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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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Proposed mechanism for microtubule sliding along the bud cortex. (Step 1) Dynein Dyn1 (red) and Pac1 (yellow) associated with the distal plus end of a cytoplasmic microtubule probe the bud cortex for attachment sites, which contain Num1 (blue) and probably other components. (Step 2) Upon attachment to a cortical site, dynein and Pac1 are offloaded from the end of microtubule and anchored to the cortex. (Step 3) The motor activity of anchored dynein is activated, causing it to walk toward the SPB. The microtubule slides, and the spindle is pulled into the bud neck. See Video 16 (available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1) and text for further discussion.
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fig8: Proposed mechanism for microtubule sliding along the bud cortex. (Step 1) Dynein Dyn1 (red) and Pac1 (yellow) associated with the distal plus end of a cytoplasmic microtubule probe the bud cortex for attachment sites, which contain Num1 (blue) and probably other components. (Step 2) Upon attachment to a cortical site, dynein and Pac1 are offloaded from the end of microtubule and anchored to the cortex. (Step 3) The motor activity of anchored dynein is activated, causing it to walk toward the SPB. The microtubule slides, and the spindle is pulled into the bud neck. See Video 16 (available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1) and text for further discussion.

Mentions: 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.


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

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

Proposed mechanism for microtubule sliding along the bud cortex. (Step 1) Dynein Dyn1 (red) and Pac1 (yellow) associated with the distal plus end of a cytoplasmic microtubule probe the bud cortex for attachment sites, which contain Num1 (blue) and probably other components. (Step 2) Upon attachment to a cortical site, dynein and Pac1 are offloaded from the end of microtubule and anchored to the cortex. (Step 3) The motor activity of anchored dynein is activated, causing it to walk toward the SPB. The microtubule slides, and the spindle is pulled into the bud neck. See Video 16 (available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1) and text for further discussion.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172672&req=5

fig8: Proposed mechanism for microtubule sliding along the bud cortex. (Step 1) Dynein Dyn1 (red) and Pac1 (yellow) associated with the distal plus end of a cytoplasmic microtubule probe the bud cortex for attachment sites, which contain Num1 (blue) and probably other components. (Step 2) Upon attachment to a cortical site, dynein and Pac1 are offloaded from the end of microtubule and anchored to the cortex. (Step 3) The motor activity of anchored dynein is activated, causing it to walk toward the SPB. The microtubule slides, and the spindle is pulled into the bud neck. See Video 16 (available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1) and text for further discussion.
Mentions: 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.

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