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Address correspondence to Anthony Bretscher, Dept. of Molecular Biology and Genetics, 351 Biotechnology Bldg., Cornell University, Ithaca, NY 14853. Tel.: (607) 255-5713. Fax: (607) 255-2428. E-mail: apb5{at}cornell.edu
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Abstract |
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Key Words: exocytosis; Saccharomyces; molecular motors; myosin; cell polarity
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Introduction |
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Active transport of membranes by myosin-V has been questioned in fungal cells as well. The budding yeast Saccharomyces cerevisiae tightly concentrates post-Golgi secretory vesicles to polarized sites at the cell surface, a process that requires the myosin-V heavy chain Myo2p (Johnston et al., 1991; Govindan et al., 1995; Schott et al., 1999) and polarized cables of actin filaments (Pruyne et al., 1998). Because nearly all of the Myo2p is at these sites, and at least some can concentrate there independently of actin (Ayscough et al., 1997), Myo2p has been proposed to target vesicles by passive capture (Reck-Peterson et al., 2000).
We investigated this by following the itinerary of secretory vesicles in live cells. The ease with which we could genetically modify Myo2p's motor properties not only allowed us to determine whether Myo2p is the motor for vesicle movement, but also allowed us to test models of how myosin produces force. The swinging lever arm model predicts that the light chain binding region of the myosin heavy chain forms a rigid rod, or lever arm, that amplifies small movements of the actin-binding globular domain (Uyeda et al., 1996; Geeves and Holmes, 1999; Houdusse and Sweeney, 2001; Fig. 1). If so, the step size and therefore the sliding velocity are predicted to be proportional to the length of this rod. Although this model has recently been disputed (Yanagida et al., 2000b; Yanagida and Iwane, 2000), studies of purified myosin-II, which normally has a short predicted lever arm of two light chain binding (IQ) repeats, have shown that velocity and step size are proportional to the predicted lever arm length up to one and a half times the natural length (Uyeda et al., 1996; Ruff et al., 2001). In contrast, an experiment on myosin-V, which normally has six IQ repeats (Fig. 1 A), showed a similar velocity for a 2-IQ version as for wild-type version (Trybus et al., 1999). Such in vitro studies are complicated by interactions between the myosin and the surface it is attached to, and by the difficulty of duplicating the physiological orientation of the myosin molecule (Yanagida et al., 2000a). Therefore, an in vivo assay would be an important tool.
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Results and discussion |
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The number of IQ repeats in myosin-V determines secretory vesicle velocity
The active movement of vesicles in living, intact cells allows us to test models of the myosin motor mechanism in a truly physiological setting. As mentioned above, lever arm models predict that velocity should be proportional to the lever arm length under ideal conditions for motility. Therefore, we made a series of otherwise identical yeast strains containing zero, two, four, six, and eight IQ repeats in the sole copy of the MYO2 gene (Fig. 4 A). Growth rates for these strains are identical to wild-type strains, except for the 0IQ variant that grows 10% slower, as reported by Stevens and Davis (1998). This indicates that all of the constructs are functional, as yeast cannot survive without MYO2 (Johnston et al., 1991). Myo2p protein levels are also the same as wild-type except for the 8-IQ variant which has 75% of the wild-type level, and all variants allow slightly more frequent linear GFPSec4p movements than wild-type (Fig. 4, A, B, and C).
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The availability of a live-cell myosin motor assay in an organism that allows easy transformation, gene replacement, and large-scale genetic screening opens up a new approach that will facilitate additional studies of how myosin produces force.
In summary, the effect of Myo2p IQ repeat number on GFPSec4p velocity unambiguously demonstrates that a myosin-V directs secretion by actively transporting membranes and shows that the IQ repeat region acts as a mechanical amplifier in vivo.
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Materials and methods |
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Yeast strains with varying numbers of IQ repeats in MYO2
We cloned MYO2+ and MYO26IQ (called 0IQ here) (Stevens and Davis, 1998) into a pRS303-integrating vector as described (Schott et al., 1999) and altered the MYO2 gene by amplifying the IQ repeat region with appropriate primers and splicing back into MYO2 on the plasmid. The uniform 48 amino acid spacing of IQ repeat pairs allowed us to perform precise deletions and duplication. Numbering the myosin-V IQ repeats 1-2-3-4-5-6, the sequences of the new alleles are as follows (with primer sequences in parentheses): 2IQ, 1-6 (AATATTACCGTAAGCAGTATTTGCAAATAAAAAAAGACACTGTTGTTGTCCAAT); 4IQ, 1-2-3-6 (GTGCCAATGTGTTCAGCGTAAAAAAAGACACTGTTGTTGTCCAAT); and 8IQ, 1-2-3-4-3-4-5-6 (AAAGACAACTGAAACAAGAACATGAAGTTAACTGTGCAACTTTATTACAGGCC). Integration of these five plasmids into the haploid yeast strain CRY1 (Stevens and Davis, 1998) resulted in a series of five isogenic strains, differing only in the number of IQ repeats in MYO2, and containing the pRS303-integrating plasmid inserted between the MYO2 and SNC2 genes. All strains express mut3GFPSec4p from a YOP1 promoter on the centromere plasmid pRC556 described above. We confirmed that only one version of MYO2 is present in each of the five strains by electrophoresis of amplified MYO2 DNA, and confirmed the new alleles to be error-free by sequencing the amplified genomic DNA.
Observation of live cells
We mounted yeast cells on agarose containing 4% glucose synthetic medium, and observed them at 2224°C under a conventional fluorescence microscope with a 100x objective (NA 1.3) and a CCD detector, collecting 50-ms exposures at a rate of nine exposures per second. We recorded and viewed the digital images using the MetaMorph® software package (Universal Imaging Corporation). We measured velocities as displacement of particle images over time, excluding movements close to the bud neck where the particles tend to slow down. Velocities may be slight underestimates because we did not take into account movement perpendicular to the focal plane and pauses shorter than 0.5 s.
Temperature-sensitive strains
We introduced pRC556 (the mut3GFPSec4p expression plasmid) into ABY536 (myo216), ABY531 (MYO2), ABY988 (tpm12 tpm2), and ABY987 (TPM1 tpm2
) (Pruyne et al., 1998; Schott et al., 1999), and used a thermostat-controlled objective heater to perform temperature shifts.
Measurement of relative Myo2p levels
To determine semiquantitative Myo2p levels in the IQ variant strains relative to wild-type, we grew the five yeast strains to an optical density of 0.4 units in minimal medium, chilled the cultures on ice, washed two optical density units of each strain in water, resuspended them in 150 µl chilled buffer (50 mM Tris-HCl, 5 mM Na-EDTA, and 5% SIGMA yeast protease inhibitor cocktail in DMSO), added 500-µl glass beads, agitated them for 5 min in a bead beater at 4°C to open >90% of the cells, added SDS-PAGE sample buffer, boiled them for 3 min, centrifuged out the cell walls, and immediately loaded on a 6% polyacrylamide gel. We loaded two or three dilutions of each sample to determine the approximate relationship between the amount of sample and the amount of signal. We blotted the gel onto nitrocellulose and stained with Ponceau S to confirm equal loads for each strain. We then detected Myo2p using an affinity-purified antibody to the Myo2p COOH-terminal tail, a peroxidase-coupled secondary antibody, and the 3,3'-diaminobenzidine reaction to detect the peroxidase. We performed the experiment, from sample preparation to immunodetection, twice to confirm results.
Online supplemental material
Video 1 shows the transit of a GFPSec4p particle across a mother cell to the bud neck. Video 2 shows several GFPSec4p particles taking the same path across a mother cell and into the bud. These particles also follow defined paths across the bud to the bud cortex, and at least two can be seen to reach the bud tip. Video 3 shows Brownian motion of GFPSec4p particles in a tpm12 tpm2 and two myo216 cells shifted to the restrictive temperature. Video 4 shows a comparison of GFPSec4p movement in wild-type and 0IQ cells. Videos are available at http://www.jcb.org/cgi/content/full/jcb.200110086/DC1.
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Footnotes |
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Acknowledgments |
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This study was funded by the National Institutes of Health (GM39066 to A. Bretscher).
Submitted: 16 October 2001
Revised: 28 November 2001
Accepted: 28 November 2001
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References |
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---|
Ayscough, K.R., J. Stryker, N. Pokala, M. Sanders, P. Crews, and D. Drubin. 1997. High rates of actin filament turnover in budding yeast and roles for actin in establishment and maintenance of cell polarity revealed using the actin inhibitor latrunculin-A. J. Cell Biol. 137:399416.
Cormack, B.P., G. Bertram, M. Egerton, N.A.R. Gow, S. Falkow, and A.J.P. Brown. 1997. Yeast-enhanced green fluorescent protein (yEGFP): a reporter of gene expression in Candida albicans. Microbiology. 143:303311.[Abstract]
Corti, B. 1774. Saggio d'osservazioni sulla circolazione del fluido scoperto in una pianta acquajuola appellata cara. In Osservazione de fluido in una pianta acquajuola. Appresso Giuseppe Rocchi, Lucca, Italy. 125200.
Govindan, B., R. Bowser, and P. Novick. 1995. The role of Myo2, a yeast class V myosin, in vesicular transport. J. Cell Biol. 128:10551068.[Abstract]
Johnston, G.C., J.A. Prendergast, and R.A. Singer. 1991. The Saccharomyces cerevisiae MYO2 gene encodes an essential myosin for vectorial transport of vesicles. J. Cell Biol. 113:539551.[Abstract]
Kashiyama, T., N. Kimura, T. Mimura, and K. Yamamoto. 2000. Cloning and characterization of a myosin from characean alga, the fastest motor protein in the world. J. Biochem. 127:10651070.[Abstract]
Mehta, A. 2001. Myosin learns to walk. J. Cell Sci. 114:19811998.
Mulholland, J., A. Wesp, H. Riezman, and D. Botstein. 1997. Yeast actin cytoskeleton mutants accumulate a new class of Golgi-derived secretory vesicle. Mol. Biol. Cell. 8:14811499.[Abstract]
Pruyne, D.W., D.H. Schott, and A. Bretscher. 1998. Tropomyosin-containing actin cables direct the Myo2p-dependent polarized delivery of secretory vesicles in budding yeast. J. Cell Biol. 143:19311945.
Rief, M., R.S. Rock, A.D. Mehta, M.S. Mooseker, R.E. Cheney, and J.A. Spudich. 2000. Myosin-V stepping kinetics: a molecular model for processivity. Proc. Natl. Acad. Sci. USA. 97:94829486.
Sakamoto, T., I. Amitani, E. Yokota, and T. Ando. 2000. Direct observation of processive movement by individual myosin V molecules. Biochem. Biophys. Res. Commun. 272:586590.[CrossRef][Medline]
Schott, D., J. Ho, D. Pruyne, and A. Bretscher. 1999. The COOH-terminal domain of Myo2p, a yeast myosin V, has a direct role in secretory vesicle targeting. J. Cell Biol. 147:791808.
Sikorski, R.S., and P. Hieter. 1989. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 122:1927.
Stevens, R.C., and T.N. Davis. 1998. Mlc1p is a light chain for the unconventional myosin Myo2p in Saccharomyces cerevisiae. J. Cell Biol. 142:711722.
Trybus, K.M., E. Krementsova, and Y. Freyzon. 1999. Kinetic characterization of a monomeric unconventional myosin V construct. J. Biol. Chem. 274:2744827456.
Uyeda, T.Q., P.D. Abramson, and J.A. Spudich. 1996. The neck region of the myosin motor domain acts as a lever arm to generate movement. Proc. Natl. Acad. Sci. USA. 93:44594464.
Walch-Solimena, C., R.N. Collins, and P.J. Novick. 1997. Sec2p mediates nucleotide exchange on Sec4p and is involved in polarized delivery of post-Golgi vesicles. J. Cell Biol. 137:14951509.
Wu, X., B. Bowers, K. Rao, Q. Wei, and J.A. Hammer, III. 1999. Visualization of melanosome dynamics within wild-type and dilute melanocytes suggests a paradigm for myosin V function in vivo. J. Cell Biol. 143:18991918.
Yanagida, T., and A.H. Iwane. 2000. A large step for myosin. Proc. Natl. Acad. Sci. USA. 97:93579359.
Yanagida, T., K. Kitamura, H. Tanaka, A. Hikikoshi-Iwane, and S. Esaki. 2000b. Single molecule analysis of the actomyosin motor. Curr. Opin. Cell Biol. 12:2025.[CrossRef][Medline]