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ASSAYS FOR A C T I N S L I D I N G M O V E M E N T
[33] A s s a y s f o r A c t i n S l i d i n g M o v e m e n t Coated Surfaces
By STEPHENJ. KRON, YOKO Y.
TOYOSHIMA,
399
over Myosin-
TARO Q. P. UYEDA,and
JAMES A. SPUDICH Introduction One important result from in vitro studies of the interaction of the major proteins of muscle, actin and myosin, has been the growing recognition that nearly any aspect of muscle mechanics can be studied in a model system consisting of purified proteins. The consensus that has emerged is that actin and myosin, when placed in an appropriate geometry, can produce movement and force coupled to the hydrolysis of ATP in much the same way that stimulated muscle uses ATP to contract. The following is a compilation of our experience with techniques for purified in vitro motility assays for actin sliding movement over myosin) We limit our focus to studies using skeletal muscle proteins, but only slight modification of these protocols may be necessary for proteins derived from smooth muscle and nonmuscle sources. Protein Reagents General Considerations The properties of the protein preparations used are critical to reproducibility of actin sliding movement assays. The following methods are presented as trustworthy preparations but are not singularly successful. However, in particular it should be noted that myosin subfragment preparations that work well in solution experiments may not be optimal for use in movement assays. Myosin and Its Fragments Several forms of myosin, including filaments, monomers, and soluble proteolytic fragments, have been found to work well in aetin sliding movement assays. A desirable characteristic of myosin preparations for motility assays is the absence of contamination by irreversible "rigor heads" that show ATP-insensitive binding to actin filaments. Such heads will inevita1 S. J. Kron and J. A. Spudich, Proc. Natl. Acad. Sci. U.S.A. 83, 6272 (1986).
METHODS IN ENZYMOLOGY, VOL. 196
Copyright © 1991 by Academic Press, Inc. All fights of reproduction in any form reserved.
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bly "load down" actin filaments, inhibiting their movement. Use of freshly purified proteins, freshly made buffers, careful handling of myosin to avoid unnecessary contact with fluid-air interfaces, and liberal use of reducing agents are important. Chymotryptically derived subfragment 1 (S 1), the isolated myosin head domain, is commonly used in biochemical and structural studies. However, unless used at high concentrations, chymotryptic S1 as it is commonly prepared does not support movement in the assay. Somewhat higher molecular weight forms of S 1, prepared by papain digestion in the presence of Mg2+ or EDTA, work very well in the movement assay. Each form of myosin has a characteristic sliding speed, ranging from 1 to 2 Ftm/sec for S 1 to 4 to 8/~m/sec for heavy meromyosin (HMM) when measured in assay buffer at 30 ° .2 The basis for these differences remains uncharacterized. The following preparations give yidds o f - 70% of theoretical for HMM and - 4 0 % for S1. Modifications to increase yield result in considerable proteolytic nicking of the heavy chain at sites within the myosin head (and of the light chains), which is associated with an increase in the measured average sliding speed.
Buffers MSB: 25 m M imidazole hydrochloride, pH 7.4, 0.6 M KCI, 1 m M dithiothreitol (DTT) BED: 0.1 m M NaHCO3, 0. l m M EGTA, 1 m M DTT 2 × CHB: 20 m M imidazole hydrochloride, pH 7.0, 1 M KCI, 4 m M MgCI:, 10 m M D T T PMSF stock: 0.2 M phenylmethylsulfonyl fluoride in ethanol PMB: 25 mMimidazole hydrochloride, pH 7.4, 100 mMNaC1, 5 m M MgCl2, 1 m_MDTT E64 stock: 1 mg/ml in dimethyl sulfoxide (DMSO) SB: l0 mMimidazole hydrochloride, pH 7.4, 0.5 MKC1, 1 m M D T T
Methods. Myosin preparation and storage: Prepare myosin from skeletal muscle according to any standard protocol (e.g., Hynes et al.3), with the use of 0.1 to 1 m M DTT in all buffers. Do not use ammonium sulfate precipitation as a step in the preparation, as this is associated with the production of irreversible rigor heads. To store myosin, dissolve pelleted filaments in an equal volume of 1.2 M KCI and 1 m M DTT, add MSB to dilute the myosin to a final concentration of 30 to 60 mg/ml as determined 2 y. y. Toyoshima, S. J. Kron, E. M. McNally, K. R. Niebling, C. Toyoshima, and J. A. Spudich, Nature (London) 328, 536 (1987). 3 T. R. Hynes, S. M. Block, B. T. White, and J. A. Spudich, Cell (Cambridge, Mass.) 48, (1987).
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by its extinction coefficient, A28o = 0.53 cm2/mg,4 and then add glycerol to 50% (v/v). Centrifuge the myosin to clear it of suspended air bubbles and then aliquot into microfuge tubes, which are topped off to avoid trapping air. Store the myosin at - 2 0 °. It is stable for several months.
Heavy meromyosin preparation, based on method of Okamoto and SekineS: 1. Add 9 vol cold BED to - 2 0 mg stock myosin in a centrifuge tube and mix to precipitate filaments. After > l0 min of incubation on ice, centrifuge at 4 ° at low speed in a swinging bucket rotor to sediment the filaments (e.g., l0 min at 13,000 rpm in JS-13 rotor; Beckman, Palo Alto, CA). 2. Dissolve the pellet in 2× CHB and BED as needed to achieve a final concentration of 15 mg/ml myosin in CHB. 3. Incubate the myosin solution l0 min at 25 °. 4. Add N-tosyl-L-lysine-chloromethyl ketone (TLCK)-treated a-chymotrypsin to 12.5/tg/ml, gently mix the reaction, and incubate 7.5 to l0 rain at 25 o. 5. Add 9 vol ice-cold BED with 3 m M MgC12 and 0.1 m M PMSF to the reaction and mix. After > 1 hr on ice, centrifuge the suspension at high speed (e.g., 15 min at 75,000 rpm in TL100.3 rotor, Beckman). 6. Store the supernatant, which is typically - 0 . 7 mg/ml and > 90% pure HMM, on ice. It can be used in motility assays for 3 to 5 days. Extinction coefficient, A28o ----0.60 cm2/mg. +
Papain Mg-SI preparation2: 1. Process 20 mg myosin to a pellet as described above. 2. Dissolve the pellet in an equal volume (-0.25 ml) of 1.2 M KC1 and 20 m M DTT in BED, and incubate l0 min at 25 o. 3. Add 19 vol ice-cold BED (to - 10-ml final volume), incubate on ice > 10 rain, and centrifuge at low speed. 4. Resuspend the pellet in PMB to a final concentration of 10-12 mg/ml and incubate 10 rain at 25 °. 5. Add papain to 12.5/~g/ml, mix gently, and incubate 7.5- l0 min at 25 ° . 6. Stop the reaction with greater than an equal volume of ice-cold BED with 5 m M MgC12 and 5/~g/ml E64 (Peninsula Laboratories, Belmont, CA). Incubate > 1 hr on ice before high-speed centrifugation. 7. Store the supernatant, typically - 1.1 mg/ml S1, on ice. It can be used for 3 to 5 days in the movement assay. The level of contamination by HMM may be significant for some experiments. Extinction coefficient, A280 = 0.81 cm2/mg. 4 4 S. S. Margossian and S. Lowey, this series, Vol. 85, p. 55. s y. Okamoto and T. Sekine, J. Biochem. (Tokyo) 98, 1143 (1985).
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Gel-filtration purification of HMM and SI: Further purification of myosin soluble fragments is often desirable and is accomplished by highperformance gel filtration [e.g., Superose 6 or 12 HR FPLC columns (Pharmacia LKB, Piscataway NJ), 0.25 ml/min] using a high ionic strength buffer such as SB. Concentrate the HMM or S1 supernatant, prepared as described above, in a Centricon or Centriprep 30 (Amicon, Danvers MA) ultrafiltration device to < 0.5 ml. Centrifuge the retentate at high speed (5 min at 75,000 rpm in TL100.2, Beckman), and load the supernatant onto the column. Pool protein peaks by OD28o or by Coomassie SDS-PAGE analysis. Myosin fragments can be used directly as eluted from the column. Actin affinity purification: Soluble myosin fragments can be treated to precipitate irreversible rigor heads, before use in the motility assay. Add filamentous actin to 0.15 mg/ml and 1 m M ATP to an aliquot of the stock HMM or S 1. After a short incubation on ice, centrifuge the mixture at high speed (10 min at 75,000 rpm in a TL100.2, Beckman) to sediment the actin. Actin preparations can have an associated proteolytic activity. Thus, use the supernatant within 2 to 3 hr. Actin Buffers GB: 2.5 m M imidazole hydrochloride, pH 7.4, 0.2 m M CaC12, 0.2 m M ATP, 0.2 m M DTT 10× AB: 225 m M imidazole hydrochloride, pH 7.4, 250 m M KC1, 40 m M MgCI2, 10 m M EGTA AB: 25 m M imidazole hydrochloride, pH 7.4, 25 m M KC1, 4 m M MgCI2, 1 m M EGTA, 1 m M DTT
Methods. Actin preparation: To prepare rabbit skeletal muscle actin, we use a modification of the method of Pardee and Spudich. 6 Further purification of actin by anion-exchange chromatography may be useful, but is often complicated by polymerization of actin in the chromatographic media as it elutes. 1. Prepare the acetone powder as described and store at - 20 °. Starting with - 10 g acetone powder, perform the extraction, polymerization, KC1 cut, and centrifugation essentially as described. 2. After leaving the actin pellets overnight on ice softening in buffer A 6, resuspend the pellets in GB to 10- 15 ml and dialyze against one change of buffer GB, > 48 hr at 4 °. 3. Change the dialysis to GB with 0.05 m M CaC12 and dialyze overnight at 4 °. 6 j. D. Pardee and J. A. Spudich, Methods CellBiol. 24, 271 (1982).
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4. Centrifuge the actin at high speed and polymerize the supernatant by adding 1/9 vol of 10X AB, made up in the last dialysis buffer. The final polymerization leaves the actin as filaments in motility assay buffer (AB) with 0.2 m M ATP. 5. Store the actin on ice. It can be used in the movement assay or for purification of myosin soluble fragments for several weeks. Extinction coefficient A29o_3t0= 0.62 cm2/mg.7 Stored actin rejuvenated by "recycling. '" 1. Pellet an aliquot of the actin by high-speed centrifugation (15 min at 100,000 rpm in TlI00.3, Beckman). Wash the pellet with GB and then soak under GB overnight on ice. 2. Resuspend the actin to less than 5 mg/ml in GB and dialyze against one change of GB, > 24 hr at 4 °. 3. Change the dialysis to GB with 0.05 m M CaC12 overnight. 4. Centrifuge the actin at high speed and polymerize the supernatant with 1/9 vol 10X AB made up in the last change of dialysis buffer. Fluorescent actin filaments: The diameter of actin filaments precludes direct imaging by transmitted light microscopy. Thus the filaments must be labeled to be imaged. The original description of imaging of fluorescent actin filaments in the microscope by Asakura and colleagues was of actin covalently modified with a fluorescent group and stabilized by phaUoidin. This approach offers the widest range of possible fluorescent probes. However, the most convenient label for actin filaments is tetramethylrhodamine phalloidin (RhPh), a fluorescent analog of the Amanita phalloides toxin, phalloidin. Phalloidin binds with high affinity (Kd -- 10-8) to actin filaments and stabilizes them against depolymerization. Significantly, phalloidin has no effect on actin activation of myosin ATPase in vitro. Among the advantages of RhPh are its high affinity for actin, and the stability and efficiency of the tetramethylrhodamine group,s RhPh has an excitation maximum of 550 nm, very close to the 546-nm Hg emission line, and an emission maximum at 575 nm. Also available commercially are phallotoxins labeled with fluorescein, coumarin, and N-(7-nitrobenz-2oxa- 1,3-diazol-4-yl) (NBD). Prepare the stock solution of RhPh actin filaments as follows.t 1. Dry 94/tl of 3.3 a M RhPh in methanol (Molecular Probes, Eugene, OR) to a pellet, while protected from light, in a Speed Vac concentrator (Savant, Hicksville, NY) or by an N2 stream. 7 D. J. Gordon, Y.-Z. Yang, and E. D. Korn, J. Biol. Chem. 251, 7474 (1976). 8 H. Faulstich, S. Zobeley, G. Rinnerthaler, and J. V. Small, J. Muscle Res. Cell Motil. 9, 370 (1988).
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2. Dissolve the pellet in 2 #1 ethanol. 3. Add 290 #1 AB, and vortex approximately 30 sec. 4. Add 10/tl of 1 mg/ml actin in AB and mix well. 5. Incubate overnight on ice in the dark. Test the extent of labeling by fluorescence microscopy (see below). If the filaments are inhomogeneously labeled, RhPh was not in excess over actin and/or the actin was not adequately mixed into the labeling solution. If the background fluorescence noticeably obscures the filament fluorescence, then too little actin was added to the labeling solution. RhPh labeled actin is stable on ice in the dark for at least a week.
Filament fragments: The filament length of unsheared actin is typically 5 to 40 #m. We have found that treating RhPh-labeled actin with the Ca2+-dependent actin severing and capping protein severin 9 or with sonication reproducibly shortens filaments. To use severin to fragment actin, make RhPh-labeled actin as above, except in AB with 0.1 mM EGTA. To an aliquot, add an appropriate molar ratio of severin diluted in AB with 0.1 mM EGTA, with the consideration that actin filaments have - 350 monomers/#m length. To fragment the actin, add an equal volume of AB without EGTA but with 0.2 mM CaC12. Alternatively, sonicate RhPh-labeled actin at 0 ° with a small probe. In either case, the extent of fragmentation is monitored by negative stain electron microscopy or by fluorescence microscopy. Fluorescence Microscopy
General Considerations The principles of fluorescence microscopy have been reviewed elsewhere.~° Important aspects of fluorescence microscopy specific to the motility assay are related to the challenge of nondestructively obtaining a continuous image from a small number of fluorescent groups. This demands optimization of both the optical and electrooptical components of the microscope system.
Instrument Specifications Microscope. The assay requires a research photomicroscope with epifluorescence illuminator [e.g. Nikon (Garden City, NY) Optiphot], 9 K. Yamamoto, J. D. Pardee, J. Reidler, L. Stryer, and J. A. Spudich, J. Cell Biol. 95, 711 (1982). io D. L. Taylor and E. D. Salmon, Methods CellBiol. 29, 207 (1989).
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stripped of all unnecessary optical components which may absorb or scatter light (Fig. 1). A C-mount (video standard) adaptor is required. Illumination. The standard epiillumination light source is the 546-nm emission line of a 100-W mercury high-pressure arc lamp (e.g., Osram, Berlin-Munich, FRG, HBO 100 W/2). Typically, the beam is attenuated with neutral density filters or an iris diaphragm to control photodamage. Optical Filters. The optical filter "cube" in an epiillumination fluorescence microscope has two complementary functions. The excitation light (short wavelength) is selected by the excitor filter (band pass) and reflected toward the back aperture of the objective by the dichroic mirror (long pass) while the fluorescence (long wavelength) passes through the dichroic mirror and the barrier filter (band or long pass) to the back aperture of the projection eyepiece. In order to collect a significant proportion of the fluorescence of the RhPh, relatively close excitation and emission bands are necessary (Fig. 2). However, imaging a few hundred fluorophores demands optical filter sets which display both high transmittance in the pass band and excellent rejection of the excitation light by the emission filter. While such filters are commercially available (Omega Optical, Brattleboro, VT), filter sets supplied by microscope manufacturers may fail both requirements and should be tested for suitability. Numerical Aperture (NA). The brightness of fluorescence in an epiillumination microscope depends on the object NA to the fourth power,
I
video camera
camera control
uni
1
optical image ~ t coupling intensifier
1
ve
ImieroseopestandI stage detail FIG. 1. Schematic of the microscope system (see text).
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MECHANOCHEMICAL PROPERTIES OF MOTOR PROTEINS 100
[33]
100
0~ 80
ao ~
60
6o ~ E
• •
40
~
20
e¢
I 525
550
575
600
625
Wavelength (nm) FIo. 2. Optimized epifluoreseence filter set for RhPh fluorescence. The dotted line shows the emission spectrum of RhPh with 540-nm excitation. The solid curve at shorter wavelengths is the transmission characteristic of the excitor filter (Omega 540 DF 23), the dashed line is that of the dichroic mirror (Omega DR 555 LP at 45 °), and the second solid curve that of the barrier filter (Omega 580 DF 30). With this filter set the desired qualities of bright illumination, high rejection of scattered light, and high transmission of fuorescence to the detector are achieved.
because the objective serves as a condenser as well. In addition, NA directly translates into sharpness of the image of the actin filament. Planapochromat and "fluorite" oil immersion objectives are available which feature a high transmittance and NA -> 1.2. One objective we have examined which is both bright and sharp is the Nikon 100×/1.4 NA CFN Plan Apochromat. Generally we use the Zeiss 63/1.4 Planapochromat (461840). The newer Zeiss Axioline Plan-Neofluar 100/1.3 and Axioline Planapochromat 63/1.4 are probably as bright or better. Immersion oil designed for very low autofluorescence (e.g., type FF; Cargille, Cedar Grove, N J) is critical. Magnification. Methods for mating microscopes to video cameras have been discussed elsewhere." System magnification will be affected by the objective, projection eyepiece, camera lens (if used), and camera magnifications. In general, magnification and image brightness are at cross-purposes. A system magnification which yields a 50- to 100-gm diameter field across the monitor is often optimal. With a sensitive enough camera and a high numerical aperture system, this diameter can be reduced to 10 to 20 ~m.
Video. While the sliding movement of actin filaments over myosin can be observed by eye, quantitation of the movement demands recording and analysis of real-time electronic images. Selecting a low-light video camera system inevitably becomes a matter of compromising sensitivity, spatial and temporal resolution, noise, linearity, or price.12 Generally, the fluores" S. Inoue, "Video Microscopy." Plenum, New York, 1986. 12 R. J. Lowy and K. R. Spring, Methods CellBiol. 29, 269 (1989).
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cence of actin filaments is too dim for a Newvicon or video rate CCD camera to image and is somewhat low for a SIT camera [e.g., C2400-08, Photonic Microscopy (Hamamatsu), Oak Brook, IU or SIT 66, Dage MTI, Michigan City, IN], though bright for an ISIT. Microchannel plate intensified Newvicon or CCD cameras [e.g., KS1381, Videoscope, Washington, DC, C2400-09, Photonic Microscopy or GENIIsys, DageMTI] offer another option. Analog contrast enhancement, as is afforded by adjustment of camera gain and offset, is generally useful. Real-time digital image processing is not necessary to achieve a usable image, but contrast enhancement by background subtraction, digital gain and offset, look-up tables, and spatial filters each give some benefit. Frame averaging should be used judiciously because of inevitable distortion of motion. Recording images in real time requires high-resolution monochrome recording on videotape (VHS, Umatic, etc.) or optical disk. In the future, high-definition video and video-rate laser confocal fluorescence imaging may become practical for very low light level microscopy. Temperature Control. Reproducibility demands careful attention to temperature control. Because oil immersion optics are used, a simple stage heater/cooler is inadequate. A temperature jacket for the objective is also necessary. A copper slide carrier and a copper sleeve for the objective, each with flow passages for fluid from a temperature bath, can be machined easily (Fig. 1). The temperature is monitored by small thermocouples (Omega, Stamford, CT) which can be placed next to or in the flow cell. Temperature cycling expensive objectives is a prescription for expensive repairs, once optical elements drop out of their positions. This has happened to our Zeiss Planapochromat 63/1.4 once in 3 years of exposure to temperature extremes of 0 to 42 °.
Methods Photobleaehing. RhPh shares with other fluorescence probes the problems of photobleaching and oxygen free radical generation, processes directly proportional to the rate of photon absorption events. The mechanism ofphotobleaching involves both intrinsic properties of the fluorescent probe and details of its environment. Quenching of excited states of fluorescent groups by molecular oxygen may lead to the production of free radicals. These contribute to photodestruction of the probe and of other reactive moieties in the locale of the probe, such as enzyme active sites. In movement assays, photodamage is observed progressively as inhibition of sliding movement, irreversible photobleaching, and then fragmentation of the actin filaments. Several approaches to prevent photodamage are applicable to in vitro motility assays. Obviously, illumination is limited to the lowest level com-
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MECHANOCHEMICAL PROPERTIES OF MOTOR PROTEINS
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patible with adequate imaging. Photobleach inhibitors commonly used for immunofluorescence may be toxic to the actin-myosin interaction. High concentrations of the reducing agents DTT (-< 100 mM) ~3 and 2-mercaptoethanol (-< 70 mM) TMdo not inhibit actin sliding movement, but dramaticaUy stabilize the fluorescence. However, degassing solutions under vacuum and then enzymaticaUy scavenging dissolved oxygen with glucose oxidase (0.1 mg/ml), catalase (0.018 mg/ml), and glucose (3 mg/ml), 15 added as 50 × stocks of enzymes and glucose a few minutes before imaging, is far and away the best approach. The enzyme stock cannot be stored on ice longer than 1 day. ImagingActin Filaments. In order to tune the fluorescence microscope, to make rational selection of optical components and cameras, and to protect the objective and the camera tube from accidental damage, it is necessary to gain facility with focusing on actin filaments. I. Center and collimate the arc and center and nearly close the field diaphragm. 2. Dilute the RhPh actin stock 100-fold with AB containing 0.1 M DTT and place a 4-/zl drop under a coverslip. 3. Generously oil the slide. Darken the room and open the shutter to 546-nm light (green). Focus down to touch the oil. 4. Turn on the camera and increase the gain until a bright background is imaged. Slowly rack the objective down until the filaments bound to the undersurface of the coverslip come sharply into focus (Fig. 3). 5. Adjust the field diaphragm until it vignettes the video frame and then open the diaphragm to just surround the video frame. Adjust the analog and digital contrast to optimize the image. It may be necessary initially to image the actin with the binocular and eyepieces in place, in order to be certain that any failure is not due to lack of sensitivity of the video camera. With the naked eye, fully labeled RhPh actin, illuminated by the full brightness of the 546-nm line of a 100-W Hg lamp, should appear as thin bright red lines through the eyepieces. Motility Assay
Equipment and Materials Microscope system: Epifluorescence microscope with camera system (see above), stage micrometer, video time/date generator, video recorder ~3H. Honda, H. Nagashima, and S. Asakura, J. Mol. Biol. 191, 131 (1986). 14T. Yanagida, M. Nakase, K. Nishiyama, and F. Oosawa, Nature (London) 307, 58 (1984). 15A. Kishino and T. Yanagida, Nature (London) 334, 74 (1988).
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FIG. 3. Image of fluorescent actin filaments. RhPh-labeled actin filaments were imaged bound to HMM immobilized on an NC film in the absence of ATP and a single frame was photographed from a monitor (Sony PVM-122, Teaneck, NJ). The optical system used to record the image was a Zeiss standard epifluorescence microscope with 100-W Hg arc, × 63 Planapochromat objective and ) 75-fold excess of nonfluorescent actin is added. We have investigated several approaches to continuous rather than end-point measurements of ATP hydrolysis in the flow cell. Fluorescence assays for a coupled NADH/NAD ÷ reaction using microscope photometry or high-pressure liquid chromatography (HPLC) analysis of samples taken from the flow cell may offer a reasonable approach. Force Measurement. One area which has only begun to be investigated is force measurement in the in vitro assay. The interaction of myosin heads with actin can be stabilized by decreasing the ATP concentration, adding a slow isoform of myosin, or by chemically modifying a fraction of the myosin heads with a sulfhydryl-modifying reagent such as N-ethylmaleimide (NEM) in order to produce "internal" forces. However, external 19 H. Hayashi, K. Takiguchi, and S. Higashi-Fujime, J. Biochem. (Tokyo) 105, 875 (1989).
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mechanical perturbations of moving filaments are necessary to measure the forces explicitly. Viscosity of the assay medium is not a practical approach to loading down the actin filament. Kishino and Yanagida 15used a glass needle attached to an actin filament moving over a myosin-coated surface to measure the forces produced by small numbers of myosin heads. The recently developed laser optical tweezer may offer another approach to applying a force to a sliding actin filament. Electron Microscopy. There are likely many experiments which require a high-resolution analysis of the myosin-coated surface or its interaction with actin filaments. It is often desirable to measure the density of heads on the surface or to assess the extent of their interaction with actin. An ideal approach to this problem might be with scanning tunneling or atomic force microscopy, either of which may not require dehydration and fixing of the specimen. However, practical considerations restrict us to considering negative stain electron microscopy (EM) techniques. Thin copper EM grids can be laid onto a floating NC or Formvar film and then sandwiched between the film and the coverslip. This technique leaves an air gap between film and glass within the grid squares, which makes imaging events occurring on the grid difficult when this is attempted through the coverslip. Alternately, the grid can be placed on the slide just below the upper coverslip and its surface imaged from above. A finder grid allows the same region imaged in fluorescence to be identified in negative stain EM. 17 In order to quench actin sliding movement, we have found that simply removing ATP by washing in ATP-free buffer results in fragmentation of the actin filaments. Instead, a 40% solution of ethylene glycol in AB/BSA/ ATP will stop the movement without fragmentation or dissociation of actin from the surface. The ATP can then be washed out with the same buffer and the surface fixed and stained with 1% (w/v) uranyl acetate. The stability of any film in the electron microscope is increased by evaporating carbon onto the surface. Immunoelectron microscopy techniques can be very useful. Using a specific antibody, secondary antibody-colloidal gold conjugates (Auroprobe; Janssen, Piscataway, NJ) can be used to label myosin heads or actin binding proteins bound to the actin filaments. After quenching the sliding movement, introduce a dilution of the primary antibody in AB/BSA into the flow cell and incubate at room temperature. After washing with AB/ BSA, infuse a 1:25 dilution of the secondary antibody-gold conjugate in AB/BSA and incubate 60 min at room temperature. Rinse the flow cell and infuse uranyl acetate to stain in situ or remove the EM grid in order to stain it. Quantitation. Quantitation to abstract velocities of sliding movement is
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potentially a frustrating exercise. Actin filaments do not all move at precisely the same speed nor does each filament move at a constant speed throughout its path, probably due to imperfections in the myosin coated surface. The filaments move in winding paths, so that measuring displacements across the video screen is not straightforward. To preserve the experimentor and to maintain objectivity, some form of automated measurement would be advised. Most likely, quite accurate analysis of sliding movement can be performed using an expensive video-based system (Celltrak; Motion Analysis, Santa Rosa, CA) which tracks centroids of moving objects. Similar results can be achieved using any image processor/microcomputer combination and an intensive programming effort. We have thus far taken a less sophisticated approach, using a cursor superimposed on the video image to trace the path of the moving filaments, while keeping track of video frames.2° This approach can be criticized as it lends itself to selecting the faster moving filaments in any sequence. Several conditions must be met before highly accurate quantitation can be performed.21 Lag in the imaging system should be minimized. The degree of spatial distortion in the imaging system can be determined and, if significant, dealt with by correction of the image in the digital domain. Digitization errors can lead to significant velocity errors, even for stationary objects. Actin Filament Length Measurement. For several types of studies, measuring the lengths of actin filaments may be important, but fixing filaments for EM analysis may be impractical. Tracing the image of individual filaments from the fluorescence image to measure their length is reproducible for longer filaments but particularly subject to error for filaments less than 1/tm in length. Integration of the fluorescence intensity over the filament image may be used if linearity of response in the imaging system can be demonstrated. Acknowledgments We thank Dr. T. Yanagida for communicating unpublished results. This work was supported by NIH Grant GM33289 to J.A.S.S.J.K. was a trainee of the Medical Scientist Training Program. T.Q.P.U. is supported by a Japan Society for the Promotion of Science Fellowship for Research Abroad.
20 M. P. Sheetz, S. M. Block, and J. A. Spudich, this series, Vol. 134, p. 531. 2~ Z. Jericevic, B. Wiese, J. Bryan, and L. C. Smith, Methods CellBiol. 30, 47 (1989).
ASSAYS FOR A C T I N S L I D I N G M O V E M E N T
[33] A s s a y s f o r A c t i n S l i d i n g M o v e m e n t Coated Surfaces
By STEPHENJ. KRON, YOKO Y.
TOYOSHIMA,
399
over Myosin-
TARO Q. P. UYEDA,and
JAMES A. SPUDICH Introduction One important result from in vitro studies of the interaction of the major proteins of muscle, actin and myosin, has been the growing recognition that nearly any aspect of muscle mechanics can be studied in a model system consisting of purified proteins. The consensus that has emerged is that actin and myosin, when placed in an appropriate geometry, can produce movement and force coupled to the hydrolysis of ATP in much the same way that stimulated muscle uses ATP to contract. The following is a compilation of our experience with techniques for purified in vitro motility assays for actin sliding movement over myosin) We limit our focus to studies using skeletal muscle proteins, but only slight modification of these protocols may be necessary for proteins derived from smooth muscle and nonmuscle sources. Protein Reagents General Considerations The properties of the protein preparations used are critical to reproducibility of actin sliding movement assays. The following methods are presented as trustworthy preparations but are not singularly successful. However, in particular it should be noted that myosin subfragment preparations that work well in solution experiments may not be optimal for use in movement assays. Myosin and Its Fragments Several forms of myosin, including filaments, monomers, and soluble proteolytic fragments, have been found to work well in aetin sliding movement assays. A desirable characteristic of myosin preparations for motility assays is the absence of contamination by irreversible "rigor heads" that show ATP-insensitive binding to actin filaments. Such heads will inevita1 S. J. Kron and J. A. Spudich, Proc. Natl. Acad. Sci. U.S.A. 83, 6272 (1986).
METHODS IN ENZYMOLOGY, VOL. 196
Copyright © 1991 by Academic Press, Inc. All fights of reproduction in any form reserved.
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bly "load down" actin filaments, inhibiting their movement. Use of freshly purified proteins, freshly made buffers, careful handling of myosin to avoid unnecessary contact with fluid-air interfaces, and liberal use of reducing agents are important. Chymotryptically derived subfragment 1 (S 1), the isolated myosin head domain, is commonly used in biochemical and structural studies. However, unless used at high concentrations, chymotryptic S1 as it is commonly prepared does not support movement in the assay. Somewhat higher molecular weight forms of S 1, prepared by papain digestion in the presence of Mg2+ or EDTA, work very well in the movement assay. Each form of myosin has a characteristic sliding speed, ranging from 1 to 2 Ftm/sec for S 1 to 4 to 8/~m/sec for heavy meromyosin (HMM) when measured in assay buffer at 30 ° .2 The basis for these differences remains uncharacterized. The following preparations give yidds o f - 70% of theoretical for HMM and - 4 0 % for S1. Modifications to increase yield result in considerable proteolytic nicking of the heavy chain at sites within the myosin head (and of the light chains), which is associated with an increase in the measured average sliding speed.
Buffers MSB: 25 m M imidazole hydrochloride, pH 7.4, 0.6 M KCI, 1 m M dithiothreitol (DTT) BED: 0.1 m M NaHCO3, 0. l m M EGTA, 1 m M DTT 2 × CHB: 20 m M imidazole hydrochloride, pH 7.0, 1 M KCI, 4 m M MgCI:, 10 m M D T T PMSF stock: 0.2 M phenylmethylsulfonyl fluoride in ethanol PMB: 25 mMimidazole hydrochloride, pH 7.4, 100 mMNaC1, 5 m M MgCl2, 1 m_MDTT E64 stock: 1 mg/ml in dimethyl sulfoxide (DMSO) SB: l0 mMimidazole hydrochloride, pH 7.4, 0.5 MKC1, 1 m M D T T
Methods. Myosin preparation and storage: Prepare myosin from skeletal muscle according to any standard protocol (e.g., Hynes et al.3), with the use of 0.1 to 1 m M DTT in all buffers. Do not use ammonium sulfate precipitation as a step in the preparation, as this is associated with the production of irreversible rigor heads. To store myosin, dissolve pelleted filaments in an equal volume of 1.2 M KCI and 1 m M DTT, add MSB to dilute the myosin to a final concentration of 30 to 60 mg/ml as determined 2 y. y. Toyoshima, S. J. Kron, E. M. McNally, K. R. Niebling, C. Toyoshima, and J. A. Spudich, Nature (London) 328, 536 (1987). 3 T. R. Hynes, S. M. Block, B. T. White, and J. A. Spudich, Cell (Cambridge, Mass.) 48, (1987).
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by its extinction coefficient, A28o = 0.53 cm2/mg,4 and then add glycerol to 50% (v/v). Centrifuge the myosin to clear it of suspended air bubbles and then aliquot into microfuge tubes, which are topped off to avoid trapping air. Store the myosin at - 2 0 °. It is stable for several months.
Heavy meromyosin preparation, based on method of Okamoto and SekineS: 1. Add 9 vol cold BED to - 2 0 mg stock myosin in a centrifuge tube and mix to precipitate filaments. After > l0 min of incubation on ice, centrifuge at 4 ° at low speed in a swinging bucket rotor to sediment the filaments (e.g., l0 min at 13,000 rpm in JS-13 rotor; Beckman, Palo Alto, CA). 2. Dissolve the pellet in 2× CHB and BED as needed to achieve a final concentration of 15 mg/ml myosin in CHB. 3. Incubate the myosin solution l0 min at 25 °. 4. Add N-tosyl-L-lysine-chloromethyl ketone (TLCK)-treated a-chymotrypsin to 12.5/tg/ml, gently mix the reaction, and incubate 7.5 to l0 rain at 25 o. 5. Add 9 vol ice-cold BED with 3 m M MgC12 and 0.1 m M PMSF to the reaction and mix. After > 1 hr on ice, centrifuge the suspension at high speed (e.g., 15 min at 75,000 rpm in TL100.3 rotor, Beckman). 6. Store the supernatant, which is typically - 0 . 7 mg/ml and > 90% pure HMM, on ice. It can be used in motility assays for 3 to 5 days. Extinction coefficient, A28o ----0.60 cm2/mg. +
Papain Mg-SI preparation2: 1. Process 20 mg myosin to a pellet as described above. 2. Dissolve the pellet in an equal volume (-0.25 ml) of 1.2 M KC1 and 20 m M DTT in BED, and incubate l0 min at 25 o. 3. Add 19 vol ice-cold BED (to - 10-ml final volume), incubate on ice > 10 rain, and centrifuge at low speed. 4. Resuspend the pellet in PMB to a final concentration of 10-12 mg/ml and incubate 10 rain at 25 °. 5. Add papain to 12.5/~g/ml, mix gently, and incubate 7.5- l0 min at 25 ° . 6. Stop the reaction with greater than an equal volume of ice-cold BED with 5 m M MgC12 and 5/~g/ml E64 (Peninsula Laboratories, Belmont, CA). Incubate > 1 hr on ice before high-speed centrifugation. 7. Store the supernatant, typically - 1.1 mg/ml S1, on ice. It can be used for 3 to 5 days in the movement assay. The level of contamination by HMM may be significant for some experiments. Extinction coefficient, A280 = 0.81 cm2/mg. 4 4 S. S. Margossian and S. Lowey, this series, Vol. 85, p. 55. s y. Okamoto and T. Sekine, J. Biochem. (Tokyo) 98, 1143 (1985).
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MECHANOCHEMICAL PROPERTIES OF MOTOR PROTEINS
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Gel-filtration purification of HMM and SI: Further purification of myosin soluble fragments is often desirable and is accomplished by highperformance gel filtration [e.g., Superose 6 or 12 HR FPLC columns (Pharmacia LKB, Piscataway NJ), 0.25 ml/min] using a high ionic strength buffer such as SB. Concentrate the HMM or S1 supernatant, prepared as described above, in a Centricon or Centriprep 30 (Amicon, Danvers MA) ultrafiltration device to < 0.5 ml. Centrifuge the retentate at high speed (5 min at 75,000 rpm in TL100.2, Beckman), and load the supernatant onto the column. Pool protein peaks by OD28o or by Coomassie SDS-PAGE analysis. Myosin fragments can be used directly as eluted from the column. Actin affinity purification: Soluble myosin fragments can be treated to precipitate irreversible rigor heads, before use in the motility assay. Add filamentous actin to 0.15 mg/ml and 1 m M ATP to an aliquot of the stock HMM or S 1. After a short incubation on ice, centrifuge the mixture at high speed (10 min at 75,000 rpm in a TL100.2, Beckman) to sediment the actin. Actin preparations can have an associated proteolytic activity. Thus, use the supernatant within 2 to 3 hr. Actin Buffers GB: 2.5 m M imidazole hydrochloride, pH 7.4, 0.2 m M CaC12, 0.2 m M ATP, 0.2 m M DTT 10× AB: 225 m M imidazole hydrochloride, pH 7.4, 250 m M KC1, 40 m M MgCI2, 10 m M EGTA AB: 25 m M imidazole hydrochloride, pH 7.4, 25 m M KC1, 4 m M MgCI2, 1 m M EGTA, 1 m M DTT
Methods. Actin preparation: To prepare rabbit skeletal muscle actin, we use a modification of the method of Pardee and Spudich. 6 Further purification of actin by anion-exchange chromatography may be useful, but is often complicated by polymerization of actin in the chromatographic media as it elutes. 1. Prepare the acetone powder as described and store at - 20 °. Starting with - 10 g acetone powder, perform the extraction, polymerization, KC1 cut, and centrifugation essentially as described. 2. After leaving the actin pellets overnight on ice softening in buffer A 6, resuspend the pellets in GB to 10- 15 ml and dialyze against one change of buffer GB, > 48 hr at 4 °. 3. Change the dialysis to GB with 0.05 m M CaC12 and dialyze overnight at 4 °. 6 j. D. Pardee and J. A. Spudich, Methods CellBiol. 24, 271 (1982).
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4. Centrifuge the actin at high speed and polymerize the supernatant by adding 1/9 vol of 10X AB, made up in the last dialysis buffer. The final polymerization leaves the actin as filaments in motility assay buffer (AB) with 0.2 m M ATP. 5. Store the actin on ice. It can be used in the movement assay or for purification of myosin soluble fragments for several weeks. Extinction coefficient A29o_3t0= 0.62 cm2/mg.7 Stored actin rejuvenated by "recycling. '" 1. Pellet an aliquot of the actin by high-speed centrifugation (15 min at 100,000 rpm in TlI00.3, Beckman). Wash the pellet with GB and then soak under GB overnight on ice. 2. Resuspend the actin to less than 5 mg/ml in GB and dialyze against one change of GB, > 24 hr at 4 °. 3. Change the dialysis to GB with 0.05 m M CaC12 overnight. 4. Centrifuge the actin at high speed and polymerize the supernatant with 1/9 vol 10X AB made up in the last change of dialysis buffer. Fluorescent actin filaments: The diameter of actin filaments precludes direct imaging by transmitted light microscopy. Thus the filaments must be labeled to be imaged. The original description of imaging of fluorescent actin filaments in the microscope by Asakura and colleagues was of actin covalently modified with a fluorescent group and stabilized by phaUoidin. This approach offers the widest range of possible fluorescent probes. However, the most convenient label for actin filaments is tetramethylrhodamine phalloidin (RhPh), a fluorescent analog of the Amanita phalloides toxin, phalloidin. Phalloidin binds with high affinity (Kd -- 10-8) to actin filaments and stabilizes them against depolymerization. Significantly, phalloidin has no effect on actin activation of myosin ATPase in vitro. Among the advantages of RhPh are its high affinity for actin, and the stability and efficiency of the tetramethylrhodamine group,s RhPh has an excitation maximum of 550 nm, very close to the 546-nm Hg emission line, and an emission maximum at 575 nm. Also available commercially are phallotoxins labeled with fluorescein, coumarin, and N-(7-nitrobenz-2oxa- 1,3-diazol-4-yl) (NBD). Prepare the stock solution of RhPh actin filaments as follows.t 1. Dry 94/tl of 3.3 a M RhPh in methanol (Molecular Probes, Eugene, OR) to a pellet, while protected from light, in a Speed Vac concentrator (Savant, Hicksville, NY) or by an N2 stream. 7 D. J. Gordon, Y.-Z. Yang, and E. D. Korn, J. Biol. Chem. 251, 7474 (1976). 8 H. Faulstich, S. Zobeley, G. Rinnerthaler, and J. V. Small, J. Muscle Res. Cell Motil. 9, 370 (1988).
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2. Dissolve the pellet in 2 #1 ethanol. 3. Add 290 #1 AB, and vortex approximately 30 sec. 4. Add 10/tl of 1 mg/ml actin in AB and mix well. 5. Incubate overnight on ice in the dark. Test the extent of labeling by fluorescence microscopy (see below). If the filaments are inhomogeneously labeled, RhPh was not in excess over actin and/or the actin was not adequately mixed into the labeling solution. If the background fluorescence noticeably obscures the filament fluorescence, then too little actin was added to the labeling solution. RhPh labeled actin is stable on ice in the dark for at least a week.
Filament fragments: The filament length of unsheared actin is typically 5 to 40 #m. We have found that treating RhPh-labeled actin with the Ca2+-dependent actin severing and capping protein severin 9 or with sonication reproducibly shortens filaments. To use severin to fragment actin, make RhPh-labeled actin as above, except in AB with 0.1 mM EGTA. To an aliquot, add an appropriate molar ratio of severin diluted in AB with 0.1 mM EGTA, with the consideration that actin filaments have - 350 monomers/#m length. To fragment the actin, add an equal volume of AB without EGTA but with 0.2 mM CaC12. Alternatively, sonicate RhPh-labeled actin at 0 ° with a small probe. In either case, the extent of fragmentation is monitored by negative stain electron microscopy or by fluorescence microscopy. Fluorescence Microscopy
General Considerations The principles of fluorescence microscopy have been reviewed elsewhere.~° Important aspects of fluorescence microscopy specific to the motility assay are related to the challenge of nondestructively obtaining a continuous image from a small number of fluorescent groups. This demands optimization of both the optical and electrooptical components of the microscope system.
Instrument Specifications Microscope. The assay requires a research photomicroscope with epifluorescence illuminator [e.g. Nikon (Garden City, NY) Optiphot], 9 K. Yamamoto, J. D. Pardee, J. Reidler, L. Stryer, and J. A. Spudich, J. Cell Biol. 95, 711 (1982). io D. L. Taylor and E. D. Salmon, Methods CellBiol. 29, 207 (1989).
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stripped of all unnecessary optical components which may absorb or scatter light (Fig. 1). A C-mount (video standard) adaptor is required. Illumination. The standard epiillumination light source is the 546-nm emission line of a 100-W mercury high-pressure arc lamp (e.g., Osram, Berlin-Munich, FRG, HBO 100 W/2). Typically, the beam is attenuated with neutral density filters or an iris diaphragm to control photodamage. Optical Filters. The optical filter "cube" in an epiillumination fluorescence microscope has two complementary functions. The excitation light (short wavelength) is selected by the excitor filter (band pass) and reflected toward the back aperture of the objective by the dichroic mirror (long pass) while the fluorescence (long wavelength) passes through the dichroic mirror and the barrier filter (band or long pass) to the back aperture of the projection eyepiece. In order to collect a significant proportion of the fluorescence of the RhPh, relatively close excitation and emission bands are necessary (Fig. 2). However, imaging a few hundred fluorophores demands optical filter sets which display both high transmittance in the pass band and excellent rejection of the excitation light by the emission filter. While such filters are commercially available (Omega Optical, Brattleboro, VT), filter sets supplied by microscope manufacturers may fail both requirements and should be tested for suitability. Numerical Aperture (NA). The brightness of fluorescence in an epiillumination microscope depends on the object NA to the fourth power,
I
video camera
camera control
uni
1
optical image ~ t coupling intensifier
1
ve
ImieroseopestandI stage detail FIG. 1. Schematic of the microscope system (see text).
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MECHANOCHEMICAL PROPERTIES OF MOTOR PROTEINS 100
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100
0~ 80
ao ~
60
6o ~ E
• •
40
~
20
e¢
I 525
550
575
600
625
Wavelength (nm) FIo. 2. Optimized epifluoreseence filter set for RhPh fluorescence. The dotted line shows the emission spectrum of RhPh with 540-nm excitation. The solid curve at shorter wavelengths is the transmission characteristic of the excitor filter (Omega 540 DF 23), the dashed line is that of the dichroic mirror (Omega DR 555 LP at 45 °), and the second solid curve that of the barrier filter (Omega 580 DF 30). With this filter set the desired qualities of bright illumination, high rejection of scattered light, and high transmission of fuorescence to the detector are achieved.
because the objective serves as a condenser as well. In addition, NA directly translates into sharpness of the image of the actin filament. Planapochromat and "fluorite" oil immersion objectives are available which feature a high transmittance and NA -> 1.2. One objective we have examined which is both bright and sharp is the Nikon 100×/1.4 NA CFN Plan Apochromat. Generally we use the Zeiss 63/1.4 Planapochromat (461840). The newer Zeiss Axioline Plan-Neofluar 100/1.3 and Axioline Planapochromat 63/1.4 are probably as bright or better. Immersion oil designed for very low autofluorescence (e.g., type FF; Cargille, Cedar Grove, N J) is critical. Magnification. Methods for mating microscopes to video cameras have been discussed elsewhere." System magnification will be affected by the objective, projection eyepiece, camera lens (if used), and camera magnifications. In general, magnification and image brightness are at cross-purposes. A system magnification which yields a 50- to 100-gm diameter field across the monitor is often optimal. With a sensitive enough camera and a high numerical aperture system, this diameter can be reduced to 10 to 20 ~m.
Video. While the sliding movement of actin filaments over myosin can be observed by eye, quantitation of the movement demands recording and analysis of real-time electronic images. Selecting a low-light video camera system inevitably becomes a matter of compromising sensitivity, spatial and temporal resolution, noise, linearity, or price.12 Generally, the fluores" S. Inoue, "Video Microscopy." Plenum, New York, 1986. 12 R. J. Lowy and K. R. Spring, Methods CellBiol. 29, 269 (1989).
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cence of actin filaments is too dim for a Newvicon or video rate CCD camera to image and is somewhat low for a SIT camera [e.g., C2400-08, Photonic Microscopy (Hamamatsu), Oak Brook, IU or SIT 66, Dage MTI, Michigan City, IN], though bright for an ISIT. Microchannel plate intensified Newvicon or CCD cameras [e.g., KS1381, Videoscope, Washington, DC, C2400-09, Photonic Microscopy or GENIIsys, DageMTI] offer another option. Analog contrast enhancement, as is afforded by adjustment of camera gain and offset, is generally useful. Real-time digital image processing is not necessary to achieve a usable image, but contrast enhancement by background subtraction, digital gain and offset, look-up tables, and spatial filters each give some benefit. Frame averaging should be used judiciously because of inevitable distortion of motion. Recording images in real time requires high-resolution monochrome recording on videotape (VHS, Umatic, etc.) or optical disk. In the future, high-definition video and video-rate laser confocal fluorescence imaging may become practical for very low light level microscopy. Temperature Control. Reproducibility demands careful attention to temperature control. Because oil immersion optics are used, a simple stage heater/cooler is inadequate. A temperature jacket for the objective is also necessary. A copper slide carrier and a copper sleeve for the objective, each with flow passages for fluid from a temperature bath, can be machined easily (Fig. 1). The temperature is monitored by small thermocouples (Omega, Stamford, CT) which can be placed next to or in the flow cell. Temperature cycling expensive objectives is a prescription for expensive repairs, once optical elements drop out of their positions. This has happened to our Zeiss Planapochromat 63/1.4 once in 3 years of exposure to temperature extremes of 0 to 42 °.
Methods Photobleaehing. RhPh shares with other fluorescence probes the problems of photobleaching and oxygen free radical generation, processes directly proportional to the rate of photon absorption events. The mechanism ofphotobleaching involves both intrinsic properties of the fluorescent probe and details of its environment. Quenching of excited states of fluorescent groups by molecular oxygen may lead to the production of free radicals. These contribute to photodestruction of the probe and of other reactive moieties in the locale of the probe, such as enzyme active sites. In movement assays, photodamage is observed progressively as inhibition of sliding movement, irreversible photobleaching, and then fragmentation of the actin filaments. Several approaches to prevent photodamage are applicable to in vitro motility assays. Obviously, illumination is limited to the lowest level com-
408
MECHANOCHEMICAL PROPERTIES OF MOTOR PROTEINS
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patible with adequate imaging. Photobleach inhibitors commonly used for immunofluorescence may be toxic to the actin-myosin interaction. High concentrations of the reducing agents DTT (-< 100 mM) ~3 and 2-mercaptoethanol (-< 70 mM) TMdo not inhibit actin sliding movement, but dramaticaUy stabilize the fluorescence. However, degassing solutions under vacuum and then enzymaticaUy scavenging dissolved oxygen with glucose oxidase (0.1 mg/ml), catalase (0.018 mg/ml), and glucose (3 mg/ml), 15 added as 50 × stocks of enzymes and glucose a few minutes before imaging, is far and away the best approach. The enzyme stock cannot be stored on ice longer than 1 day. ImagingActin Filaments. In order to tune the fluorescence microscope, to make rational selection of optical components and cameras, and to protect the objective and the camera tube from accidental damage, it is necessary to gain facility with focusing on actin filaments. I. Center and collimate the arc and center and nearly close the field diaphragm. 2. Dilute the RhPh actin stock 100-fold with AB containing 0.1 M DTT and place a 4-/zl drop under a coverslip. 3. Generously oil the slide. Darken the room and open the shutter to 546-nm light (green). Focus down to touch the oil. 4. Turn on the camera and increase the gain until a bright background is imaged. Slowly rack the objective down until the filaments bound to the undersurface of the coverslip come sharply into focus (Fig. 3). 5. Adjust the field diaphragm until it vignettes the video frame and then open the diaphragm to just surround the video frame. Adjust the analog and digital contrast to optimize the image. It may be necessary initially to image the actin with the binocular and eyepieces in place, in order to be certain that any failure is not due to lack of sensitivity of the video camera. With the naked eye, fully labeled RhPh actin, illuminated by the full brightness of the 546-nm line of a 100-W Hg lamp, should appear as thin bright red lines through the eyepieces. Motility Assay
Equipment and Materials Microscope system: Epifluorescence microscope with camera system (see above), stage micrometer, video time/date generator, video recorder ~3H. Honda, H. Nagashima, and S. Asakura, J. Mol. Biol. 191, 131 (1986). 14T. Yanagida, M. Nakase, K. Nishiyama, and F. Oosawa, Nature (London) 307, 58 (1984). 15A. Kishino and T. Yanagida, Nature (London) 334, 74 (1988).
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FIG. 3. Image of fluorescent actin filaments. RhPh-labeled actin filaments were imaged bound to HMM immobilized on an NC film in the absence of ATP and a single frame was photographed from a monitor (Sony PVM-122, Teaneck, NJ). The optical system used to record the image was a Zeiss standard epifluorescence microscope with 100-W Hg arc, × 63 Planapochromat objective and ) 75-fold excess of nonfluorescent actin is added. We have investigated several approaches to continuous rather than end-point measurements of ATP hydrolysis in the flow cell. Fluorescence assays for a coupled NADH/NAD ÷ reaction using microscope photometry or high-pressure liquid chromatography (HPLC) analysis of samples taken from the flow cell may offer a reasonable approach. Force Measurement. One area which has only begun to be investigated is force measurement in the in vitro assay. The interaction of myosin heads with actin can be stabilized by decreasing the ATP concentration, adding a slow isoform of myosin, or by chemically modifying a fraction of the myosin heads with a sulfhydryl-modifying reagent such as N-ethylmaleimide (NEM) in order to produce "internal" forces. However, external 19 H. Hayashi, K. Takiguchi, and S. Higashi-Fujime, J. Biochem. (Tokyo) 105, 875 (1989).
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mechanical perturbations of moving filaments are necessary to measure the forces explicitly. Viscosity of the assay medium is not a practical approach to loading down the actin filament. Kishino and Yanagida 15used a glass needle attached to an actin filament moving over a myosin-coated surface to measure the forces produced by small numbers of myosin heads. The recently developed laser optical tweezer may offer another approach to applying a force to a sliding actin filament. Electron Microscopy. There are likely many experiments which require a high-resolution analysis of the myosin-coated surface or its interaction with actin filaments. It is often desirable to measure the density of heads on the surface or to assess the extent of their interaction with actin. An ideal approach to this problem might be with scanning tunneling or atomic force microscopy, either of which may not require dehydration and fixing of the specimen. However, practical considerations restrict us to considering negative stain electron microscopy (EM) techniques. Thin copper EM grids can be laid onto a floating NC or Formvar film and then sandwiched between the film and the coverslip. This technique leaves an air gap between film and glass within the grid squares, which makes imaging events occurring on the grid difficult when this is attempted through the coverslip. Alternately, the grid can be placed on the slide just below the upper coverslip and its surface imaged from above. A finder grid allows the same region imaged in fluorescence to be identified in negative stain EM. 17 In order to quench actin sliding movement, we have found that simply removing ATP by washing in ATP-free buffer results in fragmentation of the actin filaments. Instead, a 40% solution of ethylene glycol in AB/BSA/ ATP will stop the movement without fragmentation or dissociation of actin from the surface. The ATP can then be washed out with the same buffer and the surface fixed and stained with 1% (w/v) uranyl acetate. The stability of any film in the electron microscope is increased by evaporating carbon onto the surface. Immunoelectron microscopy techniques can be very useful. Using a specific antibody, secondary antibody-colloidal gold conjugates (Auroprobe; Janssen, Piscataway, NJ) can be used to label myosin heads or actin binding proteins bound to the actin filaments. After quenching the sliding movement, introduce a dilution of the primary antibody in AB/BSA into the flow cell and incubate at room temperature. After washing with AB/ BSA, infuse a 1:25 dilution of the secondary antibody-gold conjugate in AB/BSA and incubate 60 min at room temperature. Rinse the flow cell and infuse uranyl acetate to stain in situ or remove the EM grid in order to stain it. Quantitation. Quantitation to abstract velocities of sliding movement is
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MECHANOCHEMICAL PROPERTIES OF MOTOR PROTEINS
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potentially a frustrating exercise. Actin filaments do not all move at precisely the same speed nor does each filament move at a constant speed throughout its path, probably due to imperfections in the myosin coated surface. The filaments move in winding paths, so that measuring displacements across the video screen is not straightforward. To preserve the experimentor and to maintain objectivity, some form of automated measurement would be advised. Most likely, quite accurate analysis of sliding movement can be performed using an expensive video-based system (Celltrak; Motion Analysis, Santa Rosa, CA) which tracks centroids of moving objects. Similar results can be achieved using any image processor/microcomputer combination and an intensive programming effort. We have thus far taken a less sophisticated approach, using a cursor superimposed on the video image to trace the path of the moving filaments, while keeping track of video frames.2° This approach can be criticized as it lends itself to selecting the faster moving filaments in any sequence. Several conditions must be met before highly accurate quantitation can be performed.21 Lag in the imaging system should be minimized. The degree of spatial distortion in the imaging system can be determined and, if significant, dealt with by correction of the image in the digital domain. Digitization errors can lead to significant velocity errors, even for stationary objects. Actin Filament Length Measurement. For several types of studies, measuring the lengths of actin filaments may be important, but fixing filaments for EM analysis may be impractical. Tracing the image of individual filaments from the fluorescence image to measure their length is reproducible for longer filaments but particularly subject to error for filaments less than 1/tm in length. Integration of the fluorescence intensity over the filament image may be used if linearity of response in the imaging system can be demonstrated. Acknowledgments We thank Dr. T. Yanagida for communicating unpublished results. This work was supported by NIH Grant GM33289 to J.A.S.S.J.K. was a trainee of the Medical Scientist Training Program. T.Q.P.U. is supported by a Japan Society for the Promotion of Science Fellowship for Research Abroad.
20 M. P. Sheetz, S. M. Block, and J. A. Spudich, this series, Vol. 134, p. 531. 2~ Z. Jericevic, B. Wiese, J. Bryan, and L. C. Smith, Methods CellBiol. 30, 47 (1989).
