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Address correspondence to Tim Huffaker, Dept. of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca, NY 14853-2703. Tel.: (607) 255-9947. Fax: (607) 255-6249. E-mail: tch4{at}cornell.edu
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Abstract |
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Key Words: cytoskeleton; microfilaments; microtubules; mitotic spindle apparatus; myosins
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Introduction |
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Results and discussion |
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We measured the ability of Myo2Bim1 to replace Kar9 in two ways. First, we examined the location of preanaphase spindles by fluorescence microscopy (Fig. 1, A and B). In wild-type cells, most preanaphase spindles are located adjacent to the bud neck. As expected, kar9 cells showed a marked defect in orienting spindles at the bud neck (Miller and Rose, 1998). Myo2Bim1 completely compensated for loss of Kar9. Spindle orientation also occurs efficiently in MYO2BIM1 kar9
bim1
cells, indicating that neither Myo2, Kar9, nor Bim1 is needed if cells express Myo2Bim1. In a portion of the MYO2BIM1 kar9
cells, the preanaphase spindle was located entirely within the bud. This latter phenotype agrees with the recently described role of Kar9 in ensuring that only one spindle pole migrates to the bud (Liakopoulos et al., 2003), a role that is not provided by Myo2Bim1.
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A Myo2Bim1 fusion is sufficient to orient cytoplasmic microtubules in mating yeast cells in the absence of Kar9
A motile process similar to spindle orientation occurs during yeast mating. When yeast cells sense mating factors secreted by cells of the opposite mating type, they form a mating projection. Cytoplasmic microtubules extend toward the tip of this projection and mediate the migration of the nucleus into it (Maddox et al., 1999). Actin is also required for migration of the nucleus into the mating projection (Read et al., 1992), suggesting that the mechanism may be similar to that used for spindle orientation. Both kar9 and bim1
mutants display defects in karyogamy during mating because they fail to orient microtubules correctly (Schwartz et al., 1997; Miller and Rose, 1998).
We wanted to determine whether Myo2 also plays a role in this process. Myo2 contains an amino-terminal motor domain and a carboxy-terminal tail domain that binds cargo. A series of temperature-sensitive myo2 tail domain mutants have been described that inhibit polarized localization of secretory vesicles at their restrictive temperature (Schott et al., 1999). In this work, we used three of these alleles, myo2-17 that has little effect on spindle orientation and myo2-18 and myo2-20 that have more significant effects on spindle orientation (Yin et al., 2000).
MATa cells were exposed to the mating pheromone -factor, causing them to arrest as unbudded cells with an elongated mating projection. After
-factor arrest, wild-type and myo2 mutant cells were shifted to 35°C for 5 min, and microtubule orientation was assayed (Fig. 2, A and B). The temperature shift had no effect on microtubule orientation in wild-type cells, whereas myo2 mutant cells showed a marked decrease in microtubule orientation at the restrictive temperature. As was the case for spindle orientation, this phenotype was most apparent in the myo2-18 and myo2-20 mutants. The rapid effect of the myo2 mutations suggests a direct role for the tail of Myo2 in this process.
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The rate of cytoplasmic microtubule tip movement depends on the intrinsic velocity of Myo2
We have shown that fusing the microtubule-binding protein Bim1 to the tail of the actin motor Myo2 eliminates the requirement for Kar9 in growing and mating yeast cells, suggesting that the cellular role of Kar9 is to cross-bridge Myo2 and Bim1. This result supports the model in which a Myo2Kar9Bim1 complex transports microtubule ends along polarized actin cables. To test this model directly, we used a slow-moving variant of Myo2. The rate at which Myo2 moves is influenced by the length of its lever arm. The wild-type Myo2 lever arm has six IQ repeats, and variants have been made that lack some or all or these IQ repeats. These mutants have been used to demonstrate that secretory vesicle movement in yeast depends on Myo2, and define a linear relationship between the number of IQ repeats in Myo2 and the rate of vesicle movement (Stevens and Davis, 1998; Schott et al., 2002).
We used cells expressing GFPTub1 to measure the rate of cytoplasmic microtubule tip movement in strains containing either zero or six IQ repeat Myo2 as their sole source of the Myo2. In budded cells, cytoplasmic microtubules emanating from the bud-proximal spindle pole commonly extend to the edge of the mother cell, and then move toward the bud neck (Liakopoulos et al., 2003). In wild-type cells, this movement occurs within a couple of seconds and without any significant change in microtubule length. Strikingly, the velocities of these movements depend on the number of IQ repeats in Myo2 (Fig. 3 and Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200302030/DC1). The average rate of microtubule movement is nearly five times slower in cells containing the Myo2 that lacks IQ repeats (1.22 ± 0.36 vs. 0.26 ± 0.09 µm/s). These results unambiguously demonstrate that Myo2 directs the movement of microtubule tips.
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To test whether this observation was significant, images of microtubules were collected and the intensity of the GFP signal along the microtubules was analyzed statistically, starting from the plus end. This analysis was restricted to microtubules that had not reached the bud neck or the bud to minimize artifacts due to the general localization of Myo2. Our analysis focused on microtubules emanating from the pole proximal to the bud. This work indicated an increase in intensity at microtubule plus ends that was highly reproducible and due to Myo2GFP because it was not observed if Myo2 was not tagged (Fig. 4 E). Interestingly, it was no longer observed in cells lacking Kar9, consistent with the idea that Kar9 is required to recruit Myo2 onto microtubules. The same analysis failed to show Myo2 staining on microtubules coming from the mother-bound pole (unpublished data). Thus, our results indicate that Myo2 localizes in a Kar9-dependent manner to the plus ends of microtubules coming from the daughter-bound pole. This recruitment of Myo2 to specific microtubules may determine which microtubules orient toward the bud.
In summary, our results support a model in which a Myo2Kar9Bim1 complex transports microtubule ends along polarized actin cables (Fig. 5). Whether this process provides a motive force for orienting spindles or simply orients cytoplasmic microtubules that subsequently act to provide force (Kusch et al., 2002) remains to be elucidated. Coordination of the orientation of the mitotic spindle with actin-based cortical structures has been observed in several systems, and may require the Bim1 homologue, EBI, that is found at microtubule ends (Rose and Kemphues, 1998; Bienz, 2001). In addition, microtubules in interphase cells have been reported to target to focal adhesions, although the molecular mechanisms remain elusive (Tepass et al., 2001). Based on the model supported here, we suspect that the mechanisms might include directed delivery of microtubule ends by myosin motors transporting along polarized actin filaments.
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Materials and methods |
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A strain containing MYO2BIM1::URA3 was constructed using the one-step PCR method for gene modification (Longtine et al., 1998) to integrate the coding sequence for BIM1 just downstream of the chromosomal MYO2 coding sequence. Thus, MYO2BIM1 encodes full-length Myo2 followed by a short linker polypeptide (Ala-Gly-Ala-Gly-Ala), followed by full-length Bim1. MYO2BIM1 was initially created in the diploid strain CUY1415 (MATa/MAT kar9
::LEU2/kar9
::LEU2 his3-
200/his3-
200 leu2-3,112/leu2-3,112 lys2-801/lys2-801 ura3-52/ura3-52). Sporulation and tetrad dissection yielded a haploid stain CUY1408 (MATa MYO2BIM1::URA3 kar9
::LEU2 his3-
200 leu2-3,112 lys2-801 ura3-52). MYO2BIM1 is the only source of MYO2 function in these cells, and it is expressed from the endogenous MYO2 promoter.
A strain containing MYO2GFP::kanMX was also constructed using the one-step PCR method for gene modification. MYO2GFP encodes full-length Myo2 followed directly by GFP. This protein fusion is also expressed from the endogenous MYO2 promoter.
Fluorescence microscopy
Visualization of microtubules by immunofluorescence microscopy was performed as described previously (Pasqualone and Huffaker, 1994). Visualization of GFP- and CFP-conjugated proteins was done in live cells. Single images were collected under a conventional fluorescence microscope with a 100x objective and a CCD detector using Openlab (Improvision) or TILLVisION (T.I.L.L. Photonics) software (Kusch et al., 2002). Time-lapse images of cytoplasmic microtubule movement were collected at 0.2- or 0.4-s intervals using a live cell imaging system (UltraVIEWTM; PerkinElmer).
Online supplemental material
Video 1 shows cytoplasmic microtubule movement in cells containing GFPTub1 and wild-type Myo2 with six IQ repeats. Video 2 shows cytoplasmic microtubule movement in cells containing GFPTub1 and a mutant Myo2 lacking IQ repeats. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200302030/DC1.
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health to T.C. Huffaker (GM40479) and from the Swiss Federal Institute of Technology (ETH) to Y. Barral and J. Kusch. Y. Barral is a member of the EMBO young investigator program.
Submitted: 5 February 2003
Revised: 26 March 2003
Accepted: 26 March 2003
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References |
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