ABSTRACT
This paper describes the procedures used to purify the microtubule motor, kinesin, from mitotic cells, namely sea urchin eggs and cleavage stage embryos, and describes methods for assaying its motor activity.
INTRODUCTION
Intracellular transport systems that move and position cytoplasmic particles such as organelles, chromosomes and perhaps macromolecules, play essential roles in the functioning of eukaryotic cells. Many particles are thought to be transported along microtubule ‘tracks’ by enzymes called ‘motors’. One such motor, kinesin, is a microtubule-activated ATPase that transports particles towards the plus ends of microtubules (Vale et al. 19856; Porter et al. 1987; Cohn et al. 1989; Howard et al. 1989). Kinesin has been isolated from a variety of eukaryotic organisms (reviewed by McIntosh and Porter, 1989) and several studies suggest that kinesin may perform a number of critical functions within the cell (Vale et al. 1986). Possible physiological functions include: anterograde transport of vesicles in neurons (Vale et al. 1985a; Schroer et al. 1988; Brady et al. 1990) and other cell types (Dabora and Sheetz, 1988a; Pfister et al. 1989), spreading of tubular membrane structures of lysosomes and the endoplasmic reticulum along microtubules (Dabora and Sheetz, 19886; Vale and Hotani, 1988; Hollenbeck, 1989; Hollenbeck and Swanson, 1990), mitosis (Scholey et al. 1985; Leslie et al. 1987; Endow et al. 1990; Enos and Morris, 1990; Meluh and Rose, 1990) and using microtubule tracks to move and organize intracellular membranes associated with interphase and mitotic asters of early sea urchin embryos (Wright et al. 1991).
Sea urchin eggs and early embryos are well suited for the study of kinesin-driven motile events in vitro and in vivo. Sufficiently large quantities of eggs and synchronized dividing embryos can easily be obtained for biochemical and cytological studies of early embryogénie events such as chromosome movement, directed transport of vesicles and organelles and pronuclear migration (see Bloom and Vallee, 1990 for a review). In addition, microtubules and motors can be isolated from eggs and embryos and used to reconstitute microtubule-based motility (e.g. Porter et al. 1987; Cohn et al. 1987,1989), and antibody microinjections into cells of the early embryo can be used to learn the function of motor proteins in vivo (Mabuchi and Okuno, 1977; Kiehart et al. 1982).
We have isolated and characterized kinesin from sea urchin eggs and early embryos (see Table 1 for a summary of the structural and functional properties of sea urchin kinesin). The motor binds microtubules in a nucleotidesensitive fashion, and this property has been exploited in the first two purification procedures described below, which make use of the fact that kinesin binds to MTs (see Appendix for abbreviations) in the absence of ATP and ADP, in the presence of the nucleotide analogue, AMP - PNP, or in the presence of the divalent cation chelators, EDTA and PPP,; subsequently, MT-bound kinesin can be released from MTs by adding MgATP. An alternative purification scheme makes use of anti-kinesin monoclonal antibodies that have been raised against sea urchin kinesin (Ingold et al. 1988); the latter were used in the last purification scheme described below which is a rapid immunoaffinity procedure using one of our anti-kinesin monoclonal antibodies covalently coupled to a Sepharose matrix. To assay kinesin mechanochemical activity, our lab routinely measures two properties of kinesin function: the MT-activated hydrolysis of ATP and the translocation of MTs over a surface coated with immobilized kinesin. The procedures used by our lab to purify and assay sea urchin kinesin will be described in detail below. Additional information can be obtained from articles by Scholey et al. (1984, 1985,1989); Porter et al. (1987); Cohn et al. (1987, 1989); Leslie et al. (1987); Ingold et al. (1988); Johnson et al. (1990).
COLLECTION OF SEA URCHIN EGGS AND EARLY EMBRYOS, AND PREPARATION OF CYTOSOLIC EXTRACTS
Most of our studies are performed with sea urchins, Strongylocentrotus purpuratus, obtained from the University of California Bodega Bay Marine Biology Laboratory or from Marinus, Inc. (Long Beach, CA). We sometimes also use S. franciscanas, S. droebrachiensis, Lytechinus pictus, and L. variegatus. S. purpuratus are transported as rapidly as possible in styrofoam boxes, packed in layers with paper well-soaked with sea water and cooled with ice packs. The urchins can be processed for gamete production immediately upon their arrival in the lab or they may be placed in well-aerated tanks and processed at leisure. Alternatively, the gametes of sea urchins (S. franciscanus or S. purpuratus) are collected at Bodega Bay and transported in jars suspended in large containers of 10–15 °C sea water.
1. Sea urchins are induced to shed their gametes by injection of approx. 2 ml of 0.56 M KC1 solution into the coelomic cavity. The yellow eggs are collected by inverting the shedding females onto beakers filled with sea water for 1–2 h. The white sperm are collected by inverting males onto dry pétri dishes. For production of HSS, perform this and subsequent steps at 0–4 °C; for developmental studies, keep the eggs at their physiological temperature of approx. 15 °C.
Note: Contamination of eggs with even traces of sperm must be rigorously avoided to prevent unexpected fertilization of the eggs. Thorough rinsing of hands, containers, and tools with tap water will kill unwanted sperm.
Decant excess sea water from the egg-containing breakers, pool the eggs, and pass the eggs 10–15 times through sea water-moistened Nitex screen (150 gm mesh; Small Parts, Inc.) to remove debris and the eggs’ jelly coats.
Production of cytosolic extracts (HSS) (steps 3–5)
3. To produce HSS, transfer the eggs to 50 ml plastic tubes and gently pellet using a low speed, short duration (lmin) spin in a clinical centrifuge. Aspirate off the supernatant and gently resuspend the egg pellets in approx. 7 vols ‘19:1 buffer’ (see Appendix for buffer recipes). Repeat the centrifugation and resuspension steps twice more to remove completely the eggs’ jelly coats and to minimize the Ca2+ concentration (many sea urchin proteases are Ca2+-activated and MT polymerization is Ca2+ sensitive). Then resuspend the eggs in sufficient PMEG buffer to reduce the NaCl concentration to less than 0.1 M and gently pellet again.
4. Resuspend the eggs in 2 vols PMEG buffer and homogenize on ice using a chilled Dounce homogenizer until intact eggs are no longer visible under a stereomicroscope (about 10–20 strokes).
5. Centrifuge the homogenate at 85000 g for 30 min at 4 °C. Discard the large pellet and the orange, top lipid layer; save and centrifuge the clear, intermediate supernatant at 175 000 g for Ih at 4 °C. Once again, save the clear, intermediate, high speed supernatant (HSS). This material is a cytosolic extract and can be stored for extended periods if immediately frozen in liquid nitrogen and then transferred to —80 °C.
Embryo production (steps 3a—6a)
3a. For developmental studies, resuspend the dejellied eggs in approx. 5 vols sea water (pH 8.1, 15 °C) containing 10 mM p-aminobenzoic acid (PABA) to soften fertilization membranes. Aspirate off the supernatant when the eggs have settled. Repeat this step 2–3 times.
4a. Resuspend the eggs in at least 10 vol of previously well-aerated sea water (containing 10mM PABA, pH8.1, 15 °C) and very gently stir with a paddle driven by an overhead electrical motor. The metabolic rate of eggs greatly increases upon fertilization. Adequate dilution of the eggs into well-aerated sea water is necessary to prevent asphyxiation.
5a. Activate sperm by diluting 2 drops of sperm into 50 ml sea water (use within 15 min). To fertilize eggs, add approx. 0.0025 vol. of diluted sperm to the diluted eggs. Caution: different sea urchin species are differentially prone to polyspermy and the quantity of sperm used must be varied accordingly (see Leslie and Wilson, 1989).
6a. The extent of successful fertilization can be assessed within a few minutes by observing an aliquot of eggs under a stereomicroscope: fertilized eggs will be ringed by a clearly visible fertilization membrane. Egg development is improved if the dilute egg suspension is occasionally gently aerated. Well-fertilized and aerated eggs remain largely synchronized in their mitotic cycles through two or three divisions.
7a. Aliquots of developing embryos can be taken at selected points in the mitotic cycle for HSS production or histological studies. For HSS production, the embryos must be passed through a sea water-moistened Nitex screen (∽ 10 times) to remove the fertilization membranes. The embryos can then be processed into HSS as described above (steps 3-5).
PURIFICATION PROCEDURES
Kinesin purification by AMP-PNP-induced MT affinity binding (Scholey et al. 1985; Cohn et al. 1987; Ingold et al. 1988)
Depletion of actomyosin and denatured protein. 1. Thaw frozen HSS (stored at —80 °C in 50 ml plastic tubes) in a beaker of tap-fed cool water. Move the thawed HSS to ice immediately.
2. Incubate HSS with hexokinase (10 units ml-1) and 50 mM glucose (to convert ATP to ADP) at room temperature for 30 min. Then centrifuge at 100 000 g (30 min, 4 °C) to pellet actomyosin and denatured protein, leaving kinesin and unpolymerized MT protein in the high speed supernatant.
Induced MT binding by kinesin. 3. Kinesin can be removed from the supernatant if kinesin is induced to bind to MT polymers, and then the kinesin-MT complex is pelleted. First, add 0.5–1 mM GTP and 20|UM taxol and incubate at room temperature (RT) for 15 min to promote MT assembly in the supernatant. Supplement the solution with ⩾1 mM AMP-PNP to enhance MT binding by kinesin and incubate at RT for 20 min. Sometimes we have used 5 units ml-1 apyrase in place of AMP-PNP to induce MT - binding by kinesin.
4. Centrifuge the solution over a 15 % sucrose cushion (containing ImM GTP, 2.5μM taxol may be added to stabilize MTs) in a swinging bucket rotor at 23000 g, 60 min, 10 °C (or 57000 g-, 20 min, 10 °C, in fixed angle rotor) to pellet the kinesin-MT complexes.
5. Wash the pellet by homogenizing in O.lmM GTP in PMEG buffer (or in 20 mM EDTA, 2 mM ATP, 0.1 mM GTP, 10 gM taxol in PMEG buffer with magnesium sulfate omitted) and let stand 10 min at 4°C. Centrifuge in a swinging bucket rotor at 45 000 g-, 20 min, 4 °C (or at 57 000g, 20 min, 4°C, in a fixed angle rotor).
6. Release kinesin from MTs in the pellet by homogenizing the pellet in 100 mM KC1,10 mM MgATP (0.1 mM GTP, 5μM taxol may be included) and holding overnight at 0·4 °C. Add a further 5 mM MgATP before centrifuging at 45000 g, 20 min, 4 °C to pellet MTs and leave MAPs in supernatant.
Biogel A5M chromatography. 7. Fractionate the extracted MAPs supernatant by gel filtration chromatography (Biogel A5M (Bio-Rad), 1.8cm×12cm) in PMEG buffer containing 1 mM ATP (ATP successfully omitted in some experiments).
8. Fractions are tested for MT-translocating activity by video microscopy (see below), and analysed by SDS-PAGE and Western blotting (using anti-kinesin monoclonal antibodies). The peak of kinesin activity constitutes those fractions that will support MT movement at 0.4gms”1. These fractions are pooled for further analysis.
MT binding and release. 9. The pooled kinesin fractions are further purified by addition of 1.5mgml-1 Pll phosphocellulose-purified bovine brain MTs (polymerized by incubation with >10μM taxol and 0.5 mM GTP for 15 min at RT; tubulin purified according to the method of Williams and Lee, 1982), 20 mM EDTA, and 2 mM ATP, and incubation for 20 min at RT. Pellet the resulting kin - esin-MT complex by centrifugation (45 000 g, 20 min, 20 °C).
10. Resuspend the kinesin—MT pellet in PMEG buffer containing 10 mM ATP, 12.5 mM magnesium sulfate, 30 pM taxol, and 0.1 mM GTP for 30 min, RT, to release kinesin from MTs. Centrifuge the solution again at 45 000 g’, 20 min, 20 °C to pellet MTs and leave purified kinesin in the supernatant.
Pll phosphocellulose chromatography. 11. Tubulin and ATP can be removed from the kinesin-containing supernatant by Pll phosphocellulose chromatography; kinesin (but not tubulin or ATP) binds to Pll phosphocellulose resin (Whatman). Activate Pll phosphocellulose resin according to manufacturer’s instructions and equilibrate in PMEG buffer. Mix 1 vol Pll to 10 vols of supernatant from step 10 (or from step 8 if required) for 30 min, 4°C.
12. Wash the resin batch-wise (by centrifuging in a clinical centrifuge) 2 times with 5 vols PMEG buffer, then 2 times with 5 vols PMEG containing 0.1 M KC1 to remove non-binding material. Pack the resin into a column and then elute kinesin using PMEG buffer containing ⩾ 0.5 M NaCl or KC1. Collect 25 drop fractions and use SDS-PAGE to locate kinesin-containing fractions. Dialyze the pooled fractions against PMEG buffer (0–4°C) to remove salt.
The purified kinesin has both ATPase and MT translocating activities and can be stored for extended periods in liquid nitrogen. Using this method, we obtain 50–500 μ g kinesin from 100 ml HSS (see Fig. 1 A).
Kinesin purification by PPPi-induced MT affinity binding (Johnson et al. 1990)
Pll phosphocellulose chromatography of PISS. 1. Thaw 150 ml of HSS (stored at −80 °C) and supplement with fresh protease inhibitors (see Appendix). Add HSS to approx. 20 ml of activated Pll phosphocellulose resin and gently mix the slurry for Ih (all steps at 0–4°C). Remove non-binding proteins by washing the Pll resin 3 times with about 4 vols of PMEG buffer, then once with 4 vols of PMEG buffer containing 0.1M KC1. A clinical centrifuge is used to pellet the resin after each wash.
2. Bound proteins (including kinesin) are eluted with 2 vols of PMEG buffer containing 0.75 M KC1. The eluate is passed through glass wool to remove any residual Pll resin - the presence of Pll resin in subsequent steps greatly decreases kinesin yield. Dilute eluate with 1 vol of PMEG buffer.
Ammonium sulfate precipitation. 3
Precipitate kinesin from the eluate by adding 0.39 g ammonium sulfate per ml eluate (60 % final concentration) and stirring overnight at 4 °C.
4. Precipitated material is pelleted by centrifugation at 17250 g- for 10min, 4°C. Resuspend the pellet in 3ml PMEG buffer and clarify by centrifugation at 17 250 g, 10 min, 4 °C.
Biogel A1.5M chromatography. 5
The clarified supernatant is fractionated and desalted by gel filtration chromatography on a Biogel A1.5M (Bio-Rad) column (2.5 cm × 15 cm) equilibrated with PMEG buffer containing 2ma ATP and 100μM (instead of 2.5mw) magnesium sulfate. Kinesin-containing fractions are identified by SDS-PAGE and immunoblotting, pooled, and then supplemented with 20 |UM (final concentration) taxol.
PPPi-induced MT binding: 6
Polymerize MTs by mixing a previously prepared, concentrated solution of Pll phosphocellulose-purified bovine brain tubulin (sufficient tubulin is added to make the tubulin final concentration 1–2 mg ml-1 after the pooled Biogel fractions are added at step 7), with roughly equimolar taxol (10μM per Imgml”1 tubulin), and 0.5mM GTP. Incubate at 37 °C for 30 min.
7. Chill and then mix the MT solution with the pooled, taxol - and kinesin-containing Biogel fractions and incubate for 10 min, 4°C. Add 20-30 mM (final concentration) PPPi (or EDTA) to promote kinesin binding to MTs and incubate for a further 10 min, 4°C. The kinesin-MT complexes are pelleted by centrifugation at 85 000 g, 20 min, 4 °C.
8. Kinesin is released from the MTs by resuspending the pellet in 2 ml of cold resuspension buffer using a chilled Dounce homogenizer and incubating for 1 h at 4°C. Pellet MTs by centrifugation at 85000 g; 20 min, 4 °C.
Pll phosphocellulose chromatography. 9
Mix the kinesin-containing supernatant with 1—2 ml of activated Pll phosphocellulose resin and gently rock the slurry at 4 °C for 1 h. Wash the Pll resin 2 times with about 8 vols of PMEG buffer, and wash twice more with PMEG buffer containing 0.1M KC1 to remove non - and weakly-binding proteins. After each wash, gently pellet the resin in a clinical centrifuge before resuspending in the next wash buffer.
10. Pack the washed Pll resin into a disposable 11ml polystyrene column (Bio-Rad), and elute kinesin by developing the column with PMEG buffer containing 0.6 M KC1 and collecting 25 drop fractions.
Kinesin-containing fractions can be identified by SDS-PAGE and dialyzed against PMEG buffer to remove KC1. This procedure yields —0.1 mg active kinesin from 100 ml HSS (see Fig. IB).
Kinesin purification by monoclonal antibody immunoaffinity chromatography (Johnson et al. 1990)
For immunoaffinity chromatography, we routinely use the monoclonal anti-kinesin, SUK4, prepared from mouse ascitic fluid using Protein A Affigel and the MAPs II kit (Bio-Rad) as described previously (Ingold et al. 1988; Johnson et al. 1990). Nonspecific mouse IgG and rat IgG purchased from Sigma are routinely used as controls for non-specifically sedimenting polypeptides. We usually immobilize the SUK4 to cyanogen bromide-activated Sepharose 4B (Pharmacia) to produce an effective immunoadsorbent. We have used SUK4 Sepharose immunoadsorbents on fresh and frozen HSS from eggs and embryos, and we have observed no significant differences in the kinesin preparations obtained. Our current immunoaffinity protocol is described below.
Preparation of HSS. 1
Thaw frozen HSS and supplement with fresh protease inhibitors (see Appendix). Clarify by centrifugation at 150000 g-, lh, 4 °C, to remove any denatured protein.
Elimination of non-specific IgG-binding proteins
2. Gently mix the HSS with 1/20 vol rat IgG-Sepharose for 1—2h to remove nonspecific IgG-binding proteins (all steps at 4°C). The resin is pelleted by brief centrifugation in a clinical centrifuge and the HSS saved. The resin is regenerated with successive washes of 1 M NaCl in 10 mM phosphate buffer, pH 8.5, then IM NaCl in 0.1M diethylamine buffer pH 11.5, and then PMEG buffer.
Immunoprecipitation. 3
The preadsorbed HSS is divided and mixed with either control (non-specific mouse IgG) or SUK4 resin that has been incubated with an equal volume of 10 mg ml-1 SBTI for 1-2 h at 4°C to saturate nonspecific protein binding sites. Approximately 1 vol of resin is incubated with 50 vols HSS for 1-2 h (4 °C) with gentle mixing.
4. Wash the resins 3 times with 30 vol PMEG buffer. Then wash with 30 vol of 1 M NaCl, 10 mM ATP (pH 7) to remove kinesin-associated proteins (see Schroer et al. 1988), and then with 30 vol IM NaCl in 10mM phosphate buffer pH 8.5 (to get the resin into a higher pH solution in preparation for elution).
5. A 50 μi sample of a resin may be boiled in SDS sample buffer (Laemmli, 1970) for PAGE analysis. The remainder of the resin (approx. 400–450 μA) is treated with at least 2 vols of 1M NaCl, 0.1 M diethylamine pH 11.5 to elute the bound kinesin. The eluate can be neutralized by immediate addition of 0.1M sodium acetate (pH4) or IM dibasic phosphate. Alternatively, kinesin can be precipitated from the eluate using 20 % trichloroacetic acid and washed with cold acetone.
Kinesin purified by this procedure lacks MT translocating activity, possibly as a result of a small amount of the function-blocking monoclonal, SUK4, leaching from the column into the eluate or because the elution conditions denature the kinesin. In some experiments using the same elution conditions, we were able to recover active kinesin using immunoadsorption with SUK2-Sepharose, although the efficiency of adsorption was lower than with SUK4 - Sepharose. Approximately 0.5-1 mg kinesin can be immunoaffinity purified from 100 ml HSS (Fig. IC). The resins can be re-used if they are immediately neutralized with PMEG buffer and regenerated with the same washing protocol as is used with the rat IgG-Sepharose (above). We have found that some kinesin (presumably denatured) becomes irreversibly bound to re-used SUK4-Sepharose resin. This residual kinesin does not elute from the resin and, therefore, presumably does not contaminate subsequent immunoaffinity purification experiments.
ASSAY PROCEDURES
Video microscopic assay of sea urchin kinesin-driven microtubule motility
We have adapted the original MT gliding assay described by Vale et al. (1985a) to allow us to measure conveniently the speed of kinesin-driven MT movement over glass surfaces in real time. This is a highly reproducible, quantitative assay that allows us to characterize motor enzymology and screen for probes that inhibit kinesin - driven transport using very small quantities of the motor (on the order of 1 μg per assay).
The motility assay (Porter et al. 1987; Cohn et al. 1987, 1989)
1. Spread a solution of purified kinesin (15 μl) onto a No. 1 coverslip and allow it to adsorb for 20 min in a humidified chamber.
2. After adsorption, 2μl of approx. 0.2 mg ml-1 MTs (bovine brain tubulin purified by Pll phosphocellulose chromatography and polymerized in 1 mM GTP and 20 μM taxol) are added, along with 0.5–2.0 μl each of nucleotide and/or inhibitor solution (made up as stock solutions of 1-100 mM) to obtain a final volume of approx. 20μl.
3. Invert the coverslip and seal the edges to a microscope slide with molten VALAP (vaseline/lanolin/ paraffin, 1:1:1 (w/w/w)).
4. The sealed slide is mounted on the microscope, taking care to avoid bubbles or dust in the immersion oil. Focus onto the coverslip surface looking through the ocular, adjust field iris and condenser with lamp set for critical illumination and differential interference contrast (DIC) optics set at extinction. Redirect image to monitor, then turn slider away from extinction until MT-covered coverslip surface is visible. ‘Fine tune’ contrast on monitor using optics and camera control box by trial and error. Use the computer mouse to track and measure velocities of 10–30 MTs, and finally command the computer to print out the velocity histogram, with the mean MT velocity and the standard deviation.
In our lab, kinesin-driven MT translocation is visualized by use of a video-enhanced microscope system consisting of a Zeiss standard research microscope with a ×100 (NA 1.25) objective, ×2 optavar, a wide-band-pass green interference filter (wavelength 546 nm), and standard DIC optics. The microscope is equipped with a 100 W mercury arc lamp set at a critical illumination and is connected to a MTI Series 68 Newvicon video camera (equipped with remote gain and black offset level) whose output is directed to a RCA monochrome monitor. Using this set up, we find that no computer background subtraction is needed to visualize the MTs (Porter et al. 1987; Cohn et al. 1987, 1989). The velocities of MTs ‘gliding’ across the coverslip are measured in real time using computer - assisted analysis (Cohn et al. 1987,1989). The video output of an Amiga 1000 computer is mixed with the output of the MTI video camera using a video mixer installed by DAGE Inc. - allowing the image of the computer’s mouse - controlled cursor (an arrow) to be superimposed on the image of the MTs. Alternatively, a Genlock-type video mixer can be connected to the Amiga computer directly, for better resolution and greater versatility. A computer program (Appendix) monitors the x and y coordinates of the cursor, as well as the time measured by an internal computer clock. In practice, the cursor is moved to the site of a MT end and the mouse input button triggered to mark its position and the time of the observation. After an arbitrary period (usually 10-30 s), the cursor is moved to the new site of the same MT end and again the mouse button triggered. The program calculates the distance between the two x,y coordinates (using scalar factors between x,y coordinates and actual distances measured previously with a stage micrometer) and divides this value by the time difference to obtain the velocity of the translocating MT. After the measurement of an arbitrary number of MT velocities (usually 10-30), the program will calculate the mean velocity and standard deviation and also print out a histogram of MT velocities.
We have also used the assay to monitor the velocity of MT-gliding induced by 21S dynein prepared by sucrose density gradient centrifugation from sea urchin sperm flagellar axonemes. We find, however, that the dynein often moves MTs too fast for us to accurately find MT ends, causing greater errors in the measured velocity. Therefore, we would recommend the restricted use of this assay for accurately measuring velocities 2μms-1. Other advantages and limitations of this assay are discussed by Cohn et al. (1989).
Use of MT motility assays for analysis of steady state kinetic parameters of kinesin
(Cohn et al. 1989) Using the procedure described above, MT motility assays and their analyses can be performed to determine the kinetic parameters of kinesin-driven MT translocation.
1. Adsorb 14–15μl kinesin to a glass coverslip for 20 min, then add the desired concentrations of substrates, inhibitors and phosphocellulose-purified bovine brain MTs (to 20 μgml-1) to a final volume of 20 μl. Nucleotide stock solutions are made up as 100 mM solutions of 0.1M Tris, pH 7. Inhibitor solutions are made up as 100 mM stocks in either 0.1 M Tris pH 7 or PMEG buffer. Mg-nucleotides (or analogs) are prepared by adding 100 mM MgSO4 to the stock solutions. Dilutions of working solutions are made in PMEG buffer.
2. Measure the velocities of at least 10 MTs for each data point and each experiment should be performed on at least two different preparations of kinesin. For determinations of Km and kinesin is used at a concentration of 250–300 μg ml-1, which is at least 4–5 times the critical concentration necessary for MT motility (Cohn et al. 1987, 1989).
Note: In order to eliminate any decrease in ATP concentrations during the motility assays due to hydrolysis, perform all experiments using ATP at concentrations sS100 μM (except those involving measurements of ADP) in the presence of an ATP regenerating system of 10 units ml-1 creatine-phosphokinase and 50 mM phosphocreatine. Samples containing creatine-phosphokinase and phosphocreatine retain MT motility for several hours, even when using ATP concentrations as low as 10–25 μM.
3. The Vmax and Km of substrates that support kinesin - driven MT-motion can be obtained from Lineweaver-Burk plots (Fig. 2). To study compounds interfering with MT translocation, the type of inhibition is determined by plotting the reciprocal of MT velocity versus the reciprocal of substrate concentration at a constant inhibitor concentration (Lineweaver and Burk, 1934) or the reciprocal of MT velocity versus inhibitor concentration at constant substrate concentration (Dixon, 1953).
Use of motility assays for identifying function-blocking kinesin antibodies (Ingold et al. 1988)
The ability of antibodies to inhibit the MT-translocating activity of kinesin can be assayed using video-enhanced microscopy and computer-assisted velocity analysis as described above.
1. Adsorb 14 μl of 100–300 μg ml-1 kinesin solutions onto coverslips for 10–20 min at RT in a humidified chamber before addition of the test antibodies. Then add antibody solutions to the coverslips for an additional 10–20 min. Routinely we use solutions of monoclonal antibodies purified from ascitic fluid. These can be obtained at sufficiently high concentrations that only small (1–2 μi) volumes are needed.
2. Add polymerized, Pll phosphocellulose-purified, bovine brain MTs (20μgml-1) and MgATP (10 mM) to the coverslip, and seal the coverslip to a slide using VALAP.
3. Determine the velocity of a gliding MT over an interval of at least 10 s and randomly select 10–20 MTs from several microscopic fields to be used for each data point. Each set of data points and standard deviations in Fig. 3 is from single typical experiments that have been reproduced many times.
The ATPase assay (Cohn et al. 1987)
ATPase assays are performed at RT by modification of the method of Seals et al. (1978). The assays themselves are quantitative and reproducible (even though we have observed variability in the specific ATPase activity of our kinesin preparations; see Cohn et al. 1987), but require larger quantities (—100 μg per assay) of kinesin. The specific ATPase activities of our tubulo-kinesin preparations (<100 nmol min-1 mg-1 at 25°C) are usually much lower than those found by other workers, but the activity can be stimulated markedly by addition of function blocking antibodies (Ingold et al. 1988) or proteolysis (Table 1).
1. Kinesin (prepared by purification procedure 1 or 2, above) in 200–300 μl PMEG buffer is mixed with 40 μl phosphocellulose-purified bovine brain MTs (10mgml-1) or with 40 μl buffer plus 1 μl of 10 mM taxol (as a control).
2. Add sufficient PMEG buffer to each assay tube to give a final volume of 380 μI, and incubate the mixture for approx. 10 min at RT.
3. To start the reaction, add 20 μl of 50 mM [γ - 32P]MgATP (specific activity 10 000–40 000 cpm nmol-1 ATP). Remove aliquots (100 pl) at a minimum of three time points (0, 5, 10, 15, 20, 30 or 60 min), and quench the reaction in the sample by addition of 30 μl 10 % SDS.
4. Add 75 pl phosphate reagent (4 vols 5N sulfuric acid/5% ammonium molybdate: 1 vol 0.1M silicotungstic acid) to react with liberated phosphate; extract the resulting 32P-labelled phosphomolybdate complex with 1ml of a 65%: 35% (v/v) xylene: isobutanol mixture.
5. After centrifugation in a microfuge to separate organic and aqueous phases, analyze a 0.7 ml aliquot of the organic phase (containing the 32P-phosphomolybdate) of each sample by scintillation counting.
6. To calculate ATPase specific activity, aliquots of the [γ-32P]MgATP stock solution are scintillation counted to determine cpm nmol-1 ATP for the stock solution. The concentration of liberated phosphate for each sample from step 5 can be calculated by dividing the sample’s cpm by the value for cpm nmol-1 ATP. The ATPase specific activity (nmol phosphate min-1 mg-1) is calculated by dividing the slope of plots of free phosphate concentration as a function of time by the protein concentration of the kinesin sample.
CONCLUSION
It is clear that kinesin performs a number of vital functions within the cell, all of which are a consequence of kinesin’s ability to translocate cellular structures along MT tracks. Sea urchin eggs and early embryos have proved to be a rich source of kinesin and also serve as amenable experimental subjects for cytological investigations of kinesin function in fixed cells and in vivo. Our lab has concentrated on isolating and characterizing sea urchin kinesin, and has developed the purification and assay procedures described here. This work has provided us with basic information on the structure and in vitro functions of the sea urchin kinesin motor molecule; Table 1 summarizes our current understanding of these properties of sea urchin kinesin. The isolation and assay protocols described in this paper may assist other workers who wish to study kinesin-like proteins isolated directly from their natural host cells, or expressed in vitro or in transformed cells from cloned and manipulated genes.
Abbreviations
AMP-PNP: 5’-adenylyl imidodiphosphate.
HSS: high speed supernatant of cytosolic extract.
MAP: microtubule associated protein.
MT: microtubule.
PAGE: polyacrylamide gel electrophoresis.
PPPi: tripolyphosphate.
SBTI: soybean trypsin inhibitor.
SUK: sea urchin kinesin (notation used for monoclonal antibodies).
Buffer solutions (all concentrations are final)
1. Sea water: 760 g Instant Ocean sea salts brought to 20 liters with tap water (check specific activity= 1.025–1.026, 4°C).
2. 19:1 buffer: 530mM NaCl, 28mM KC1, ImM EDTA, 5 mM Tris HC1 in distilled water, pH 7.
3. PMEG buffer: 0.1M potassium Pipes, 2.5mM magnesium sulfate (promotes MT assembly), 0.1 mM EDTA (chelates divalent metal ions which catalyze sulfhydryl oxidation), 5 mM EGTA (chelates Ca2+ which inhibits MT assembly and activates some proteases), 0.9 M glycerol (establishes correct osmolarity for eggs) in distilled water, pH 6.9. Stored at 4°C.
Protease inhibitors: Ipgml’1 pepstatin and leupeptin, 2(ugmU1 aprotinin, 100μgml-1 SBTI, 0.1 mM phenyl methyl sulfonyl fluoride, lmgml-1 tosyl arginine methyl ester, ImM dithiothreitol, 20μgml-1 benzamidine, ImM sodium azide. Protease inhibitors are added to PMEG buffer immediately before use.
4. Resuspension buffer: 0.5 mM GTP, 30 μM taxol, 10 mM ATP, 15 mM magnesium sulfate, 0.33 mg ml-1 bovine serum albumin, 0.1M KC1 in PMEG buffer. Made immediately before use.
The computer programs used to analyze MT translocation were developed by Dr S. A. Cohn while working in the lab of J.M.S. Current updates of the programs can be obtained from SAC (current address: De Paul Univ., Dept of Biological Sciences, 1036 W. Belden Ave, Chicago, IL 60614, USA). SAC was recipient of an American Cancer Society Postdoctoral Fellowship no. PF-2925.
ACKNOWLEDGEMENTS
This work was supported by American Cancer Society Grant no. BE-46C and March of Dimes Birth Defects Foundation Grant no. 1-1188 to J.M.S. Thanks to Dr S. A. Cohn for reviewing an early version of this paper.