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Chapter 9 Tracking Nanometer Movements of Single Motor Molecules
CHAPTER 9
Tracking Nanometer Movements of Single Motor Molecules Michael P. Sheetz and Scot C. Kuo Department of Cell Biology Duke University Medical Center Durham, North Carolina 27710
I. Introduction 11. Movements by Single Motor Molecules
111. Microtubule Movements on Motor-Coated Glass A. Purification and Storage of Motor Proteins for Motility B. Bead Movements on Microtubules C. Recording Nanometer-Level Movements D. Video-Enhanced DIC Microscopy of Nanometer Movements E. Analysis IV. Conclusion References
I. Introduction To understand the mechanism of motor movements it is necessary to understand how individual molecules move with sufficient spatial precision. Until recently, motility of motor proteins, such as muscle myosin, required an ensemble of motor molecules interacting with the same filament. In the last few years, the microtubule motor kinesin was demonstrated to work as single molecules (Howard et al., 1989; Block et al., 1990). Similar approaches with cytoplasmic dynein are showing promising results (Lopez and Sheetz, 1992). In these approaches, limiting dilutions of motor protein are adsorbed to an anionic surface, either acid-washed glass or carboxylate-modified polystyrene microspheres. The addition of carrier protein, such as bovine serum albumin, cytochrome c, METHODS IN CELL BIOLOGY. VOL 39 Copyright 8 1YY3 by Academic Prcrr. Inc. All rights ofreproduction
111
m y form rrserved
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Michael P. Sheetz and Scot C . Kuo
and casein, increases the range of active motor dilutions at least 50-fold during surface adsorption. Although specific activation of the motor protein cannot be excluded, the number of carrier proteins that preserve motor activity suggests that surface denaturation of the adsorbed motor protein is a major cause of reduced motor activity. As described in the later protocols, a number of carrier protein protocols have been published (Howard et al., 1989; Block et al., 1990), but our best activity for both kinesin and cytoplasmic dynein from either chicken or squid is obtained using casein alone. A variety of different technologies have been used to follow particle movements with high precision. A complete description of these technologies, including the necessary microscopy, is beyond the scope of this chapter, but the general principles are described. Approaches based on video are most convenient; however, improved temporal resolution and spatial sensitivity are achieved by custom photodiode detection systems with custom illumination.
11. Movements by Single Motor Molecules In general, the greatest obstacle to performing studies of single motor molecules is proving that the motility is indeed produced by single and not multiple molecules. As most motility assays often show less than a tenth of the motor molecules as active, arguments based on adsorbed motor density are ineffective and statistical arguments must be used. In the case of microtubules gliding on motor-coated glass, however, translocation while pivoting around a nodal point is a strong indicator of motility by a single motor molecule. The absence of nodal translocation clearly indicates multiple attachments to the glass. The presence of nodal translocation still requires confirmation by careful statistics to prove translocation by a single molecule. In the case of bead movements, again, statistical arguments have been used to show that the movements observed are driven by single motors (Block et al., 1990). The use of the optical tweezers in those assays is important to make an unbiased sample of the population of beads.
111. Microtubule Movements on Motor-Coated Glass A. Purification and Storage of Motor Proteins for Motility
Many procedures have been published for the purification of either kinesin or cytoplasmic dynein (see Schroer et al., 1989; Kuznetsov et al., 1988; Vale et al., 1985; Paschal er al., 1987). In general, the motor activity is best within the first day, but the addition of reducing agents and storage on ice have preserved detectable activity for weeks. It is important to titrate the activity each time an experiment is done to be in a single motor range.
131
9. Nanometer Movements of Single Motor Molecules
1. Kinesin Coating the glass surface with other proteins that block the denaturation of kinesin has greatly improved the motility of kinesin attached to glass surfaces. Originally, a sequential coating of dimeric tubulin and cytochrome c was found to preserve motility at dramatically lower concentrations of kinesin, exhibiting linear dependence (Hill coefficient = 1) of probability of surface attachment and movement of microtubules with the concentration of kinesin (Howard et al., 1989). This protocol (see Table I, bottom) has worked for many other kinesins, but even better results have been obtained with a-casein (see Table I). In some cases there is still a threshold phenomenon in which motility will not occur unless there is a minimum concentration of motor. This appears to be dependent on the age of the motor, and the freshest preparations of motor have no detectTable I Microtubule Motility on Motor-Coated Glass" Time
Volume
Solution
Acid-washed coverslips 21 h l0-20% Hydrochloric or nitric acid Rinsing in a stream of filtered, distilled water 220 min Spin dry or rinse in 50% ethyl alcohol and air-dry Optional Adsorption of Fidircial Markers 1:20,000 dilution in PEM of 2% 150-nm latex microspheres I5 pI 5 min Motor dilution in CP (typically 1:5000 or greater) 2 min 30 pl 30 p1 CP Rinse Microtubules in CP with nucleotides and 5 p M taxol 2-15 min 30pl Observe under microscope Buffers and Solitlions Pipes (KOH), pH 6.8 EGTA MgS04 a-casein in PEM Alternate Prrbli~hedCompositions of Carrier Protein C P 10 mg/ml BSA in 80 mM Pipes, 50 mM KCI, 0.5 m M DTT. 1 mM MgCI2, 1 mM EGTA, pH 6.8 (Block et a / . . 1990) 50 pg/ml casein and 50 pg/ml cytochrome C in 80 mM Pipes, 50 mM KCI, 0.5 mM DTT, I mM MgCI2. 1 mM EGTA, pH 6.8 (Block e f a / . , 1990) 50 pglml cytochrome C in 80 mM Pipes (KOH), 1 mM EGTA, 2 mM MgC12, pH 6.85 (Howard r t a l . . 1989) Published protocols Blocking protein: CP (Block et a / . , 1990) or tubulin (Howard et a / . , 1 min 1989) 2 min Motor dilution in CP Microtubules in CP with 1-2 m M ATP and 5 p M (10 pg/ml?) taxol 2-15 min
PEM
100mM 1 mM ImM CP, 150 pg/ml
I' Pipes, I ,4-piperazinediethanesulfonicacid; EGTA, ethylene glycol bis (@-aminoethyl ether) N,N'-tetraacetic acid; DTT. dithiothreitol
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Michael P. Sheetz and Scot C. Kuo
able threshold. Treatment of the glass, the concentration of the surface blockers, and the motor source also affect the linearity of motor activity.
2. Cytoplasmic Dynein Cytoplasmic dynein-dependent motility also benefits from the use of blocking proteins. Although cytochrome c inhibits motility, coating with dimeric tubulin alone maintains cytoplasmic dynein motility on dilution. As for kinesin, the use of casein as the blocking protein provides the most sensitive assay for motility. Unlike kinesin, cytoplasmic dynein shows a consistent threshold effect requiring a minimal motor density for motility. Above this threshold, the probability of microtubules attaching to the surface and moving was monotonic with increasing motor concentration.
3. Attachment of Marker Particles to Microtubules Current microscope systems do not detect microtubules with sufficient contrast to allow high-precision measurements of position; however, marker particles on the surface of microtubules provide sufficient contrast. Microtubules containing biotinylated tubulin heterodimers have been described elsewhere (Hyman et af., 1991) and streptavidin-coated colloidal gold is commercially available. For 0.5-pm latex particles, however, we had to develop a sandwich technique to construct streptavidin-coated particles. Prior to particle tagging of biotinylated microtubules, all biotinylated dimeric tubulin must be removed by centrifugation or washing in a flow cell, even when microtubules are stabilized by taxol. The bond between bead and microtubule is stronger than multiple kinesin motors (>lo pN per bond), allowing us to characterize the force of a single kinesin molecule. With a standard video-enhanced differential interference contrast (DIC) microscope system it is possible to resolve movements of latex particles with 1- to 5-nm precision. B. Bead Movements on Microtubules Very small (200-400nm) glass beads have been coated with the same blocking proteins as coverslips, which has produced motility of a fraction of beads with less than one kinesin per particle (Block et al., 1990). A variation of this protocol is described in Table I for the microtubule gliding assay. A protocol for coating anionic latex beads with motors after adsorption with a surface blocker is provided in Table 11. In general the criterion for single-motordependent movement is that the percentage of beads capable of movement is linearly dependent on the concentration of motor on the bead surface. As previously described, optical tweezers can provide an unbiased sampling of motor-coated particles and show that the frequency of bead movement follows poissonian statistics (Block et al., 1990). At a limiting dilution of motors, if 20% of the particles attach and move on microtubules, a Poisson distribution predicts
133
9. Nanometer Movements of Single Motor Molecules
Table I1 Latex Bead Movement by Single Motors Time
Volume
Solution
Attachment of Microtubules to Coverslip Antitubulin antibody (1:800 dilution in PMEE'), should be dried at 37°C 50 pl I h on coverslip surface SB to wash lanes of coverslip sandwich (parallel lines of silicone grease I min 50pl between coverslips form the sandwich) Diluted microtubules ( - 5 ml of stock in I mi of SB) 10rnin 30pl Rinsing buffer ( I : 1 of H,O:PMEE' with I mM ATP, 9.6 pM taxol, 240 I min 50pl mM NaCI. 220 pg/mla-casein) Addition of motor-coated beads
PMEE'
SB
35 mM 5mM
ImM 5 mM 1 mM
20 uM
Buffrrs and Solutions PIPES" (KOH), pH 7.2 EGTA EDTA MgCIZ
PMEE' GTP Taxol
" See Table 1 for explanation of abbreviations.
that 1.75% of the particles (<9% of the motile beads) have more than one motor molecule. A statistical argument shows that the majority of the movements are driven by single motors. C. Recording Nanometer-Level Movements
To record the position of particles moving by single motors requires a major commitment of resources, either to a specialized system or to a high-quality video-enhanced DIC microscope. The lack of image formation in the photodiode
134
Michael P. Sheetz and Scot C. Kuo
systems is compensated by their greater temporal and spatial resolution. The limitations of the photodiode systems have been reported for hair cell bundle measurements (about 0.01 nm at 1 millisecond) (Denk et al., 1989). In contrast, the video-enhanced DIC systems have lower resolution (about 0.5 nm at 30 milliseconds but variable scan cameras can give 3 milliseconds) and give additional information about the microtubule position and allow the use of fiducial marker beads in the system. The major problem is to match the instrument to the question being asked because the acquisition of more data than is needed makes analysis cumbersome. D. Video-Enhanced DIC Microscopy of Nanometer Movements
Earlier works have dealt with some of the important parameters in videoenhanced DIC recording of nanometer movements (Schnapp et al., 1988; Gelles et al., 1988; Kuo et al., 1991). Resolution of the position measurement is affected by the size of the recorded field, the intensity of the light source, particle contrast, and the mode of recording. These measurements require stationary reference beads in the same field. Time resolution in standard video systems is limited to 30 milliseconds, and standard Newvicon cameras have a frame-to-frame carryover of 15-20%. Solid-state cameras (CCD or CID) have variable scan features that allow portions of the video field to be scanned as rapidly as 3 milliseconds; however, solid-state cameras require digital contrast enhancement to approach the performance of Newvicon cameras on lowcontrast DIC images, such as microtubules. The recording medium is important as it often limits the final resolution obtained. In comparing direct digital video recorders (a real-time disk system from Applied Memory Technologies that records up to 3000 digitized frames), optical memory disk recorders, and S-VHS tape recorders, we have found that the final noise contributed by the recording medium is a small factor in routine measurements. The noise contributed by the S-VHS tape players is as low as 0.04 pixel (typically 2 nm). Optical memory disk recorders, when frameaveraged to reduce playback noise, perform better and introduce only 0.01 pixel (typically 0.5 nm). As a result, it is only justifiable to use the more expensive media when the experimental system requires improved spatial precision. E. Analysis
Several commercial analysis routines are available and most investigators share their software with other researchers; however, efficient analysis requires a hardware-dependent system, which usually involves a major commitment of money or time. Two modes of analysis have commonly been employed: a simple centroid calculation or a cross-correlation analysis followed by a centroid calculation of the cross-correlation peak. With either fluorescence microscopy of labeled particles (Gross and Webb, 1988) or bright-field microscopy of gold
9. Nanometer Movements of Single Motor Molecules
135
particles (DeBrabander et al., 1988) the simple centroid calculation should give maximal precision. In general, the signal-to-noise ratio for particle detection is greatest for DIC microscopy, and analysis of only the dark or light portion of the particle image gives only half of the possible precision. To obtain the greatest possible precision we have used a cross-correlation analysis (Gelles et al., 1988). Stage drift is a major problem, particularly for high-precision or long-term measurements. Latex particles will bind strongly to the glass surface and can be used as reference particles to compensate for drift. Several problems with reference particles will actually increase the error of the final motion analyses. The simplest is that the particles, although stationary to the eye, are actually moving about their attachment site. This is often evident in the analysis and can be overcome by averaging the positions of several stationary particles. The other problem is inherent in the use of reference particles in that the noise of the position measurement of the reference particle is added to the noise of the position measurement of the moving particle. As the measurement noise is high frequency, it is possible to use a sliding-window temporal average of the position of the reference particle to diminish the error of position measurement.
IV.Conclusion The detailed analysis of motor movements at the molecular level has great promise for aiding in the understanding of the basic mechanisms of motor function. The technology is now available to perform these analyses routinely at the nanometer level. References Block, S . M., Goldstein, L . S. B., and Schnapp, B. J. (1990). Bead movement by single kinesin molecules studied with optical tweezers. Nature (London) 348,348-352. DeBrabander, M., Nuydens, R., Geerts, H.. and Hopkins, C. R. (1988). Dynamic behavior of the transferrin receptor followed in living epidermoid carcinoma (A43I ) cells with nanovid microscopy. Cell Motil. Cytoskel. 9,30-47. Denk, W., Webb, W. W., and Hudspeth, A. J . (1989). Mechanical properties ofsensory hair bundles are reflected in their Brownian motion measured with a laser differential interferometer. Proc. Natl. Acad. Sci. U . S . A . 86,5371-5375. Gelles, J., Schnapp, B. J., and Sheetz, M. P. (1988). Tracking kinesin-driven movements with nanometer precision. Nature (London) 331,450-453. Gross, D. J. and Webb, W. W. (1988). Cell surface clustering and mobility of the liganded LDL receptor measured by digital fluorescence microscopy. In “Spectroscopic Membrane Probes” (L. M. Leow, ed.), Vol. 1 I , pp. 19-48. CRC Press Inc. Boca Raton, Florida. Howard, J., Hudspeth, A. J.. and Vale, R. D. (1989). Movement of microtubules by single kinesin molecules. Nature (London) 342, 154-158. Hyman, A.. Drechsel, D., Kellogg, D., Salser, S., Sawin, K., Steffen, P., Wordernan, L., and Mitchison, T. (1991). Preparation of modified tubulins. In “Methods in Enzymology” (R. B. Vallee. ed.). Vol. 196, pp. 478-485. Academic Press, San Diego.
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Kuo, S. C., Gelles, J., Steuer, E., and Sheetz, M. P. (1991). A model for kinesin movement from nanometer level movements of kinesin and cytoplasmic dynein and force measurements. J . Cell Sci.,Suppl. 14, 135-138. Kuznetsov, S . A., Vaisberg, Y. A., Shanina, N . A., Magretova, N. N., Chernyak, V. Y., and Gelfand, V. I. (1988). The quaternary structure of bovine brain kinesin. EMBO J . 7 , 353-356. Lopez, L. A., and Sheetz, M. P. (1992). Inhibition of dynein and kinesin motility by MAP2. Cell Motil. Cyroskel. (in press). Paschal, B. M., Shpetner, H. S., and Vallee, R. B. (1987). MAP 1C is a microtubule-activated ATPase which translocates microtubules in v i m and has dynein-like properties. J . Cell Biol. 105, 1273- 1282. Schnapp, B. J., Gelles, J., and Sheetz, M. P. (1988). Nanometer-scale measurements using video light microscopy. Cell Motil. Cyroskel. 10,47-53. Schroer, T. A., Steuer, E. R., and Sheetz, M. P. (1989). Cytoplasmic dynein is a minus-end directed motor for membranous organelles. Cell (Cambridge, Mass.) 56,937-946. Vale, R. D., Reese, T. S . , and Sheetz, M. P. (1985). Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility. Cell (Cambridge, Mass.) 42, 39-50.
Tracking Nanometer Movements of Single Motor Molecules Michael P. Sheetz and Scot C. Kuo Department of Cell Biology Duke University Medical Center Durham, North Carolina 27710
I. Introduction 11. Movements by Single Motor Molecules
111. Microtubule Movements on Motor-Coated Glass A. Purification and Storage of Motor Proteins for Motility B. Bead Movements on Microtubules C. Recording Nanometer-Level Movements D. Video-Enhanced DIC Microscopy of Nanometer Movements E. Analysis IV. Conclusion References
I. Introduction To understand the mechanism of motor movements it is necessary to understand how individual molecules move with sufficient spatial precision. Until recently, motility of motor proteins, such as muscle myosin, required an ensemble of motor molecules interacting with the same filament. In the last few years, the microtubule motor kinesin was demonstrated to work as single molecules (Howard et al., 1989; Block et al., 1990). Similar approaches with cytoplasmic dynein are showing promising results (Lopez and Sheetz, 1992). In these approaches, limiting dilutions of motor protein are adsorbed to an anionic surface, either acid-washed glass or carboxylate-modified polystyrene microspheres. The addition of carrier protein, such as bovine serum albumin, cytochrome c, METHODS IN CELL BIOLOGY. VOL 39 Copyright 8 1YY3 by Academic Prcrr. Inc. All rights ofreproduction
111
m y form rrserved
129
130
Michael P. Sheetz and Scot C . Kuo
and casein, increases the range of active motor dilutions at least 50-fold during surface adsorption. Although specific activation of the motor protein cannot be excluded, the number of carrier proteins that preserve motor activity suggests that surface denaturation of the adsorbed motor protein is a major cause of reduced motor activity. As described in the later protocols, a number of carrier protein protocols have been published (Howard et al., 1989; Block et al., 1990), but our best activity for both kinesin and cytoplasmic dynein from either chicken or squid is obtained using casein alone. A variety of different technologies have been used to follow particle movements with high precision. A complete description of these technologies, including the necessary microscopy, is beyond the scope of this chapter, but the general principles are described. Approaches based on video are most convenient; however, improved temporal resolution and spatial sensitivity are achieved by custom photodiode detection systems with custom illumination.
11. Movements by Single Motor Molecules In general, the greatest obstacle to performing studies of single motor molecules is proving that the motility is indeed produced by single and not multiple molecules. As most motility assays often show less than a tenth of the motor molecules as active, arguments based on adsorbed motor density are ineffective and statistical arguments must be used. In the case of microtubules gliding on motor-coated glass, however, translocation while pivoting around a nodal point is a strong indicator of motility by a single motor molecule. The absence of nodal translocation clearly indicates multiple attachments to the glass. The presence of nodal translocation still requires confirmation by careful statistics to prove translocation by a single molecule. In the case of bead movements, again, statistical arguments have been used to show that the movements observed are driven by single motors (Block et al., 1990). The use of the optical tweezers in those assays is important to make an unbiased sample of the population of beads.
111. Microtubule Movements on Motor-Coated Glass A. Purification and Storage of Motor Proteins for Motility
Many procedures have been published for the purification of either kinesin or cytoplasmic dynein (see Schroer et al., 1989; Kuznetsov et al., 1988; Vale et al., 1985; Paschal er al., 1987). In general, the motor activity is best within the first day, but the addition of reducing agents and storage on ice have preserved detectable activity for weeks. It is important to titrate the activity each time an experiment is done to be in a single motor range.
131
9. Nanometer Movements of Single Motor Molecules
1. Kinesin Coating the glass surface with other proteins that block the denaturation of kinesin has greatly improved the motility of kinesin attached to glass surfaces. Originally, a sequential coating of dimeric tubulin and cytochrome c was found to preserve motility at dramatically lower concentrations of kinesin, exhibiting linear dependence (Hill coefficient = 1) of probability of surface attachment and movement of microtubules with the concentration of kinesin (Howard et al., 1989). This protocol (see Table I, bottom) has worked for many other kinesins, but even better results have been obtained with a-casein (see Table I). In some cases there is still a threshold phenomenon in which motility will not occur unless there is a minimum concentration of motor. This appears to be dependent on the age of the motor, and the freshest preparations of motor have no detectTable I Microtubule Motility on Motor-Coated Glass" Time
Volume
Solution
Acid-washed coverslips 21 h l0-20% Hydrochloric or nitric acid Rinsing in a stream of filtered, distilled water 220 min Spin dry or rinse in 50% ethyl alcohol and air-dry Optional Adsorption of Fidircial Markers 1:20,000 dilution in PEM of 2% 150-nm latex microspheres I5 pI 5 min Motor dilution in CP (typically 1:5000 or greater) 2 min 30 pl 30 p1 CP Rinse Microtubules in CP with nucleotides and 5 p M taxol 2-15 min 30pl Observe under microscope Buffers and Solitlions Pipes (KOH), pH 6.8 EGTA MgS04 a-casein in PEM Alternate Prrbli~hedCompositions of Carrier Protein C P 10 mg/ml BSA in 80 mM Pipes, 50 mM KCI, 0.5 m M DTT. 1 mM MgCI2, 1 mM EGTA, pH 6.8 (Block et a / . . 1990) 50 pg/ml casein and 50 pg/ml cytochrome C in 80 mM Pipes, 50 mM KCI, 0.5 mM DTT, I mM MgCI2. 1 mM EGTA, pH 6.8 (Block e f a / . , 1990) 50 pglml cytochrome C in 80 mM Pipes (KOH), 1 mM EGTA, 2 mM MgC12, pH 6.85 (Howard r t a l . . 1989) Published protocols Blocking protein: CP (Block et a / . , 1990) or tubulin (Howard et a / . , 1 min 1989) 2 min Motor dilution in CP Microtubules in CP with 1-2 m M ATP and 5 p M (10 pg/ml?) taxol 2-15 min
PEM
100mM 1 mM ImM CP, 150 pg/ml
I' Pipes, I ,4-piperazinediethanesulfonicacid; EGTA, ethylene glycol bis (@-aminoethyl ether) N,N'-tetraacetic acid; DTT. dithiothreitol
132
Michael P. Sheetz and Scot C. Kuo
able threshold. Treatment of the glass, the concentration of the surface blockers, and the motor source also affect the linearity of motor activity.
2. Cytoplasmic Dynein Cytoplasmic dynein-dependent motility also benefits from the use of blocking proteins. Although cytochrome c inhibits motility, coating with dimeric tubulin alone maintains cytoplasmic dynein motility on dilution. As for kinesin, the use of casein as the blocking protein provides the most sensitive assay for motility. Unlike kinesin, cytoplasmic dynein shows a consistent threshold effect requiring a minimal motor density for motility. Above this threshold, the probability of microtubules attaching to the surface and moving was monotonic with increasing motor concentration.
3. Attachment of Marker Particles to Microtubules Current microscope systems do not detect microtubules with sufficient contrast to allow high-precision measurements of position; however, marker particles on the surface of microtubules provide sufficient contrast. Microtubules containing biotinylated tubulin heterodimers have been described elsewhere (Hyman et af., 1991) and streptavidin-coated colloidal gold is commercially available. For 0.5-pm latex particles, however, we had to develop a sandwich technique to construct streptavidin-coated particles. Prior to particle tagging of biotinylated microtubules, all biotinylated dimeric tubulin must be removed by centrifugation or washing in a flow cell, even when microtubules are stabilized by taxol. The bond between bead and microtubule is stronger than multiple kinesin motors (>lo pN per bond), allowing us to characterize the force of a single kinesin molecule. With a standard video-enhanced differential interference contrast (DIC) microscope system it is possible to resolve movements of latex particles with 1- to 5-nm precision. B. Bead Movements on Microtubules Very small (200-400nm) glass beads have been coated with the same blocking proteins as coverslips, which has produced motility of a fraction of beads with less than one kinesin per particle (Block et al., 1990). A variation of this protocol is described in Table I for the microtubule gliding assay. A protocol for coating anionic latex beads with motors after adsorption with a surface blocker is provided in Table 11. In general the criterion for single-motordependent movement is that the percentage of beads capable of movement is linearly dependent on the concentration of motor on the bead surface. As previously described, optical tweezers can provide an unbiased sampling of motor-coated particles and show that the frequency of bead movement follows poissonian statistics (Block et al., 1990). At a limiting dilution of motors, if 20% of the particles attach and move on microtubules, a Poisson distribution predicts
133
9. Nanometer Movements of Single Motor Molecules
Table I1 Latex Bead Movement by Single Motors Time
Volume
Solution
Attachment of Microtubules to Coverslip Antitubulin antibody (1:800 dilution in PMEE'), should be dried at 37°C 50 pl I h on coverslip surface SB to wash lanes of coverslip sandwich (parallel lines of silicone grease I min 50pl between coverslips form the sandwich) Diluted microtubules ( - 5 ml of stock in I mi of SB) 10rnin 30pl Rinsing buffer ( I : 1 of H,O:PMEE' with I mM ATP, 9.6 pM taxol, 240 I min 50pl mM NaCI. 220 pg/mla-casein) Addition of motor-coated beads
PMEE'
SB
35 mM 5mM
ImM 5 mM 1 mM
20 uM
Buffrrs and Solutions PIPES" (KOH), pH 7.2 EGTA EDTA MgCIZ
PMEE' GTP Taxol
" See Table 1 for explanation of abbreviations.
that 1.75% of the particles (<9% of the motile beads) have more than one motor molecule. A statistical argument shows that the majority of the movements are driven by single motors. C. Recording Nanometer-Level Movements
To record the position of particles moving by single motors requires a major commitment of resources, either to a specialized system or to a high-quality video-enhanced DIC microscope. The lack of image formation in the photodiode
134
Michael P. Sheetz and Scot C. Kuo
systems is compensated by their greater temporal and spatial resolution. The limitations of the photodiode systems have been reported for hair cell bundle measurements (about 0.01 nm at 1 millisecond) (Denk et al., 1989). In contrast, the video-enhanced DIC systems have lower resolution (about 0.5 nm at 30 milliseconds but variable scan cameras can give 3 milliseconds) and give additional information about the microtubule position and allow the use of fiducial marker beads in the system. The major problem is to match the instrument to the question being asked because the acquisition of more data than is needed makes analysis cumbersome. D. Video-Enhanced DIC Microscopy of Nanometer Movements
Earlier works have dealt with some of the important parameters in videoenhanced DIC recording of nanometer movements (Schnapp et al., 1988; Gelles et al., 1988; Kuo et al., 1991). Resolution of the position measurement is affected by the size of the recorded field, the intensity of the light source, particle contrast, and the mode of recording. These measurements require stationary reference beads in the same field. Time resolution in standard video systems is limited to 30 milliseconds, and standard Newvicon cameras have a frame-to-frame carryover of 15-20%. Solid-state cameras (CCD or CID) have variable scan features that allow portions of the video field to be scanned as rapidly as 3 milliseconds; however, solid-state cameras require digital contrast enhancement to approach the performance of Newvicon cameras on lowcontrast DIC images, such as microtubules. The recording medium is important as it often limits the final resolution obtained. In comparing direct digital video recorders (a real-time disk system from Applied Memory Technologies that records up to 3000 digitized frames), optical memory disk recorders, and S-VHS tape recorders, we have found that the final noise contributed by the recording medium is a small factor in routine measurements. The noise contributed by the S-VHS tape players is as low as 0.04 pixel (typically 2 nm). Optical memory disk recorders, when frameaveraged to reduce playback noise, perform better and introduce only 0.01 pixel (typically 0.5 nm). As a result, it is only justifiable to use the more expensive media when the experimental system requires improved spatial precision. E. Analysis
Several commercial analysis routines are available and most investigators share their software with other researchers; however, efficient analysis requires a hardware-dependent system, which usually involves a major commitment of money or time. Two modes of analysis have commonly been employed: a simple centroid calculation or a cross-correlation analysis followed by a centroid calculation of the cross-correlation peak. With either fluorescence microscopy of labeled particles (Gross and Webb, 1988) or bright-field microscopy of gold
9. Nanometer Movements of Single Motor Molecules
135
particles (DeBrabander et al., 1988) the simple centroid calculation should give maximal precision. In general, the signal-to-noise ratio for particle detection is greatest for DIC microscopy, and analysis of only the dark or light portion of the particle image gives only half of the possible precision. To obtain the greatest possible precision we have used a cross-correlation analysis (Gelles et al., 1988). Stage drift is a major problem, particularly for high-precision or long-term measurements. Latex particles will bind strongly to the glass surface and can be used as reference particles to compensate for drift. Several problems with reference particles will actually increase the error of the final motion analyses. The simplest is that the particles, although stationary to the eye, are actually moving about their attachment site. This is often evident in the analysis and can be overcome by averaging the positions of several stationary particles. The other problem is inherent in the use of reference particles in that the noise of the position measurement of the reference particle is added to the noise of the position measurement of the moving particle. As the measurement noise is high frequency, it is possible to use a sliding-window temporal average of the position of the reference particle to diminish the error of position measurement.
IV.Conclusion The detailed analysis of motor movements at the molecular level has great promise for aiding in the understanding of the basic mechanisms of motor function. The technology is now available to perform these analyses routinely at the nanometer level. References Block, S . M., Goldstein, L . S. B., and Schnapp, B. J. (1990). Bead movement by single kinesin molecules studied with optical tweezers. Nature (London) 348,348-352. DeBrabander, M., Nuydens, R., Geerts, H.. and Hopkins, C. R. (1988). Dynamic behavior of the transferrin receptor followed in living epidermoid carcinoma (A43I ) cells with nanovid microscopy. Cell Motil. Cytoskel. 9,30-47. Denk, W., Webb, W. W., and Hudspeth, A. J . (1989). Mechanical properties ofsensory hair bundles are reflected in their Brownian motion measured with a laser differential interferometer. Proc. Natl. Acad. Sci. U . S . A . 86,5371-5375. Gelles, J., Schnapp, B. J., and Sheetz, M. P. (1988). Tracking kinesin-driven movements with nanometer precision. Nature (London) 331,450-453. Gross, D. J. and Webb, W. W. (1988). Cell surface clustering and mobility of the liganded LDL receptor measured by digital fluorescence microscopy. In “Spectroscopic Membrane Probes” (L. M. Leow, ed.), Vol. 1 I , pp. 19-48. CRC Press Inc. Boca Raton, Florida. Howard, J., Hudspeth, A. J.. and Vale, R. D. (1989). Movement of microtubules by single kinesin molecules. Nature (London) 342, 154-158. Hyman, A.. Drechsel, D., Kellogg, D., Salser, S., Sawin, K., Steffen, P., Wordernan, L., and Mitchison, T. (1991). Preparation of modified tubulins. In “Methods in Enzymology” (R. B. Vallee. ed.). Vol. 196, pp. 478-485. Academic Press, San Diego.
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