Article |
Address correspondence to John A. Cooper, Department of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Ave., St. Louis, MO 63110. Tel.: (314) 362-3964. Fax: (314) 362-0098. E-mail: jcooper{at}cellbio.wustl.edu
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Abstract |
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Key Words: Pac1; dynein; Num1; microtubule; nuclear migration
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Introduction |
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In yeast, nuclear migration and spindle movement occur predominantly in two steps: (1) movement of the nucleus to a position adjacent to the neck, followed by (2) movement of the nucleus into the neck. The first step of nuclear movement involves cytoplasmic microtubules, the kinesin-related protein Kip3, the cortical protein Kar9, and other proteins (Bim1, Bni1, Bud6, Myo2, and actin) that control Kar9 localization or its interaction with microtubules (Cottingham and Hoyt, 1997; DeZwaan et al., 1997; Miller and Rose, 1998; Lee et al., 1999; Miller et al., 1999; Beach et al., 2000; Yin et al., 2000). Early in the cell cycle, a cortical attachment site composed of Kar9 and associated proteins forms at the emerging bud tip. If a growing cytoplasmic microtubule encounters this site, it can be captured. Subsequent shrinkage of the captured microtubule pulls the nucleus toward the nascent bud and orients the preanaphase spindle along the motherbud axis.
The second step of nuclear movement moves the mitotic spindle into the neck. Cytoplasmic microtubules from the spindle pole body (SPB)* associate laterally with and slide along the bud cortex, pulling the nucleus and the elongating spindle into the neck (Adames and Cooper, 2000). Microtubule sliding depends on the heavy chain of the microtubule-based motor dynein Dyn1, its regulator dynactin complex (which consists of Arp1, Jnm1, and Nip100), and the cortical attachment protein Num1 (Adames and Cooper, 2000; Heil-Chapdelaine et al., 2000). The mechanism of microtubule sliding is poorly understood, but a favored hypothesis is that dynein is anchored in the bud cortex and pulls on the microtubules by walking in the minus end direction toward the SPB (Carminati and Stearns, 1997).
Several known genes of the dynein/dynactin pathway (DYN1, JNM1, NIP100, and NUM1) were isolated in a synthetic lethal screen with the kinesin motor gene cin8; they are called pac (perish in the absence of CIN8) mutants (Geiser et al., 1997). The screen identified four additional genes (PAC1, PAC10, PAC11, and PAC14/BIK1) hypothesized to perform dynein-related functions, based on phenotypic similarity of the mutants with dyn1 mutant.
In this study, we evaluate the function of Pac1 with respect to dynein-mediated nuclear migration. Movies of living individual pac1 cells revealed defects in moving the mitotic spindle into the motherbud neck and microtubule sliding along the bud cortex. We found that Pac1 recruits dynein Dyn1 to the dynamic plus end of microtubules. Analysis of Pac1 and dynein localization in cells lacking other components of the dynein/dynactin pathway revealed novel aspects of the mechanism for how microtubules slide along the bud cortex and move the nucleus.
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Results |
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To test whether Pac1 is required for microtubule plastering and sliding, we examined cytoplasmic microtubule behavior during movement of the mitotic spindle into the neck in wild type and pac1 mutants expressing GFPtubulin (GFPTub1). Movies of living cells were viewed by two independent blinded observers, who evaluated cells in which cytoplasmic microtubules were observed during penetration of the spindle into the neck. In 10 of 30 wild-type cells, microtubules slid along the bud cortex (Fig. 1 A; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). This frequency is consistent with previous published data (Adames and Cooper, 2000). In 27 pac1
cells, no cases of microtubule sliding along the bud cortex were observed (Fig. 1 B; Videos 2 and 3, available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). Instead, microtubules in pac1
cells swept laterally in the bud, rotating about the SPB. The distal ends of the microtubules occasionally encountered the cortex and appeared attached, but only for a short time (11 ± 6 s; n = 11 events in eight cells). These microtubules then bent, grew, or shrunk, but did not slide, as observed for dynein and dynactin mutants (Adames and Cooper, 2000). Microtubule growth and shrinkage rates were similar in pac1
and wild-type mitotic cells. Growth rates were 4.92 µm/min (n = 11) and 4.67 µm/min (n = 13) for pac1
and wild-type cells, respectively; and shrinkage rates were 5.59 µm/min (n = 13) and 4.91 µm/min (n = 16). No qualitative differences were observed in the frequency of microtubule catastrophe and rescue for pac1
versus wild-type cells. Interestingly, cytoplasmic microtubules laterally plastered along the bud cortex in a few pac1
cells. These microtubules did not slide but dissociated from the bud cortex after a short time (10 s; n = 2).
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PAC13GFP and DYN13GFP are functional fusion genes
To understand how dynein and Pac1 move the spindle into the neck, we determined their cellular localizations. In previous studies, dynein was found at the SPB and along cytoplasmic microtubules (Shaw et al., 1997), but these studies used truncated and overexpressed tagged versions of dynein heavy chain, so they may not reflect the physiological location of dynein. We designed a tagging vector to integrate three tandem copies of GFP at the 3' end of the endogenous chromosomal locus of the dynein heavy chain gene DYN1 and PAC1. Multiple copies of GFP were necessary to detect the fusion proteins at endogenous levels. We included a GlyAlaGlyAlaGlyAla linker between the tagged gene and the triple GFP moiety. We performed three assays to evaluate the function of DYN13GFP and PAC13GFP.
First, we assayed nuclear segregation in DYN13GFP and PAC13GFP strains (YJC2772 and YJC2770). Loss of DYN1 or PAC1 function causes accumulation of cells with two nuclei in the mother (binucleate cells), more so at lower temperatures (Eshel et al., 1993; Li et al., 1993; Geiser et al., 1997). At 12°C, pac1 and dyn1
strains in mid-log phase had elevated levels of binucleate cells (Fig. 2). In contrast, strains carrying PAC13GFP or DYN13GFP as their sole source of Pac1 or dynein, respectively, had a level of binucleate cells similar to that of wild type (Fig. 2). Second, in a more stringent test, PAC13GFP and DYN13GFP rescued the phenotype of synthetic lethality with bim1
(Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). Tetrad dissection produced viable bim1
PAC13GFP haploids (7 from 7 tetratypes) and bim1
DYN13GFP haploids (12 from 10 tetratypes and 1 nonparental ditype). PAC13GFP also rescued synthetic lethality with kar9
in a similar analysis (unpublished data). Third, in liquid rich media (YPD) at 30°C, PAC13GFP (YJC2770) and DYN13GFP (YJC2772) strains grew with doubling times identical to that of the parental wild-type strain (YJC2296): 106.6 min (n = 2) for PAC13GFP; 106.5 min (n = 3) for DYN13GFP; and 106.7 min (n = 3) for parental wild type. These results show that the triple GFP tag did not interfere with Pac1 or Dyn1 function.
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If Pac1 functions in the bud to assist dynein to move the spindle into the neck, then its localization to distal ends of microtubules may depend on other components of the dynein pathway. To test for such dependence, we examined Pac13GFP localization in isogenic mutants carrying deletions of genes in the dynein pathway. The video camera and computer settings for collecting the fluorescence images were the same in all cases, allowing one to compare the intensity of fluorescence between strains. In cells deleted for the cortical attachment molecule Num1, we observed a twofold increase in the intensity of Pac13GFP cytoplasmic dots in the bud (P < 0.0001, based on measurements by a blinded observer). Fig. 5 shows the intensities. Videos 6 and 7 (available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1) are representative of wild-type and num1 strains, respectively. Pac1 dots in num1
cells moved rapidly and sometimes formed streaks across the bud cytoplasm, indicating that they likely correspond to the distal ends of cytoplasmic microtubules. Cytoplasmic microtubules in num1
cells are known to have dynamics consistent with these observations (Geiser et al., 1997; Heil-Chapdelaine et al., 2000; Farkasovsky and Kuntzel, 2001). The fluorescence intensity of Pac13GFP dots was also increased in the dynactin mutants nip100
(P < 0.0001; Fig. 5; Video 8, available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1) and arp1
(unpublished data). In contrast, Pac13GFP dot intensity was slightly reduced in dyn1
cells, but by an insignificant margin compared with wild-type cells (P = 0.015; Fig. 5; Video 9, available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1).
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Stationary dots of Dyn13GFP were observed at the mother cortex in some pac1 cells; however, the intensity was quite low (unpublished data). These stationary dots were not observed in num1
or arp1
cells. The origin and function of stationary dynein dots at the mother cortex remain unclear at this point.
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Discussion |
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Pac1 functions in nuclear migration
PAC1 was isolated in a synthetic lethal screen with cin8, which encodes a kinesin motor involved in spindle elongation (Geiser et al., 1997). This screen identified many components of the dynein/dynactin pathway. Our genetic analysis here supports the conclusion that Pac1 functions in the dynein pathway for nuclear migration. Many of the genetic interactions exhibited by pac1 are similar to those observed with mutants in the dynein/dynactin pathway, including strong synthetic interactions with kar9
and bim1
, two components of the microtubule capture/shrinkage pathway for nuclear migration. A weak synthetic interaction of pac1
with bud6
, another component of the microtubule capture/shrinkage pathway, was observed, as expected given the relatively mild defects in nuclear migration and Kar9 localization in bud6
cells (Miller et al., 1999). Previous studies also found the dyn1
bud6
double mutant to be viable with a mild growth defect (Miller et al., 1999), as seen here for the pac1
bud6
mutant. On the other hand, the weak synthetic interactions that we observed for pac1
with bni1
and kip3
(two more components of the microtubule capture/shrinkage pathway) were unexpected, as dyn1
bni1
and dyn1
kip3
double mutants were lethal in previous studies (Cottingham and Hoyt, 1997; DeZwaan et al., 1997; Miller and Rose, 1998; Miller et al., 1999). Perhaps a low level of residual dynein function is sufficient for viability in pac1
bni1
and pac1
kip3
cells. Alternatively, differences in genetic backgrounds of the strains used for the respective studies may account for the different level of synthetic interaction.
Mechanism of dynein-mediated microtubule sliding
To move the mitotic spindle into the neck, dynein appears to generate force between cytoplasmic microtubules and the bud cortex. As a motor, dynein presumably moves with respect to the microtubule; therefore, we expect that dynein is anchored at the bud cortex as the microtubule slides. We anticipated that dynein and Pac1 might be localized to the bud cortex before cytoplasmic microtubules plaster and slide along the cortex. Instead, we found that Pac1 and dynein localize to the distal ends of microtubules, which appear to grow and shrink in search of attachment sites on the bud cortex. The lack of stationary Pac13GFP and Dyn13GFP dots along the bud suggests that anchoring of dynein to the bud cortex may be transient or at a low level.
We hypothesize that microtubule sliding along the bud cortex occurs in the following steps (Fig. 8; Video 16, available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). First, dynein and Pac1 are targeted to plus ends of microtubules. Second, plus ends of microtubules are captured by cortical attachment sites, which contain Num1 and probably other components. Third, dynein and Pac1 are offloaded from the end of the microtubule to the cortex and anchored there. Fourth, the motor activity of dynein is activated, causing it to walk toward the minus end of the microtubule at the SPB. Because dynein is anchored, the microtubule slides, and the spindle is pulled into the bud neck. One intriguing observation in support of this model is that the levels of Pac1 and Dyn1 are higher at the plus ends of microtubules in cells lacking Num1. This observation suggests that a productive interaction of the microtubule end with the bud cortex is required to offload dynein and Pac1 from the microtubule end.
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Our results here with dynactin mutants provide insight into the function of dynactin. Dynein and Pac1 accumulate at the plus ends of microtubules in cells lacking dynactin. Therefore, dynactin is not required for targeting of dynein to plus ends. Instead, dynactin appears to function later, in microtubule capture or offloading, anchoring, or activating dynein at the bud cortex. In studies in other systems, dynactin promotes dynein-based movements along microtubules in vitro by increasing the processivity of the motor (King and Schroer, 2000). The NH2-terminal CAP-Gly domain of the dynactin p150Glued subunit binds microtubules, which may tether the cargo to the microtubule during the mechanochemical cycle of dynein, preventing diffusional loss of the cargo (King and Schroer, 2000). The presence of the conserved CAP-Gly domain in the NH2-terminal region of Nip100, the yeast p150Glued homologue, favors this hypothesis (Kahana et al., 1998). Nip100 has been localized at overexpressed, but not endogenous, levels, so its site of action remains somewhat uncertain.
Comparison of Pac1 with Aspergillus NUDF and vertebrate LIS1
Pac1 belongs to the conserved family of LIS1 lissencephaly proteins consisting of members from various organisms, including Schizosaccharomyces pombe, Aspergillus nidulans, Drosophila melanogaster, Caenorhabditis elegans, and human (see phylogenetic analysis in Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). LIS1 proteins have a predicted coiled-coil region in the NH2 terminus and seven tandem WD40 repeats in the COOH-terminal two thirds of the molecule (Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). LIS1 proteins are found at the plus ends of cytoplasmic microtubules in several organisms (Han et al., 2001; Coquelle et al., 2002).
The role of LIS1 proteins in dynein targeting appears to differ from organism to organism. In Aspergillus, deletion of the LIS1 homologue NUDF did not affect the localization of a GFP fusion of NUDA, the dynein heavy chain homologue, to the distal ends of cytoplasmic microtubules at hyphal tips (Zhang et al., 2002). In cultured mouse fibroblasts, reduction of LIS1 expression appeared to cause redistribution of dynein heavy chain to regions around the nucleus (Sasaki et al., 2000). It is not clear, however, whether this redistribution was due to mislocalization of dynein to microtubule ends or a result of enrichment of microtubules near the nucleus in these LIS1 heterozygous null cells (Smith et al., 2000). We found here, in budding yeast, that pac1 mutants show loss of dynein Dyn1 localization to the distal ends of cytoplasmic microtubules, which probably leads to loss of dynein delivery to cortical attachment sites and an inability to carry out productive microtubulecortex interactions. These results may reflect differences in the approaches used for the respective studies in different organisms. They may also reflect a loss or gain of particular LIS1ligand interactions subsequent to the divergence of these organisms, as suggested by the weak sequence similarity between vertebrate and yeast dynein/dynactin components (McMillan and Tatchell, 1994; Geiser et al., 1997; Kahana et al., 1998).
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Materials and methods |
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We deleted various genes in the PAC13GFP or DYN13GFP strains by oligonucleotide-mediated disruption (Baudin et al., 1993). Stable transformants were tested for appropriate disruption by PCR from genomic DNA. Two independent transformants were chosen for each disruption for subsequent localization studies.
Complementation analysis
We tested triple GFPtagged Pac1 and Dyn1 for function by assaying nuclear segregation and synthetic lethality with bim1 and kar9
. To assay for nuclear segregation, mid-log cells grown in YPD at 12°C were harvested, fixed in 70% ethanol, and stained with DAPI. Images of random fields (>80) of cells were collected on an IX70 Olympus fluorescence microscope with a cooled CCD camera (CCD-300T, Dage-MTI). The fraction of mitotic cells with two nuclei in the mother was plotted for each strain. To test for rescue of synthetic lethality, we crossed YJC2770 (PAC13GFP) or YJC2772 (DYN13GFP) to YJC1550 (bim1
) or YJC2225 (kar9
). Tetrad analysis of two independent heterozygous diploids from each cross was performed as above.
Fluorescence microscopy
We used GFPTUB1 (pAFS92, a gift from A. Straight and A. Murray (University of California San Francisco, San Francisco, CA; Straight et al., 1997) and CFPTUB1 (pAFS125C, a gift from D. Beach and K. Bloom, University of North Carolina at Chapel Hill, Chapel Hill, NC) to visualize microtubules. For analysis of microtubule sliding events, cells expressing GFPTUB1 were observed with a 100x objective on a BX60 Olympus fluorescence microscope, and images were collected with NIH Image software at two frames per second at a single focal plane using an intensified video camera with a digital image processor (ISIT68 and DSP2000; Dage-MTI) (Heil-Chapdelaine et al., 2000). Living cells from an asynchronous culture were observed during mitosis. When the mitotic spindle moved into the neck, we scored the presence of microtubule sliding along the bud cortex for cells in which cytoplasmic microtubules of the bud were in focus across their entire length. For localization studies, mid-log cells were grown in YPD or selective synthetic defined media (Bio101), washed with nonfluorescent media, and visualized directly on an agarose pad containing nonfluorescent media (Heil-Chapdelaine et al., 2000). Movies of Pac13GFP or Dyn13GFP were made using QED software (QED Imaging Inc.) by collecting five 1-µm slices at 10-s intervals with an intensified video camera (Dage ISIT68) or an intensified CCD camera (XR-Mega10; Stanford Photonics, Inc.). The fluorescence of Pac13GFP and Dyn13GFP cytoplasmic dots was measured (NIH image software) and corrected for the background fluorescence from an adjacent region next to each dot.
Cell lysis and immunoblotting
Yeast cultures were grown to mid-log phase in 5 ml YPD or selective media and harvested. Cell pellets were resuspended in 0.5 ml of ice cold lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1.5% Triton X-100, 1 mM PMSF, plus protease inhibitor cocktail tablet [Roche Applied Science]) and lysed by bead beating at 4°C in round-bottom glass tubes, five times for 1 min each, with 2 min on ice between each beating. Crude lysate was clarified at 500 g for 25 min and separated by 415% gradient or 4% linear SDS-PAGE. Proteins were electroblotted to nitrocellulose in 20 mM CAPS, pH 11.0, supplemented with 0.05% SDS for 30 h at 4°C. Mouse anti-GFP monoclonal antibody (BD Biosciences; CLONTECH) and HRP-conjugated goat antimouse antibody (Jackson ImmunoResearch Laboratories) were used at 1:1,000 and 1:10,000 dilutions, respectively.
Online supplemental material
The online version of this article (available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1) includes additional figures showing synthetic interactions of pac1 with kip3
, bud6
, and bni1
, complementation of synthetic lethality with bim1
by PAC13GFP and DYN13GFP, immunoblot analysis of Dyn13GFP protein in wild-type and pac1
lysates, phylogenetic analysis of Pac1/LIS1 sequences, domain organization of Pac1, movies of microtubule behavior during movement of the spindle into the motherbud neck, and movies of Pac13GFP and Dyn13GFP localization in wild-type or mutant backgrounds.
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Footnotes |
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* Abbreviations used in this paper: DIC, differential interference contrast; SPB, spindle pole body.
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Acknowledgments |
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Wei-Lih Lee was supported by a Damon Runyon Cancer Research Foundation Fellowship, DRG-1671. This work was supported by National Institutes of Health grant GM 47337.
Submitted: 4 September 2002
Revised: 3 December 2002
Accepted: 23 December 2002
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