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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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Pac1–3GFP colocalizes with the distal ends of cytoplasmic microtubules. DIC, Pac1–3GFP, and CFP–Tub1 wide-field fluorescence images of wild-type cells. The merged images on the right show Pac1–3GFP in red and CFP–Tub1 in blue. Cytoplasmic dots of Pac1–3GFP colocalize with the distal ends of microtubules at different stages of the cell cycle. Localization of Pac1–3GFP to microtubule ends was observed in the bud before the mitotic spindle moved into the neck (rows 4 and 5 from top) and after the mitotic spindle moved into the neck (row 6). Sometimes Pac1–3GFP was observed along cytoplasmic microtubules (rows 5 and 6). Strain: PAC1–3GFP CFP–TUB1, YJC2814.
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fig4: Pac1–3GFP colocalizes with the distal ends of cytoplasmic microtubules. DIC, Pac1–3GFP, and CFP–Tub1 wide-field fluorescence images of wild-type cells. The merged images on the right show Pac1–3GFP in red and CFP–Tub1 in blue. Cytoplasmic dots of Pac1–3GFP colocalize with the distal ends of microtubules at different stages of the cell cycle. Localization of Pac1–3GFP to microtubule ends was observed in the bud before the mitotic spindle moved into the neck (rows 4 and 5 from top) and after the mitotic spindle moved into the neck (row 6). Sometimes Pac1–3GFP was observed along cytoplasmic microtubules (rows 5 and 6). Strain: PAC1–3GFP CFP–TUB1, YJC2814.

Mentions: Pac1–3GFP dots colocalized with the distal ends of cytoplasmic microtubules (Fig. 4). Imaging of live cells expressing CFP–tubulin and Pac1–3GFP revealed that ∼49% of cytoplasmic microtubules had a Pac1 dot at their distal end, ∼8% had Pac1 along the distal portion of the microtubule, and ∼43% had no Pac1–3GFP (n = 300 microtubules counted). The microtubules and Pac1–3GFP dots were moving during image collection, on a time scale comparable to the interval between images. Thus, these measurements underestimate the extent of colocalization, perhaps by a significant amount.


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

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

Pac1–3GFP colocalizes with the distal ends of cytoplasmic microtubules. DIC, Pac1–3GFP, and CFP–Tub1 wide-field fluorescence images of wild-type cells. The merged images on the right show Pac1–3GFP in red and CFP–Tub1 in blue. Cytoplasmic dots of Pac1–3GFP colocalize with the distal ends of microtubules at different stages of the cell cycle. Localization of Pac1–3GFP to microtubule ends was observed in the bud before the mitotic spindle moved into the neck (rows 4 and 5 from top) and after the mitotic spindle moved into the neck (row 6). Sometimes Pac1–3GFP was observed along cytoplasmic microtubules (rows 5 and 6). Strain: PAC1–3GFP CFP–TUB1, YJC2814.
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Related In: Results  -  Collection

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

fig4: Pac1–3GFP colocalizes with the distal ends of cytoplasmic microtubules. DIC, Pac1–3GFP, and CFP–Tub1 wide-field fluorescence images of wild-type cells. The merged images on the right show Pac1–3GFP in red and CFP–Tub1 in blue. Cytoplasmic dots of Pac1–3GFP colocalize with the distal ends of microtubules at different stages of the cell cycle. Localization of Pac1–3GFP to microtubule ends was observed in the bud before the mitotic spindle moved into the neck (rows 4 and 5 from top) and after the mitotic spindle moved into the neck (row 6). Sometimes Pac1–3GFP was observed along cytoplasmic microtubules (rows 5 and 6). Strain: PAC1–3GFP CFP–TUB1, YJC2814.
Mentions: Pac1–3GFP dots colocalized with the distal ends of cytoplasmic microtubules (Fig. 4). Imaging of live cells expressing CFP–tubulin and Pac1–3GFP revealed that ∼49% of cytoplasmic microtubules had a Pac1 dot at their distal end, ∼8% had Pac1 along the distal portion of the microtubule, and ∼43% had no Pac1–3GFP (n = 300 microtubules counted). The microtubules and Pac1–3GFP dots were moving during image collection, on a time scale comparable to the interval between images. Thus, these measurements underestimate the extent of colocalization, perhaps by a significant amount.

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