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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 Pac1–3GFP. Differential interference contrast (DIC) and Pac1–3GFP wide-field fluorescence images of wild-type cells. (A) Pac1–3GFP is observed in the cytoplasm as dots (arrowhead), which move rapidly and sometimes form linear streaks (arrows). (B) Pac1–3GFP dots move toward and away from the bud (arrows). Pac1–3GFP is also observed in the nucleus, with a diffuse distribution. The time elapsed in seconds is indicated. Strain: PAC1–3GFP, YJC2770. See Videos 4 and 5 (available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1).
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fig3: Localization of Pac1–3GFP. Differential interference contrast (DIC) and Pac1–3GFP wide-field fluorescence images of wild-type cells. (A) Pac1–3GFP is observed in the cytoplasm as dots (arrowhead), which move rapidly and sometimes form linear streaks (arrows). (B) Pac1–3GFP dots move toward and away from the bud (arrows). Pac1–3GFP is also observed in the nucleus, with a diffuse distribution. The time elapsed in seconds is indicated. Strain: PAC1–3GFP, YJC2770. See Videos 4 and 5 (available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1).

Mentions: Pac1–3GFP was found in several locations in wild-type cells from asynchronous log-phase cultures. First, Pac1–3GFP localized as dots in the cytoplasm (Fig. 3). Most cells contained one to two dots; the range was zero to four. The dots moved rapidly (3.9 ± 0.7 μm/min, n = 7) and sometimes formed linear streaks (Fig. 3 A, arrows; Videos 4 and 5, available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). In budded cells, Pac1–3GFP dots moved toward and away from the bud cortex. Once at the cortex, Pac1–3GFP dots were never observed to be stationary for more than 10 s, which was the interval between image acquisitions in the time-lapse movie.


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 Pac1–3GFP. Differential interference contrast (DIC) and Pac1–3GFP wide-field fluorescence images of wild-type cells. (A) Pac1–3GFP is observed in the cytoplasm as dots (arrowhead), which move rapidly and sometimes form linear streaks (arrows). (B) Pac1–3GFP dots move toward and away from the bud (arrows). Pac1–3GFP is also observed in the nucleus, with a diffuse distribution. The time elapsed in seconds is indicated. Strain: PAC1–3GFP, YJC2770. See Videos 4 and 5 (available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172672&req=5

fig3: Localization of Pac1–3GFP. Differential interference contrast (DIC) and Pac1–3GFP wide-field fluorescence images of wild-type cells. (A) Pac1–3GFP is observed in the cytoplasm as dots (arrowhead), which move rapidly and sometimes form linear streaks (arrows). (B) Pac1–3GFP dots move toward and away from the bud (arrows). Pac1–3GFP is also observed in the nucleus, with a diffuse distribution. The time elapsed in seconds is indicated. Strain: PAC1–3GFP, YJC2770. See Videos 4 and 5 (available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1).
Mentions: Pac1–3GFP was found in several locations in wild-type cells from asynchronous log-phase cultures. First, Pac1–3GFP localized as dots in the cytoplasm (Fig. 3). Most cells contained one to two dots; the range was zero to four. The dots moved rapidly (3.9 ± 0.7 μm/min, n = 7) and sometimes formed linear streaks (Fig. 3 A, arrows; Videos 4 and 5, available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). In budded cells, Pac1–3GFP dots moved toward and away from the bud cortex. Once at the cortex, Pac1–3GFP dots were never observed to be stationary for more than 10 s, which was the interval between image acquisitions in the time-lapse movie.

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