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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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Function of PAC1–3GFP and DYN1–3GFP. Cells grown to mid-log phase at 12°C were fixed, stained for nuclei with DAPI, and imaged. The fraction of budded mitotic cells (Heil-Chapdelaine et al., 2000) with two nuclei in the mother is plotted. Error bars represent standard error (n > 750 cells counted for each strain). Strains: wild type, YJC2296; pac1Δ, YJC1629; dyn1Δ, YJC2007; PAC1–3GFP, YJC2770; DYN1–3GFP, YJC2772.
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fig2: Function of PAC1–3GFP and DYN1–3GFP. Cells grown to mid-log phase at 12°C were fixed, stained for nuclei with DAPI, and imaged. The fraction of budded mitotic cells (Heil-Chapdelaine et al., 2000) with two nuclei in the mother is plotted. Error bars represent standard error (n > 750 cells counted for each strain). Strains: wild type, YJC2296; pac1Δ, YJC1629; dyn1Δ, YJC2007; PAC1–3GFP, YJC2770; DYN1–3GFP, YJC2772.

Mentions: First, we assayed nuclear segregation in DYN1–3GFP and PAC1–3GFP strains (YJC2772 and YJC2770). Loss of DYN1 or PAC1 function causes accumulation of cells with two nuclei in the mother (binucleate cells), more so at lower temperatures (Eshel et al., 1993; Li et al., 1993; Geiser et al., 1997). At 12°C, pac1Δ and dyn1Δ strains in mid-log phase had elevated levels of binucleate cells (Fig. 2). In contrast, strains carrying PAC1–3GFP or DYN1–3GFP as their sole source of Pac1 or dynein, respectively, had a level of binucleate cells similar to that of wild type (Fig. 2). Second, in a more stringent test, PAC1–3GFP and DYN1–3GFP rescued the phenotype of synthetic lethality with bim1Δ (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). Tetrad dissection produced viable bim1Δ PAC1–3GFP haploids (7 from 7 tetratypes) and bim1Δ DYN1–3GFP haploids (12 from 10 tetratypes and 1 nonparental ditype). PAC1–3GFP also rescued synthetic lethality with kar9Δ in a similar analysis (unpublished data). Third, in liquid rich media (YPD) at 30°C, PAC1–3GFP (YJC2770) and DYN1–3GFP (YJC2772) strains grew with doubling times identical to that of the parental wild-type strain (YJC2296): 106.6 min (n = 2) for PAC1–3GFP; 106.5 min (n = 3) for DYN1–3GFP; and 106.7 min (n = 3) for parental wild type. These results show that the triple GFP tag did not interfere with Pac1 or Dyn1 function.


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

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

Function of PAC1–3GFP and DYN1–3GFP. Cells grown to mid-log phase at 12°C were fixed, stained for nuclei with DAPI, and imaged. The fraction of budded mitotic cells (Heil-Chapdelaine et al., 2000) with two nuclei in the mother is plotted. Error bars represent standard error (n > 750 cells counted for each strain). Strains: wild type, YJC2296; pac1Δ, YJC1629; dyn1Δ, YJC2007; PAC1–3GFP, YJC2770; DYN1–3GFP, YJC2772.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172672&req=5

fig2: Function of PAC1–3GFP and DYN1–3GFP. Cells grown to mid-log phase at 12°C were fixed, stained for nuclei with DAPI, and imaged. The fraction of budded mitotic cells (Heil-Chapdelaine et al., 2000) with two nuclei in the mother is plotted. Error bars represent standard error (n > 750 cells counted for each strain). Strains: wild type, YJC2296; pac1Δ, YJC1629; dyn1Δ, YJC2007; PAC1–3GFP, YJC2770; DYN1–3GFP, YJC2772.
Mentions: First, we assayed nuclear segregation in DYN1–3GFP and PAC1–3GFP strains (YJC2772 and YJC2770). Loss of DYN1 or PAC1 function causes accumulation of cells with two nuclei in the mother (binucleate cells), more so at lower temperatures (Eshel et al., 1993; Li et al., 1993; Geiser et al., 1997). At 12°C, pac1Δ and dyn1Δ strains in mid-log phase had elevated levels of binucleate cells (Fig. 2). In contrast, strains carrying PAC1–3GFP or DYN1–3GFP as their sole source of Pac1 or dynein, respectively, had a level of binucleate cells similar to that of wild type (Fig. 2). Second, in a more stringent test, PAC1–3GFP and DYN1–3GFP rescued the phenotype of synthetic lethality with bim1Δ (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). Tetrad dissection produced viable bim1Δ PAC1–3GFP haploids (7 from 7 tetratypes) and bim1Δ DYN1–3GFP haploids (12 from 10 tetratypes and 1 nonparental ditype). PAC1–3GFP also rescued synthetic lethality with kar9Δ in a similar analysis (unpublished data). Third, in liquid rich media (YPD) at 30°C, PAC1–3GFP (YJC2770) and DYN1–3GFP (YJC2772) strains grew with doubling times identical to that of the parental wild-type strain (YJC2296): 106.6 min (n = 2) for PAC1–3GFP; 106.5 min (n = 3) for DYN1–3GFP; and 106.7 min (n = 3) for parental wild type. These results show that the triple GFP tag did not interfere with Pac1 or Dyn1 function.

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