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The role of the lissencephaly protein Pac1 during nuclear migration in budding yeast.

Lee WL, Oberle JR, Cooper JA - J. Cell Biol. (2003)

Bottom Line: Second, cells lacking Pac1 failed to display microtubule sliding in the bud, resulting in defective mitotic spindle movement and nuclear segregation.This localization did not depend on the dynein heavy chain Dyn1.Dynein must remain inactive until microtubule ends interact with the bud cortex, at which time dynein and Pac1 appear to be offloaded from the microtubule to the cortex.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA.

ABSTRACT
During mitosis in Saccharomyces cerevisiae, the mitotic spindle moves into the mother-bud neck via dynein-dependent sliding of cytoplasmic microtubules along the cortex of the bud. Here we show that Pac1, the yeast homologue of the human lissencephaly protein LIS1, plays a key role in this process. First, genetic interactions placed Pac1 in the dynein/dynactin pathway. Second, cells lacking Pac1 failed to display microtubule sliding in the bud, resulting in defective mitotic spindle movement and nuclear segregation. Third, Pac1 localized to the plus ends (distal tips) of cytoplasmic microtubules in the bud. This localization did not depend on the dynein heavy chain Dyn1. Moreover, the Pac1 fluorescence intensity at the microtubule end was enhanced in cells lacking dynactin or the cortical attachment molecule Num1. Fourth, dynein heavy chain Dyn1 also localized to the tips of cytoplasmic microtubules in wild-type cells. Dynein localization required Pac1 and, like Pac1, was enhanced in cells lacking the dynactin component Arp1 or the cortical attachment molecule Num1. Our results suggest that Pac1 targets dynein to microtubule tips, which is necessary for sliding of microtubules along the bud cortex. Dynein must remain inactive until microtubule ends interact with the bud cortex, at which time dynein and Pac1 appear to be offloaded from the microtubule to the cortex.

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Localization of Dyn1–3GFP in living wild-type cells. (A) DIC and movie frames of Dyn1–3GFP fluorescence in a wild-type cell (YJC2772). Each fluorescence image is a two-dimensional projection of a 4-μm Z-stack of confocal images. The time elapsed in seconds is indicated. Dyn1–3GFP is observed as a dot that moves away from and toward the bud (arrows). Dyn1–3GFP sometimes appears as a linear streak (t = 28, 84, and 147 s). Dyn1–3GFP is also observed as stationary cortical dots, but only in the mother (see Videos 10 and 11, available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). (B) Dyn1–3GFP colocalizes with the distal ends of cytoplasmic microtubules. DIC, Dyn1–3GFP, and CFP–Tub1 wide-field fluorescence images of wild-type cells (YJC2914) at G1 (top), preanaphase (middle), and anaphase (bottom). The merged images show cytoplasmic Dyn1–3GFP dots (red) at the distal ends of microtubules (blue).
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fig6: Localization of Dyn1–3GFP in living wild-type cells. (A) DIC and movie frames of Dyn1–3GFP fluorescence in a wild-type cell (YJC2772). Each fluorescence image is a two-dimensional projection of a 4-μm Z-stack of confocal images. The time elapsed in seconds is indicated. Dyn1–3GFP is observed as a dot that moves away from and toward the bud (arrows). Dyn1–3GFP sometimes appears as a linear streak (t = 28, 84, and 147 s). Dyn1–3GFP is also observed as stationary cortical dots, but only in the mother (see Videos 10 and 11, available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). (B) Dyn1–3GFP colocalizes with the distal ends of cytoplasmic microtubules. DIC, Dyn1–3GFP, and CFP–Tub1 wide-field fluorescence images of wild-type cells (YJC2914) at G1 (top), preanaphase (middle), and anaphase (bottom). The merged images show cytoplasmic Dyn1–3GFP dots (red) at the distal ends of microtubules (blue).

Mentions: Dynein Dyn1–3GFP also localized to the distal ends of cytoplasmic microtubules (Fig. 6 B). In wild-type cells, Dyn1–3GFP was observed as dots that moved rapidly in the cytoplasm (Fig. 6 A; Videos 10 and 11, available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). Dyn1–3GFP dots moved at a rate of 3.8 ± 1.6 μm/min (n = 12) and sometimes formed linear streaks, as seen for Pac1–3GFP. Imaging of live cells expressing CFP–tubulin and Dyn1–3GFP revealed that ∼47% of all observed cytoplasmic microtubules had a Dyn1 dot at their distal end, ∼10% had Dyn1 along the distal portion of the microtubule, and ∼43% had no Dyn1–3GFP (n = 127 microtubules counted). As seen for the case of Pac1–3GFP, the microtubules and Dyn1–3GFP dots were moving on a relatively rapid time scale during image collection. Thus, these measurements represent the lower limit for colocalization of Dyn1–3GFP with microtubule ends. In striking contrast to Pac1–3GFP, some dots of Dyn1–3GFP did not move (>6 min). These stationary dynein dots were found only at the cortex, and only in unbudded cells or the mother of budded cells (Video 11, middle cell).


The role of the lissencephaly protein Pac1 during nuclear migration in budding yeast.

Lee WL, Oberle JR, Cooper JA - J. Cell Biol. (2003)

Localization of Dyn1–3GFP in living wild-type cells. (A) DIC and movie frames of Dyn1–3GFP fluorescence in a wild-type cell (YJC2772). Each fluorescence image is a two-dimensional projection of a 4-μm Z-stack of confocal images. The time elapsed in seconds is indicated. Dyn1–3GFP is observed as a dot that moves away from and toward the bud (arrows). Dyn1–3GFP sometimes appears as a linear streak (t = 28, 84, and 147 s). Dyn1–3GFP is also observed as stationary cortical dots, but only in the mother (see Videos 10 and 11, available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). (B) Dyn1–3GFP colocalizes with the distal ends of cytoplasmic microtubules. DIC, Dyn1–3GFP, and CFP–Tub1 wide-field fluorescence images of wild-type cells (YJC2914) at G1 (top), preanaphase (middle), and anaphase (bottom). The merged images show cytoplasmic Dyn1–3GFP dots (red) at the distal ends of microtubules (blue).
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fig6: Localization of Dyn1–3GFP in living wild-type cells. (A) DIC and movie frames of Dyn1–3GFP fluorescence in a wild-type cell (YJC2772). Each fluorescence image is a two-dimensional projection of a 4-μm Z-stack of confocal images. The time elapsed in seconds is indicated. Dyn1–3GFP is observed as a dot that moves away from and toward the bud (arrows). Dyn1–3GFP sometimes appears as a linear streak (t = 28, 84, and 147 s). Dyn1–3GFP is also observed as stationary cortical dots, but only in the mother (see Videos 10 and 11, available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). (B) Dyn1–3GFP colocalizes with the distal ends of cytoplasmic microtubules. DIC, Dyn1–3GFP, and CFP–Tub1 wide-field fluorescence images of wild-type cells (YJC2914) at G1 (top), preanaphase (middle), and anaphase (bottom). The merged images show cytoplasmic Dyn1–3GFP dots (red) at the distal ends of microtubules (blue).
Mentions: Dynein Dyn1–3GFP also localized to the distal ends of cytoplasmic microtubules (Fig. 6 B). In wild-type cells, Dyn1–3GFP was observed as dots that moved rapidly in the cytoplasm (Fig. 6 A; Videos 10 and 11, available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). Dyn1–3GFP dots moved at a rate of 3.8 ± 1.6 μm/min (n = 12) and sometimes formed linear streaks, as seen for Pac1–3GFP. Imaging of live cells expressing CFP–tubulin and Dyn1–3GFP revealed that ∼47% of all observed cytoplasmic microtubules had a Dyn1 dot at their distal end, ∼10% had Dyn1 along the distal portion of the microtubule, and ∼43% had no Dyn1–3GFP (n = 127 microtubules counted). As seen for the case of Pac1–3GFP, the microtubules and Dyn1–3GFP dots were moving on a relatively rapid time scale during image collection. Thus, these measurements represent the lower limit for colocalization of Dyn1–3GFP with microtubule ends. In striking contrast to Pac1–3GFP, some dots of Dyn1–3GFP did not move (>6 min). These stationary dynein dots were found only at the cortex, and only in unbudded cells or the mother of budded cells (Video 11, middle cell).

Bottom Line: Second, cells lacking Pac1 failed to display microtubule sliding in the bud, resulting in defective mitotic spindle movement and nuclear segregation.This localization did not depend on the dynein heavy chain Dyn1.Dynein must remain inactive until microtubule ends interact with the bud cortex, at which time dynein and Pac1 appear to be offloaded from the microtubule to the cortex.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA.

ABSTRACT
During mitosis in Saccharomyces cerevisiae, the mitotic spindle moves into the mother-bud neck via dynein-dependent sliding of cytoplasmic microtubules along the cortex of the bud. Here we show that Pac1, the yeast homologue of the human lissencephaly protein LIS1, plays a key role in this process. First, genetic interactions placed Pac1 in the dynein/dynactin pathway. Second, cells lacking Pac1 failed to display microtubule sliding in the bud, resulting in defective mitotic spindle movement and nuclear segregation. Third, Pac1 localized to the plus ends (distal tips) of cytoplasmic microtubules in the bud. This localization did not depend on the dynein heavy chain Dyn1. Moreover, the Pac1 fluorescence intensity at the microtubule end was enhanced in cells lacking dynactin or the cortical attachment molecule Num1. Fourth, dynein heavy chain Dyn1 also localized to the tips of cytoplasmic microtubules in wild-type cells. Dynein localization required Pac1 and, like Pac1, was enhanced in cells lacking the dynactin component Arp1 or the cortical attachment molecule Num1. Our results suggest that Pac1 targets dynein to microtubule tips, which is necessary for sliding of microtubules along the bud cortex. Dynein must remain inactive until microtubule ends interact with the bud cortex, at which time dynein and Pac1 appear to be offloaded from the microtubule to the cortex.

Show MeSH
Related in: MedlinePlus