Report |
Address correspondence to Martin Bähler, Institute for General Zoology and Genetics, Westfälische Wilhelms-University, Schlossplatz 5, 48149 Münster, Germany. Tel.: 49-251-83-23874. Fax: 49-251-83-24723. E-mail: baehler{at}uni-muenster.de; or Edgar Meyhöfer, Department of Mechanical Engineering, University of Michigan, 2350 Hayward Street, Ann Arbor, MI 48109-2125. Tel.: (734) 647-7856. Fax: (734) 615-6647. E-mail: meyhofer{at}umich.edu
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Abstract |
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Key Words: myosin 1d; motor molecule; calmodulin; IQ motif; single molecule measurement
E. Meyhöfer's present address is Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109-2125.
* Abbreviation used in this paper: Myo1d, myosin 1d.
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Introduction |
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However, a comparison of reported step sizes for chicken smooth muscle myosin II, Dictyostelium discoideum myosin II, chicken myosin 1a (brush border myosin I), rat myosin 1b, myosin V, and myosin VI revealed that they are not directly related to the proposed lever arm length (Veigel et al., 1999, 2002; Warshaw et al., 2000; Rock et al., 2001; Ruff et al., 2001; Tanaka et al., 2002). Therefore, to explain these different myosin step sizes, either one or more determinant in addition to lever arm length or a completely different mechanism, as proposed by Yanagida and coworkers (Kitamura et al., 1999; Tanaka et al., 2002), have to be invoked. A comparison of the step sizes of different myosins and an analysis of how they are controlled will lead to the identification of the determinants for these differences. Such experiments should also resolve the ambiguity about the role of the light chain binding domain as lever arm.
Rat myosin 1d (Myo1d),* formerly called myr 4, belongs to a still poorly characterized myosin I subclass (Bähler et al., 1994). Mammalian Myo1d has been implicated to play a role in the endocytic pathway at a step between the early endosome and the recycling endosome (Huber et al., 2000). It consists of a myosin head domain, a light chain binding domain with two IQ motifs each binding the Ca2+-sensor protein calmodulin, and a relatively short tail domain (Bähler et al., 1994). Here, we report on the analysis of the mechanical properties of various Myo1d constructs by single molecule measurements. We could show that this motor translocates actin filaments with an unexpectedly large step size due to a large deduced angular rotation (90°) of its light chain binding domain.
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Results and discussion |
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A comparison of crystal structures of class II myosin motor domains in different nucleotide states revealed four major subdomains (NH2-terminal region, lower 50-kD domain, upper-50 kD domain, and converter) that are rearranged relative to each other by three flexible loops or "joints," termed switch II, relay, and SH1 helix, respectively (Houdusse et al., 1999). We propose that subtle differences in either of the flexible loops will cause differences in the movement and rotation of the converter and the extended -helix of the attached light chain binding domain. Indeed, a recently reported crystal structure of a class I myosin, D. discoideum myosin-IE, showed that the overall structure of the motor domain is almost identical to myosin II and that subtle differences occur in loop regions connecting the main structural elements. Due to differences in the relay loop, the lever arm position in the prepower stroke state extended
30° further up toward the pointed end of the actin filament than in myosin II (Kollmar et al., 2002). The movement of the Dd myosin-IE lever arm from the pre-power stroke position found in the crystal structure to a similar post-power stroke position as observed in crystal structures of class II myosins (Fisher et al., 1995; Houdusse et al., 1999, 2000) would result in an
90° rotation along the longitudinal axis of the actin filament. These observations strongly support our model of a large angular rotation during the power stroke of Myo1d.
As recently shown, step size measurements for processively moving myosins include, in addition to the working stroke, a diffusive movement to the next preferred myosin binding position (target zone; Rock et al., 2001; Veigel et al., 2002). In the backward moving myosin VI, which contains a unique 53-aa residue insert between the converter and light chain binding domain, this diffusive component appears to be quite large (Rock et al., 2001; Spudich and Rock, 2002). Although details of the processive movements of myosin V and VI still need to be worked out, we believe that these observations are not in conflict with the lever arm role of the light chain binding domain of myosins as suggested by Tanaka et al. (2002).
We conclude that the degree of lever arm rotation varies considerably among different myosins, and that it is an important determinant for myosin step size. Both determinants together, lever arm length and degree of lever arm rotation, are sufficient to explain the reported ATP-induced conformational changes or working strokes (often referred to as step size).
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Materials and methods |
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Cell culture
HtTA-1 HeLa cells (Gossen and Bujard, 1992) were cultured at 37°C and 5% CO2 in DME supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were transfected by calcium phosphate precipitation of plasmid DNA. Single colonies were isolated after selection in 200 µg/ml hygromycin for 2 wk and were subcloned. Cells were analyzed for Myo1d construct expression by immunoblotting with the rat Myo1d antibody SA 522. Selection by hygromycin was maintained continuously.
Protein purifications
For purification of recombinant proteins, cells were grown in 1020 culture dishes (Ø 150 mm) to near confluency. Cells were washed twice with PBS (150 mM NaCl, 3 mM Na2HPO4, and 1.5 mM KH2PO4) and collected by scraping. They were lysed in lysis buffer (150 mM NaCl, 20 mM Hepes, pH 7.4, 2 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100, 2 mM ATP, 0.1 mg/ml Pefabloc®, 0.01 mg/ml leupeptin, and 0.02 U/ml aprotinin) for 1 h on ice, and cleared by centrifugation at 40,000 g for 30 min followed by ultracentrifugation at 170,000 g for 45 min. The supernatant was mixed with 1 ml FLAG-antibody agarose (Sigma-Aldrich) and incubated for 2 h at 6°C. Agarose beads were washed twice with buffer WP containing 50 mM KCl, 10 mM Hepes, pH 7.4, 2 mM MgCl2, 1 mM EGTA, 1 mM 2-mercaptoethanol, and 2 mM NaN3. Bound protein was eluted by supplementing buffer WP with 125 µg/ml soluble FLAG peptide (Sigma-Aldrich). The eluted protein was dialyzed for 36 h with two buffer changes against buffer WP. Purified proteins were sometimes concentrated using microcon filters (cut-off 10 kD; Millipore). Densitometric analysis of Coomassie-stained protein bands on SDS gels was performed with a laser densitometer (Ultrascan; LKB). Values represent the mean from at least three different preparations. Purified calmodulin was purchased from Sigma-Aldrich. Actin was purified from rabbit skeletal muscle as described elsewhere (Pardee and Spudich, 1982).
ATPase assays
Steady-state ATPase activities at 37°C were determined by quantitation of Pi release from [32P]ATP as described previously (Stöffler and Bähler, 1998). Assay mixtures contained 30 mM KCl, 10 mM Hepes, pH 7.4, 2 mM MgCl2, 2 mM ATP, 2 mM EGTA, 1 mM 2-mercaptoethanol, 2 mM NaN3, and various actin concentrations. Purified Myo1d constructs were used in a concentration range between 25 and 200 nM. Kinetic values represent averaged numbers derived from at least three different preparations.
Step size measurements and data analysis
The combined microneedle laser trap transducer that was used to measure step sizes has been described in detail previously (Ruff et al., 2001). In brief, the actin filament is pulled taught between a small latex bead (d = 1.0 µm) captured in a laser trap and the tip of a fine glass microneedle. The needle serves as sensor for the transient interactions of the filament with single motor molecules attached to the substrate. All single molecule measurements were performed at 23°C (± 0.5°C) in 25 mM imidazol, pH 7.4, 25 mM KCl, 1 mM EGTA, 4 mM MgCl2, 1 µM ATP, 10 mM DTT, 100 µg/ml glucose oxidase, 18 µg/ml catalase, and 3 mg/ml glucose. Recombinant rat Myo1d proteins were adsorbed directly to the nitrocellulose-coated surface of the recording chamber using 10-min incubations. Alternatively, they were coupled via an anti-FLAG mAb (M2; Sigma-Aldrich) to the nitrocellulose surface. In these experiments, nonspecific binding was reduced by blocking the surface with BSA.
Data analysis was performed with custom-written software as described in detail previously (Ruff et al., 2001). The running variance was calculated with a 10-ms window, and individual events were identified based on the reduction of the variance. The positions of all events relative to the zero position of the free needle were plotted in a histogram and oriented. Measurements obtained with several actin filaments and different protein preparations were pooled to obtain large datasets for each construct. To establish if Myo1d moves in discrete substeps, like Myo1a, we followed the analysis approach outlined by Veigel et al. (1999), and determined the average position at the beginning and end of the actomyosin interactions. In brief, we detected the beginning and end of individual actomyosin interactions by monitoring the reduction in the variance of a 5-ms running window, and averaged many record segments at the beginning (consisting of 100 ms before and 15 ms into the detected interaction) and separately averaged the end of many individual actomyosin interactions (last 15 ms of the actomyosin event and the 100 ms segment directly after the detachment of myosin). If the step displacement is generated in distinct substeps, positions of synchronized, average beginning and ending event positions will differ. The analysis is principally limited by the bandwidth of the microneedle laser trap detector and the ability to precisely determine the time of the start and end of each interaction in the thermal fluctuations of free actinmicroneedle laser trap complex. For the experiments and analysis reported here, we can confidently resolve substeps separated by >35 ms.
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Acknowledgments |
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This work was supported by grants of the Deutsche Forschungsgemeinschaft (BA1354/6-1 to M. Bähler, and ME1414/4-2 to E. Meyhöfer).
Submitted: 4 December 2002
Revised: 11 March 2003
Accepted: 11 March 2003
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