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Chapter 19 An Assay for the Activity of Microtubule-Based Motors on the Kinetochores of Isolated Chinese Hamster Ovary Chromosomes
CHAPTER 19
An Assay for the Activity of Microtubule-Based Motors on the Kinetochores of Isolated Chinese Hamster Ovary Chromosomes A. A. Hyman and T. J. Mitchison Department of Pharmacology University of California, San Francisco San Francisco, California 94143
1. Introduction 11. Methods tor Assaying Motor Activity on the Kinetochore A. Chromosome Preparation B. Microscope Requirements C. Setup D. Recording Minus-End-Directed Movement E. Recording Plus-End-Directed Movement F. Variables in the Assay III. Conclusions References
I. Introduction During mitosis, chromosomes attach to mitotic spindle microtubules via specific structures called kinetochores. Following microtubule attachment, the kinetochore-microtubule attachment mediates complicated patterns of chromosome movement which lead eventually to correct chromosome movement. Many recent experiments have suggested that microtubule-based motors may be responsible in part for driving the movements of kinetochores on microtuMETHODS IN CELL BIOLOGY, VOL. 39 Copyrighr 0 lYY3 by Academic Press. Inc. All nghrs of reproducrloo in any form reserved
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bules (McIntosh and Pfarr, 1991); however, the complexity of mitosis in vivo has meant that it is difficult to examine the detailed role and function of microtubule-based motors in mitosis. This chapter describes a detailed method derived from a published procedure for examining the behavior of microtubulebased motors on the kinetochores of isolated chromosomes (Hyman and Mitchison, 1991b). In brief, chromosomes are isolated from Chinese hamster ovary (CHO) cells arrested at prometaphase using microtubule inhibitors, by swelling followed by digitonin lysis, and are then collected on a sucrose gradient in a low-ionicstrength buffer. This buffer minimizes extraction of loosely bound kinetochore components. The isolated chromosomes are adsorbed to the glass slide of a perfusion chamber. To assay the microtubule-based motor activity of the kinetochores of these isolated chromosome, polarity-marked microtubules (see Chapter 7) are attached to the kinetochores. After addition of ATP, the movement of these microtubules over the kinetochores of the isolated chromosomes is followed by video microscopy.
11. Methods for Assaying Motor Activity on the Kinetochore A. Chromosome Preparation
This procedure is based on Mitchison and Kirschner (1985).
I . Take 20 plates of CHO cells that are one division before confluence growing in minimum essential medium (MEM) + nucleosides + 10% supplemented calf serum (University of California, San Francisco, cell culture facility) and add 10 pg/ml vinblastin final concentration to each plate. Leave cells in drug for 8 hours. 2 . Rinse the mitotically arrested cells from the plates using gentle squirts of medium with a Pasteur pipet. Overvigorous washing will remove cells in interphase. 3. Spin down the cells at lOOOg for 5 minutes at room temperature. The cells will form a loose pellet. 4. Remove all the medium, and resuspend in swelling buffer (5 mM K-Pipes, 5 mM NaCI, 5 mM MgCI2, 1 mM EGTA, pH 6.8), at 30°C for 5 minutes. We find that swelling at 30°C improves the release of chromosomes on lysis. 5. Spin down the cells at lOOOg for 5 minutes at 30°C. Remove excess swelling buffer and resuspend in 7 ml lysis buffer [ 10 mM potassium, 1,4-piperazinediethanesulfonic acid (K-Pipes), 1 mM MgC12, I mM ethylene glycol bis(P-
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aminoethyl ether) N,N’-tetraacetic acid (EGTA), 0.1% digitonin from a 10% stock in dimethylsulfoxide (DMSO), pH 7.21 on ice. Immediately transfer to a 10-ml glass Dounce homogenizer with a tight pestle and administer 20 rapid strokes on ice. The assay of kinetochore activity relies on specific capture of microtubules by kinetochores, and trapping by chromosome arms obscures the assay. This trapping appears to be caused by adhesion of cytoskeletal fragments to the chromosome arms, not to chromatin. In general, the cleaner the chromosome preparation, the fewer the number of microtubules attached to the chromosome arms. We find that clean chromosomes are obtained only if the cells lyse within the first few strokes of the pestle. 6. Transfer the lysate to a sucrose step gradient made with 2-ml steps of 30, 40, 50, and 60% (w/v) sucrose in 10 mM Pipes, pH 7.2, 5 mM MgCI2, 1 mM EGTA, prepared in a 15-ml glass Corex tube. Spin at 5K for 15 minutes at 4°C in a Sorval HB4 swinging bucket rotor. The chromosomes accumulate as flocculent white material at the 40-50 and 50-60% interfaces. Collect them in a minimum volume using a Pasteur pipet. Freeze in liquid nitrogen in 10-pl aliquots. Store at -80°C. Chromosomes remain functional for 3 months at this temperature.
B. Microscope Requirements We use a Zeiss standard microscope equipped for epifluorescence, because simple microscopes such as these give the greatest light throughput. Simple Nikon or Olympus upright scopes should perform equivalently. The objective was a 60x DPlanApo 1.4 Olympus lens, but the equivalent Nikon lens also works. Increased magnification to the video camera is provided by a Zeiss 4 X tube. The magnification is chosen so that one chromosome with its microtubules (ca. 40 pm wide) fills a field on the video camera, ensuring high resolution and allowing us to distinguish plus- from minus-end-directed movement. A siliconintensified-target (SIT) camera is used to generate the images, four to eight frames are averaged with an image processor, and a Panasonic OMDR is used to record images. Kinetochores with rhodamine-labeled microtubules attached are very sensitive to light damage, and shuttering of the light is essential to record movement of the microtubules on the kinetochores. A fully functional antifade system is absolutely necessary to prevent oxygen radical-induced damage. We usually record images every 3 seconds. The whole system can be coordinated using an Image-] AT image processor (Universal Imaging Corporation) or a different image processor with homewritten shutter control software. It is fairly easy even for beginning programmers to write shutter control and OMDR control into software that controls a simple averaging device, such as a Hamamatsu Argus 10. We use a Data Translation I 0 board (DT-2817) together with a Screw terminal plate (DT-758c) for controlling shutters and filter wheels.
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C.Setup We first describe a basic motility assay and then consider some of the variables associated with the assay. The assay is very sensitive to variables such as time of incubation and washing, so great care and attention must be paid these factors to obtain reproducible results. We try not to let the time vary more than 5 seconds at each stage. It is further recommended that all reagents be stored as small frozen aliquots which can be used fresh for each experiment. We are standardly able to prepare four individual chambers in one experiment using this protocol. 1. Make perfusion chambers using two strips of double-stick tape (3M No. 665) on a glass slide to support a 22-mm2coverslip. Cool the chambers to 0-4°C on a metal block placed on ice. 2. Thaw the chromosomes and dilute 1/300 in PME (10 mM K-Pipes, pH 7.4, 5 mM4 mM MgC12,l mMEGTA, 100 pglml leupeptin, 100 pglml pepstatin, 100 pg/ml chymostatin, 5 mM dithiothreitol) containing 10% bovine serum albumin (BSA, Sigma No. A7638). Polyamines were used previously to stabilize chromosome arms, but in the real-time assay they cause the seeds to stick to the glass surface of the perfusion chamber and therefore must be avoided. The PME is warmed to 37°C for 10 minutes and returned to ice before addition of chromosomes, as this appears to help the BSA subsequently in blocking the binding of other proteins to the glass. 3. The diluted chromosomes are perfused into the chamber and left for 5 minutes (step 1, Fig. 1). The chromosomes tend to stick preferentially to the glass slide over the coverslip, and all subsequent experiments describe complexes attached to the glass slide. BSA prevents most other proteins from binding subsequently to the glass, although some free microtubule motors will bind to the glass. The slide, which forms the bottom surface of the chamber, gives inferior optical visualization compared with the coverslip undersurface, but it was used in all our assays because of its more favorable surface chemistry. 4. Stable, rhodamine-labeled microtubule seeds are made. Three steps (4-6) result in the attachment of segmented, polarity-marked microtubules (see Chapter 7) to the chromosomes attached to the glass slide. First, the brightly labeled seeds are attached to the kinetochores. To survive the washing steps, in which the unattached seeds are removed from the chamber, the seeds must be stabilized. Taxol cannot be used in these experiments because it will interfere with the subsequent growth of GTP-tubulin from the seeds. In our experiments, the seeds are made from GMPCPP-tubulin. Microtubules polymerized in the presence of GMPCPP are stable to dilution (Hyman et al., 1992) and can therefore be attached to kinetochores and washed without having to worry about stabilization as with GTP microtubules. A protocol to synthesize GMPCPP has been described (Hyman et af., 1992). To make the GMPCPP seeds, 40 pLM rhodamine-tubulin and 0.15 mM GMPCPP are mixed together
19. Microtubule-Based Motor Activity on CHO Chromosome Kinetochores
f
1)
MITOTIC CHROMOSOMES, FOLLOWED BY BSA TO BLOCK GLASS SURFACE
2)
STABLE RHODAMINE LABELED MICROTUBULES SEEDS AT HIGH CONCENTRATION, INCUBATION TO ALLOW CAPTURE.
27 1
3) WASH OUT UNCAPTURED SEEDS
f
0 0
4,
UNLABELED TUBULIN SUBUNITS + GTP + ATP
5)
PERFUSE IN ANTIFADE BUFFER
OO0
Fig. 1 Diagram to show the perfusion steps required in setting up the assay for motor activity on the kinetochores. For details see text.
(Hyman el al., 1991). This is most conveniently made up as a large volume on ice and spun 30 psi in the airfuge at 4°C to remove aggregates of tubulin formed on thawing; the supernatant is frozen in 5-p1 aliquots. Prior to use an aliquot is thawed and polymerized for 15 minutes at 37°C. 5. To attach the seeds to the kinetochores of the chromosomes that have been attached to the glass slide, the chamber is washed once (one wash is one chamber volume), in PMD (80 mM K-Pipes, pH 6.8, 1 mM EGTA, 4 mM MgC12, 5 mM dithiothreitol) + lx protease inhibitors (10 pg/ml leupeptin, pepstatin, and chymostatin), and then the slide is transferred to an aluminum block at 37°C for 30 seconds. The seeds, diluted 1/30 into PMD + l x protease inhibitors at 37"C, are then perfused into the chamber. The reaction is allowed to proceed for
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5 minutes so that the seeds are captured by the kinetochores (step 2, Fig. 1). Uncaptured seeds are washed out by perfusing the chamber with PMD + 1 rnM GTP four times at 37°C (step 3, Fig. 1). 6. To elongate more dimly labeled microtubule segments off the ends of the GMPCPP seeds, the chamber is washed twice with PMD 1 mM GTP 0. lx protease inhibitors + 3% polyethylene glycol (PEG) 3360, containing oncecycled tubulin (Hyman et al., 1991) at 15 $Wand rhodamine-tubulin (Hyman et af., 1991) at 1.5 pM.The final stochiometry is approximately one rhodamine per 10 tubulin subunits. The 3% PEG 3360 is included in the polymerization buffer to ensure growth from the minus end and also to prevent adsorption of the rhodamine-tubulin to the glass surface of the chamber. Polymerization is allowed to proceed for 10 minutes at 37°C (step 4, Fig. 1). 7. The microtubules are stabilized by washing five times in an antifade buffer containing taxol (PMD 0.2% 2-mercaptoethanol, 10 pM taxol, 5 pg/ml glucose oxidase, 10 pg/ml catalase, 10 mM glucose, 0.Ix protease inhbitors) + 1 pg/ml Hoescht 33342 (step 5, Fig. 1). The glucose oxidase and catalase are most conveniently stored as a lOOX stock in 50% glycerol at -20°C. The antifade buffer is essential to the assay. It works by scavenging oxygen, but a number of problems are associated with its use. First, the glucose tends to be used up if solutions are not kept in sealed tubes. Second, it essential that the temperature of solutions only increase, not decrease, subsequent to addition of the antifade component, because oxygen redisolving at lower temperatures uses up the glucose. Third, as the glucose oxidase works, its FAD cofactor becomes oxidized. This greatly reduces the strength of the fluorescent signal by an unknown mechanism, and may also increase the fluorescence background. It is therefore preferred that the antifade system be kept at 0°C until just prior to use. Fourth, glucose is converted to gluconic acid, potentially changing the pH of the buffer in weakly buffered solutions. Lowering of pH is particularly prevalent around bubbles if these are sealed under the coverslip. 8. If successful, the chromosomes should be attached to the glass slide, and their arms and primary constriction should be visible by Hoechst fluorescence. The degree of swelling of the chromatin arms depends on the concentration of MgCI2 in the assay. Excessive swelling reduced our ability to discriminate the primary constriction from the rest of the chromosome by Hoechst fluorescence; however we also found that swelling tended to reduce background binding of microtubule to chromosomes arms and made kinetochore visualization in the rhodamine channel easier. Thus, our buffer represents a compromise. At the primary constriction, the two kinetochores should be visible as clusters of brightly labeled seeds, and in addition the kinetochores may bind some rhodamine-tubulin directly. If seeds are present all over the slide, then too many seeds were used in the capture reaction. The more dimly labeled portions of the microtubules should radiate away from the kinetochores. If rhodaminetubulin is adsorbed to the glass surface, try increasing the concentration of PEG
+
+
+
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slightly. The most common problem is sticking of multiple microtubules to chromosome arms. This is caused by cytoskeletal debris on the arms, and must be addressed by improving the chromosome isolation as discussed above. The chromosomes should be fairly sparse, one to four per field with a 60x objective, so that individual chromosomes do not obscure each other.
D. Recording Minus-End-Directed Movement Minus-end-directed movement is the most difficult to record. The amount of minus-end growth rarely exceeds 10 pm, and because the minus-end motor moves at 40 p m per minute, only about 20 seconds elapses between addition of ATP and the microtubule running off the kinetochore. Although it is possible with practice to record minus-end-directed movement following perfusion of ATP into the chamber, for routine measurement it is easier to initiate movement by uncaging caged ATP. 1. Wash the chamber with 1 mM caged ATP in antifade buffer. It is important that caged ATP be protected from room light at all times, and we perform these manipulations under a sodium vapor safelight. 2 . Using rhodamine fluorescence, select a nice chromosome, that is, a chromosome in which the two kinetochores can be distinguished as clusters of brightly labeled seeds and no microtubules are attached to the arms of the chromosomes. 3. With the video recording sequence running, locally activate the caged ATP. The easiest way to uncage the ATP on a simple microscope system is to employ a filter used in a Hoechst or DAPI filter set. With a typical 360-nm bandpass filter, a 100-W mercury arc illuminator, and a bright 60X, 1.4NA lens, complete uncaging in a small area takes about 0.3 second. Start recording the chromosomes at 3-second intervals with a 0.3-second shutter-open time during each recording. During the 3-second wait time between recordings, switch the filter holder to the Hoechst position, so that the next time the shutter opens, the field is irradiated with 360-nm light for0.3 second, which uncages the ATP. Then switch back to rhodamine fluorescence to record any subsequent movement of the microtubules. Alternatively, more sophisticated systems using filter wheels can be employed. E. Recording Plus-End-Directed Movement
The plus-end-directed motor will not move in an ATP concentration below 100 pM.It is difficult to observe this activity by activation of caged ATP because the activated ATP rapidly diffuses away from the observation field; however, because the movement is slower and the plus-end segments are longer, it is easy to record plus-end-directed movement after perfusing 1 mM ATP in antifade buffer into the chamber, refocusing, and beginning the recording.
A. A. Hyman and T. J. Mitchison
274 F. Variables in the Assay
1. Switching between Plus- and Minus-End-Directed Movement at the Kinetochore Using ATPyS The addition of ATPyS during the capture reaction (step A, Fig. 2 ) is sufficient to switch the kinetochores from plus- to minus-end-directed movement (Hyman and Mitchison, 1991b). Different chromosome preparations have different levels of endogenous plus- or minus-end-directed movement, although movement tends to be more minus end directed in the absence of ATPyS. Therefore, for each chromosome preparation, a dose-response curve must be constructed to determine the concentration of ATPyS required to fully turn on plus-enddirected movement. The concentration required for full switching varied between 10 and 500 pM. One reason that plus-end-directed movement was routinely observed in early work (Mitchison and Kirschner, 1985) may be that GTPyS was routinely included in the assay buffer because it was used in the microtubule seed stabilization step.
2. Washing the Kinetochores Prior to the Capture Reaction A key variable in the assay is the wash between steps A and B (Fig. 2), as illustrated in Hyman and Mitchison (1991a). The relationship between washing and kinetochore motor activity varies from chromosome preparation to preparation. The general rule is that incubation of chromosomes at 37"C, without microtubules attached to their kinetochores, tends to inactivate the plus-enddirected movement system. A second reason that plus-end-directed movement was routinely observed in early work (Mitchison and Kirschner, 1985) may be that the chromosomes were not washed between microtubule capture and ATP addition. Presumably, the washing removes some weakly bound kinetochore components necessary for plus-end movement. We do not know whether this is due to inactivation of the motor itself or of the phosphorylation machinery.
3. Variation in Microtubule Number at the Kinetochore The number of microtubules attached to the kinetochore determines the concentration of ATPyS required to activate the plus-end-directed motor (Hyman and Mitchison, 1991a). At fewer than three microtubules per kinetochore it is very difficult to initiate plus-end-directed movement, even in the presence of 1 mM ATPyS. At more than six microtubules per kinetochore, plus-enddirected movement may be observed even in the absence of ATPyS. The number of microtubules attached per kinetochore can be varied by varying the number of microtubules in the capture reaction (step B, Fig. 2 ) while keeping the time constant.
0
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rhodamine
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mitotic chromosomes
labeled microtubules
Capture reaction
I+RTPySl
I
Creation o f Poiarit -marked microtubules
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11 tubulin subunits llaur leuel of label) 21 taxoi
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ATPyS step A
mlnus-enddirected mouement
directed mauement
Fig. 2 Diagram of the results of ATP addition to the kinetochores of isolated chromosomes. Summary of the movements of microtubules on the kinetochores of isolated chromosomes. (A) The capture of the microtubule seeds by the kinetochores. (B) Tubulin grows from the ends of the captured seeds. After addition of ATP, microtubules move either by minus-end directed (C) or plus-end directed (D) movement.
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111. Conclusions We have described an assay in which the motor activity of the kinetochores of isolated mammalian chromosomes can be examined by video microscopy. The assay is technically challenging: Only small numbers of chromosomes can be assayed in each experiment, and the assay is very sensitive to the buffers used and the incubation times. Multiple perfusions must be performed in reproducible time intervals, and the sequence of temperature changes cannot be varied. Under a defined set of conditions, however, the assay can be made quite reproducible. It should prove very useful for testing specific inhibitory reagents to probe the identities of the motors and kinases involved in mammalian kinetochore function. The assay demonstrates the utility of modern fluorescence microscopy in the study of subcellular reactions and obtains quantitative biochemical information on their molecular mechanisms. References Hyman, A. A., and Mitchison, T. J. (1991a). Regulation of the direction ofchromosome movement. Cold Spring Harbor Symp. Quant. Biol. 56,745-750. Hyman, A. A., and Mitchison, T. J. (1991b). Two different microtubule-based motor activities with opposite polarites in kinetochores. Nature (London) 351,206-21 1. Hyman, A. A., Drexel, D., Kellog. D., Salser, S . , Sawin, K., Steffen, P., Wordeman, L., and Mitchison, T. J. (1991). Preparation of modified tubulins. In "Methods of Enzymology" (R.B. Vallee, ed.), Vol. 196, pp. 478-485. Academic Press, San Diego. Hyman, A. A., Salser, S., Drechsel, D., Unwin, N., and Mitchison, T. J. (1992). The role of GTP hydrolysis in microtubule dynamics: Information from a slowly hydrolyseable analogue GMPCPP. Mol. Biol. Cell 3, 1155-1 167. McIntosh, J. R.,and Pfarr,C. M.,(1991). Mitotic motors. J . Cell Biol. 115,577-587. Mitchison, T. J., and Kirschner, M. W. (1985). Properties ofthe kinetochore in vitro. 2. Microtubule capture and ATP dependent translocation. J. Cell Biol. 101,767-777.
An Assay for the Activity of Microtubule-Based Motors on the Kinetochores of Isolated Chinese Hamster Ovary Chromosomes A. A. Hyman and T. J. Mitchison Department of Pharmacology University of California, San Francisco San Francisco, California 94143
1. Introduction 11. Methods tor Assaying Motor Activity on the Kinetochore A. Chromosome Preparation B. Microscope Requirements C. Setup D. Recording Minus-End-Directed Movement E. Recording Plus-End-Directed Movement F. Variables in the Assay III. Conclusions References
I. Introduction During mitosis, chromosomes attach to mitotic spindle microtubules via specific structures called kinetochores. Following microtubule attachment, the kinetochore-microtubule attachment mediates complicated patterns of chromosome movement which lead eventually to correct chromosome movement. Many recent experiments have suggested that microtubule-based motors may be responsible in part for driving the movements of kinetochores on microtuMETHODS IN CELL BIOLOGY, VOL. 39 Copyrighr 0 lYY3 by Academic Press. Inc. All nghrs of reproducrloo in any form reserved
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bules (McIntosh and Pfarr, 1991); however, the complexity of mitosis in vivo has meant that it is difficult to examine the detailed role and function of microtubule-based motors in mitosis. This chapter describes a detailed method derived from a published procedure for examining the behavior of microtubulebased motors on the kinetochores of isolated chromosomes (Hyman and Mitchison, 1991b). In brief, chromosomes are isolated from Chinese hamster ovary (CHO) cells arrested at prometaphase using microtubule inhibitors, by swelling followed by digitonin lysis, and are then collected on a sucrose gradient in a low-ionicstrength buffer. This buffer minimizes extraction of loosely bound kinetochore components. The isolated chromosomes are adsorbed to the glass slide of a perfusion chamber. To assay the microtubule-based motor activity of the kinetochores of these isolated chromosome, polarity-marked microtubules (see Chapter 7) are attached to the kinetochores. After addition of ATP, the movement of these microtubules over the kinetochores of the isolated chromosomes is followed by video microscopy.
11. Methods for Assaying Motor Activity on the Kinetochore A. Chromosome Preparation
This procedure is based on Mitchison and Kirschner (1985).
I . Take 20 plates of CHO cells that are one division before confluence growing in minimum essential medium (MEM) + nucleosides + 10% supplemented calf serum (University of California, San Francisco, cell culture facility) and add 10 pg/ml vinblastin final concentration to each plate. Leave cells in drug for 8 hours. 2 . Rinse the mitotically arrested cells from the plates using gentle squirts of medium with a Pasteur pipet. Overvigorous washing will remove cells in interphase. 3. Spin down the cells at lOOOg for 5 minutes at room temperature. The cells will form a loose pellet. 4. Remove all the medium, and resuspend in swelling buffer (5 mM K-Pipes, 5 mM NaCI, 5 mM MgCI2, 1 mM EGTA, pH 6.8), at 30°C for 5 minutes. We find that swelling at 30°C improves the release of chromosomes on lysis. 5. Spin down the cells at lOOOg for 5 minutes at 30°C. Remove excess swelling buffer and resuspend in 7 ml lysis buffer [ 10 mM potassium, 1,4-piperazinediethanesulfonic acid (K-Pipes), 1 mM MgC12, I mM ethylene glycol bis(P-
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aminoethyl ether) N,N’-tetraacetic acid (EGTA), 0.1% digitonin from a 10% stock in dimethylsulfoxide (DMSO), pH 7.21 on ice. Immediately transfer to a 10-ml glass Dounce homogenizer with a tight pestle and administer 20 rapid strokes on ice. The assay of kinetochore activity relies on specific capture of microtubules by kinetochores, and trapping by chromosome arms obscures the assay. This trapping appears to be caused by adhesion of cytoskeletal fragments to the chromosome arms, not to chromatin. In general, the cleaner the chromosome preparation, the fewer the number of microtubules attached to the chromosome arms. We find that clean chromosomes are obtained only if the cells lyse within the first few strokes of the pestle. 6. Transfer the lysate to a sucrose step gradient made with 2-ml steps of 30, 40, 50, and 60% (w/v) sucrose in 10 mM Pipes, pH 7.2, 5 mM MgCI2, 1 mM EGTA, prepared in a 15-ml glass Corex tube. Spin at 5K for 15 minutes at 4°C in a Sorval HB4 swinging bucket rotor. The chromosomes accumulate as flocculent white material at the 40-50 and 50-60% interfaces. Collect them in a minimum volume using a Pasteur pipet. Freeze in liquid nitrogen in 10-pl aliquots. Store at -80°C. Chromosomes remain functional for 3 months at this temperature.
B. Microscope Requirements We use a Zeiss standard microscope equipped for epifluorescence, because simple microscopes such as these give the greatest light throughput. Simple Nikon or Olympus upright scopes should perform equivalently. The objective was a 60x DPlanApo 1.4 Olympus lens, but the equivalent Nikon lens also works. Increased magnification to the video camera is provided by a Zeiss 4 X tube. The magnification is chosen so that one chromosome with its microtubules (ca. 40 pm wide) fills a field on the video camera, ensuring high resolution and allowing us to distinguish plus- from minus-end-directed movement. A siliconintensified-target (SIT) camera is used to generate the images, four to eight frames are averaged with an image processor, and a Panasonic OMDR is used to record images. Kinetochores with rhodamine-labeled microtubules attached are very sensitive to light damage, and shuttering of the light is essential to record movement of the microtubules on the kinetochores. A fully functional antifade system is absolutely necessary to prevent oxygen radical-induced damage. We usually record images every 3 seconds. The whole system can be coordinated using an Image-] AT image processor (Universal Imaging Corporation) or a different image processor with homewritten shutter control software. It is fairly easy even for beginning programmers to write shutter control and OMDR control into software that controls a simple averaging device, such as a Hamamatsu Argus 10. We use a Data Translation I 0 board (DT-2817) together with a Screw terminal plate (DT-758c) for controlling shutters and filter wheels.
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C.Setup We first describe a basic motility assay and then consider some of the variables associated with the assay. The assay is very sensitive to variables such as time of incubation and washing, so great care and attention must be paid these factors to obtain reproducible results. We try not to let the time vary more than 5 seconds at each stage. It is further recommended that all reagents be stored as small frozen aliquots which can be used fresh for each experiment. We are standardly able to prepare four individual chambers in one experiment using this protocol. 1. Make perfusion chambers using two strips of double-stick tape (3M No. 665) on a glass slide to support a 22-mm2coverslip. Cool the chambers to 0-4°C on a metal block placed on ice. 2. Thaw the chromosomes and dilute 1/300 in PME (10 mM K-Pipes, pH 7.4, 5 mM4 mM MgC12,l mMEGTA, 100 pglml leupeptin, 100 pglml pepstatin, 100 pg/ml chymostatin, 5 mM dithiothreitol) containing 10% bovine serum albumin (BSA, Sigma No. A7638). Polyamines were used previously to stabilize chromosome arms, but in the real-time assay they cause the seeds to stick to the glass surface of the perfusion chamber and therefore must be avoided. The PME is warmed to 37°C for 10 minutes and returned to ice before addition of chromosomes, as this appears to help the BSA subsequently in blocking the binding of other proteins to the glass. 3. The diluted chromosomes are perfused into the chamber and left for 5 minutes (step 1, Fig. 1). The chromosomes tend to stick preferentially to the glass slide over the coverslip, and all subsequent experiments describe complexes attached to the glass slide. BSA prevents most other proteins from binding subsequently to the glass, although some free microtubule motors will bind to the glass. The slide, which forms the bottom surface of the chamber, gives inferior optical visualization compared with the coverslip undersurface, but it was used in all our assays because of its more favorable surface chemistry. 4. Stable, rhodamine-labeled microtubule seeds are made. Three steps (4-6) result in the attachment of segmented, polarity-marked microtubules (see Chapter 7) to the chromosomes attached to the glass slide. First, the brightly labeled seeds are attached to the kinetochores. To survive the washing steps, in which the unattached seeds are removed from the chamber, the seeds must be stabilized. Taxol cannot be used in these experiments because it will interfere with the subsequent growth of GTP-tubulin from the seeds. In our experiments, the seeds are made from GMPCPP-tubulin. Microtubules polymerized in the presence of GMPCPP are stable to dilution (Hyman et al., 1992) and can therefore be attached to kinetochores and washed without having to worry about stabilization as with GTP microtubules. A protocol to synthesize GMPCPP has been described (Hyman et af., 1992). To make the GMPCPP seeds, 40 pLM rhodamine-tubulin and 0.15 mM GMPCPP are mixed together
19. Microtubule-Based Motor Activity on CHO Chromosome Kinetochores
f
1)
MITOTIC CHROMOSOMES, FOLLOWED BY BSA TO BLOCK GLASS SURFACE
2)
STABLE RHODAMINE LABELED MICROTUBULES SEEDS AT HIGH CONCENTRATION, INCUBATION TO ALLOW CAPTURE.
27 1
3) WASH OUT UNCAPTURED SEEDS
f
0 0
4,
UNLABELED TUBULIN SUBUNITS + GTP + ATP
5)
PERFUSE IN ANTIFADE BUFFER
OO0
Fig. 1 Diagram to show the perfusion steps required in setting up the assay for motor activity on the kinetochores. For details see text.
(Hyman el al., 1991). This is most conveniently made up as a large volume on ice and spun 30 psi in the airfuge at 4°C to remove aggregates of tubulin formed on thawing; the supernatant is frozen in 5-p1 aliquots. Prior to use an aliquot is thawed and polymerized for 15 minutes at 37°C. 5. To attach the seeds to the kinetochores of the chromosomes that have been attached to the glass slide, the chamber is washed once (one wash is one chamber volume), in PMD (80 mM K-Pipes, pH 6.8, 1 mM EGTA, 4 mM MgC12, 5 mM dithiothreitol) + lx protease inhibitors (10 pg/ml leupeptin, pepstatin, and chymostatin), and then the slide is transferred to an aluminum block at 37°C for 30 seconds. The seeds, diluted 1/30 into PMD + l x protease inhibitors at 37"C, are then perfused into the chamber. The reaction is allowed to proceed for
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5 minutes so that the seeds are captured by the kinetochores (step 2, Fig. 1). Uncaptured seeds are washed out by perfusing the chamber with PMD + 1 rnM GTP four times at 37°C (step 3, Fig. 1). 6. To elongate more dimly labeled microtubule segments off the ends of the GMPCPP seeds, the chamber is washed twice with PMD 1 mM GTP 0. lx protease inhibitors + 3% polyethylene glycol (PEG) 3360, containing oncecycled tubulin (Hyman et al., 1991) at 15 $Wand rhodamine-tubulin (Hyman et af., 1991) at 1.5 pM.The final stochiometry is approximately one rhodamine per 10 tubulin subunits. The 3% PEG 3360 is included in the polymerization buffer to ensure growth from the minus end and also to prevent adsorption of the rhodamine-tubulin to the glass surface of the chamber. Polymerization is allowed to proceed for 10 minutes at 37°C (step 4, Fig. 1). 7. The microtubules are stabilized by washing five times in an antifade buffer containing taxol (PMD 0.2% 2-mercaptoethanol, 10 pM taxol, 5 pg/ml glucose oxidase, 10 pg/ml catalase, 10 mM glucose, 0.Ix protease inhbitors) + 1 pg/ml Hoescht 33342 (step 5, Fig. 1). The glucose oxidase and catalase are most conveniently stored as a lOOX stock in 50% glycerol at -20°C. The antifade buffer is essential to the assay. It works by scavenging oxygen, but a number of problems are associated with its use. First, the glucose tends to be used up if solutions are not kept in sealed tubes. Second, it essential that the temperature of solutions only increase, not decrease, subsequent to addition of the antifade component, because oxygen redisolving at lower temperatures uses up the glucose. Third, as the glucose oxidase works, its FAD cofactor becomes oxidized. This greatly reduces the strength of the fluorescent signal by an unknown mechanism, and may also increase the fluorescence background. It is therefore preferred that the antifade system be kept at 0°C until just prior to use. Fourth, glucose is converted to gluconic acid, potentially changing the pH of the buffer in weakly buffered solutions. Lowering of pH is particularly prevalent around bubbles if these are sealed under the coverslip. 8. If successful, the chromosomes should be attached to the glass slide, and their arms and primary constriction should be visible by Hoechst fluorescence. The degree of swelling of the chromatin arms depends on the concentration of MgCI2 in the assay. Excessive swelling reduced our ability to discriminate the primary constriction from the rest of the chromosome by Hoechst fluorescence; however we also found that swelling tended to reduce background binding of microtubule to chromosomes arms and made kinetochore visualization in the rhodamine channel easier. Thus, our buffer represents a compromise. At the primary constriction, the two kinetochores should be visible as clusters of brightly labeled seeds, and in addition the kinetochores may bind some rhodamine-tubulin directly. If seeds are present all over the slide, then too many seeds were used in the capture reaction. The more dimly labeled portions of the microtubules should radiate away from the kinetochores. If rhodaminetubulin is adsorbed to the glass surface, try increasing the concentration of PEG
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slightly. The most common problem is sticking of multiple microtubules to chromosome arms. This is caused by cytoskeletal debris on the arms, and must be addressed by improving the chromosome isolation as discussed above. The chromosomes should be fairly sparse, one to four per field with a 60x objective, so that individual chromosomes do not obscure each other.
D. Recording Minus-End-Directed Movement Minus-end-directed movement is the most difficult to record. The amount of minus-end growth rarely exceeds 10 pm, and because the minus-end motor moves at 40 p m per minute, only about 20 seconds elapses between addition of ATP and the microtubule running off the kinetochore. Although it is possible with practice to record minus-end-directed movement following perfusion of ATP into the chamber, for routine measurement it is easier to initiate movement by uncaging caged ATP. 1. Wash the chamber with 1 mM caged ATP in antifade buffer. It is important that caged ATP be protected from room light at all times, and we perform these manipulations under a sodium vapor safelight. 2 . Using rhodamine fluorescence, select a nice chromosome, that is, a chromosome in which the two kinetochores can be distinguished as clusters of brightly labeled seeds and no microtubules are attached to the arms of the chromosomes. 3. With the video recording sequence running, locally activate the caged ATP. The easiest way to uncage the ATP on a simple microscope system is to employ a filter used in a Hoechst or DAPI filter set. With a typical 360-nm bandpass filter, a 100-W mercury arc illuminator, and a bright 60X, 1.4NA lens, complete uncaging in a small area takes about 0.3 second. Start recording the chromosomes at 3-second intervals with a 0.3-second shutter-open time during each recording. During the 3-second wait time between recordings, switch the filter holder to the Hoechst position, so that the next time the shutter opens, the field is irradiated with 360-nm light for0.3 second, which uncages the ATP. Then switch back to rhodamine fluorescence to record any subsequent movement of the microtubules. Alternatively, more sophisticated systems using filter wheels can be employed. E. Recording Plus-End-Directed Movement
The plus-end-directed motor will not move in an ATP concentration below 100 pM.It is difficult to observe this activity by activation of caged ATP because the activated ATP rapidly diffuses away from the observation field; however, because the movement is slower and the plus-end segments are longer, it is easy to record plus-end-directed movement after perfusing 1 mM ATP in antifade buffer into the chamber, refocusing, and beginning the recording.
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274 F. Variables in the Assay
1. Switching between Plus- and Minus-End-Directed Movement at the Kinetochore Using ATPyS The addition of ATPyS during the capture reaction (step A, Fig. 2 ) is sufficient to switch the kinetochores from plus- to minus-end-directed movement (Hyman and Mitchison, 1991b). Different chromosome preparations have different levels of endogenous plus- or minus-end-directed movement, although movement tends to be more minus end directed in the absence of ATPyS. Therefore, for each chromosome preparation, a dose-response curve must be constructed to determine the concentration of ATPyS required to fully turn on plus-enddirected movement. The concentration required for full switching varied between 10 and 500 pM. One reason that plus-end-directed movement was routinely observed in early work (Mitchison and Kirschner, 1985) may be that GTPyS was routinely included in the assay buffer because it was used in the microtubule seed stabilization step.
2. Washing the Kinetochores Prior to the Capture Reaction A key variable in the assay is the wash between steps A and B (Fig. 2), as illustrated in Hyman and Mitchison (1991a). The relationship between washing and kinetochore motor activity varies from chromosome preparation to preparation. The general rule is that incubation of chromosomes at 37"C, without microtubules attached to their kinetochores, tends to inactivate the plus-enddirected movement system. A second reason that plus-end-directed movement was routinely observed in early work (Mitchison and Kirschner, 1985) may be that the chromosomes were not washed between microtubule capture and ATP addition. Presumably, the washing removes some weakly bound kinetochore components necessary for plus-end movement. We do not know whether this is due to inactivation of the motor itself or of the phosphorylation machinery.
3. Variation in Microtubule Number at the Kinetochore The number of microtubules attached to the kinetochore determines the concentration of ATPyS required to activate the plus-end-directed motor (Hyman and Mitchison, 1991a). At fewer than three microtubules per kinetochore it is very difficult to initiate plus-end-directed movement, even in the presence of 1 mM ATPyS. At more than six microtubules per kinetochore, plus-enddirected movement may be observed even in the absence of ATPyS. The number of microtubules attached per kinetochore can be varied by varying the number of microtubules in the capture reaction (step B, Fig. 2 ) while keeping the time constant.
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Fig. 2 Diagram of the results of ATP addition to the kinetochores of isolated chromosomes. Summary of the movements of microtubules on the kinetochores of isolated chromosomes. (A) The capture of the microtubule seeds by the kinetochores. (B) Tubulin grows from the ends of the captured seeds. After addition of ATP, microtubules move either by minus-end directed (C) or plus-end directed (D) movement.
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111. Conclusions We have described an assay in which the motor activity of the kinetochores of isolated mammalian chromosomes can be examined by video microscopy. The assay is technically challenging: Only small numbers of chromosomes can be assayed in each experiment, and the assay is very sensitive to the buffers used and the incubation times. Multiple perfusions must be performed in reproducible time intervals, and the sequence of temperature changes cannot be varied. Under a defined set of conditions, however, the assay can be made quite reproducible. It should prove very useful for testing specific inhibitory reagents to probe the identities of the motors and kinases involved in mammalian kinetochore function. The assay demonstrates the utility of modern fluorescence microscopy in the study of subcellular reactions and obtains quantitative biochemical information on their molecular mechanisms. References Hyman, A. A., and Mitchison, T. J. (1991a). Regulation of the direction ofchromosome movement. Cold Spring Harbor Symp. Quant. Biol. 56,745-750. Hyman, A. A., and Mitchison, T. J. (1991b). Two different microtubule-based motor activities with opposite polarites in kinetochores. Nature (London) 351,206-21 1. Hyman, A. A., Drexel, D., Kellog. D., Salser, S . , Sawin, K., Steffen, P., Wordeman, L., and Mitchison, T. J. (1991). Preparation of modified tubulins. In "Methods of Enzymology" (R.B. Vallee, ed.), Vol. 196, pp. 478-485. Academic Press, San Diego. Hyman, A. A., Salser, S., Drechsel, D., Unwin, N., and Mitchison, T. J. (1992). The role of GTP hydrolysis in microtubule dynamics: Information from a slowly hydrolyseable analogue GMPCPP. Mol. Biol. Cell 3, 1155-1 167. McIntosh, J. R.,and Pfarr,C. M.,(1991). Mitotic motors. J . Cell Biol. 115,577-587. Mitchison, T. J., and Kirschner, M. W. (1985). Properties ofthe kinetochore in vitro. 2. Microtubule capture and ATP dependent translocation. J. Cell Biol. 101,767-777.