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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 arp1Δ, num1Δ, and pac1Δ cells. (A) DIC and Dyn1–3GFP fluorescence images of isogenic wild-type and mutant cells. The video camera and microscope settings were the same for the different strains. arp1Δ and num1Δ cells showed increased fluorescence intensity for Dyn1–3GFP dots in the bud. pac1Δ cells showed the absence of cytoplasmic Dyn1–3GFP motile dots. See Videos 12–15 (available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). (B) Relative fluorescence intensity of motile Dyn1–3GFP dots in wild-type, arp1Δ, and num1Δ cells. The average corrected fluorescence per dot is plotted; n = 72 dots for wild type, 91 dots for arp1Δ, 84 dots for num1Δ. Error bars represent standard error. Strains: DYN1–3GFP, YJC2772; DYN1–3GFP arp1Δ, YJC2908; DYN1–3GFP num1Δ, YJC2910; DYN1–3GFP pac1Δ, YJC2912.
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fig7: Localization of Dyn1–3GFP in living arp1Δ, num1Δ, and pac1Δ cells. (A) DIC and Dyn1–3GFP fluorescence images of isogenic wild-type and mutant cells. The video camera and microscope settings were the same for the different strains. arp1Δ and num1Δ cells showed increased fluorescence intensity for Dyn1–3GFP dots in the bud. pac1Δ cells showed the absence of cytoplasmic Dyn1–3GFP motile dots. See Videos 12–15 (available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). (B) Relative fluorescence intensity of motile Dyn1–3GFP dots in wild-type, arp1Δ, and num1Δ cells. The average corrected fluorescence per dot is plotted; n = 72 dots for wild type, 91 dots for arp1Δ, 84 dots for num1Δ. Error bars represent standard error. Strains: DYN1–3GFP, YJC2772; DYN1–3GFP arp1Δ, YJC2908; DYN1–3GFP num1Δ, YJC2910; DYN1–3GFP pac1Δ, YJC2912.

Mentions: We asked whether Dyn1 localization to the distal ends of microtubules depends on other components of the dynein pathway. We examined Dyn1–3GFP localization in isogenic arp1Δ, num1Δ, and pac1Δ mutants. In arp1Δ and num1Δ cells, Dyn1–3GFP was seen as dots in the cytoplasm, which moved rapidly and sometimes formed linear streaks, similar to what was observed in wild-type cells. Furthermore, the fluorescence intensity of the dots was increased relative to wild type, by an amount slightly greater than that seen for Pac1–3GFP dots in these mutants (Fig. 7; compare Video 11 [wild type] with Videos 12 [arp1Δ] and 13 [num1Δ], available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1).


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 arp1Δ, num1Δ, and pac1Δ cells. (A) DIC and Dyn1–3GFP fluorescence images of isogenic wild-type and mutant cells. The video camera and microscope settings were the same for the different strains. arp1Δ and num1Δ cells showed increased fluorescence intensity for Dyn1–3GFP dots in the bud. pac1Δ cells showed the absence of cytoplasmic Dyn1–3GFP motile dots. See Videos 12–15 (available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). (B) Relative fluorescence intensity of motile Dyn1–3GFP dots in wild-type, arp1Δ, and num1Δ cells. The average corrected fluorescence per dot is plotted; n = 72 dots for wild type, 91 dots for arp1Δ, 84 dots for num1Δ. Error bars represent standard error. Strains: DYN1–3GFP, YJC2772; DYN1–3GFP arp1Δ, YJC2908; DYN1–3GFP num1Δ, YJC2910; DYN1–3GFP pac1Δ, YJC2912.
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getmorefigures.php?uid=PMC2172672&req=5

fig7: Localization of Dyn1–3GFP in living arp1Δ, num1Δ, and pac1Δ cells. (A) DIC and Dyn1–3GFP fluorescence images of isogenic wild-type and mutant cells. The video camera and microscope settings were the same for the different strains. arp1Δ and num1Δ cells showed increased fluorescence intensity for Dyn1–3GFP dots in the bud. pac1Δ cells showed the absence of cytoplasmic Dyn1–3GFP motile dots. See Videos 12–15 (available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). (B) Relative fluorescence intensity of motile Dyn1–3GFP dots in wild-type, arp1Δ, and num1Δ cells. The average corrected fluorescence per dot is plotted; n = 72 dots for wild type, 91 dots for arp1Δ, 84 dots for num1Δ. Error bars represent standard error. Strains: DYN1–3GFP, YJC2772; DYN1–3GFP arp1Δ, YJC2908; DYN1–3GFP num1Δ, YJC2910; DYN1–3GFP pac1Δ, YJC2912.
Mentions: We asked whether Dyn1 localization to the distal ends of microtubules depends on other components of the dynein pathway. We examined Dyn1–3GFP localization in isogenic arp1Δ, num1Δ, and pac1Δ mutants. In arp1Δ and num1Δ cells, Dyn1–3GFP was seen as dots in the cytoplasm, which moved rapidly and sometimes formed linear streaks, similar to what was observed in wild-type cells. Furthermore, the fluorescence intensity of the dots was increased relative to wild type, by an amount slightly greater than that seen for Pac1–3GFP dots in these mutants (Fig. 7; compare Video 11 [wild type] with Videos 12 [arp1Δ] and 13 [num1Δ], available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1).

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