-induced changes in mitotic PtK1 spindle microtubule organization