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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 in living dyn1Δ, num1Δ, and nip100Δ cells. (A) DIC and a frame from movies of Pac1–3GFP fluorescence in isogenic wild-type and mutant cells. The video camera and microscope settings were the same, allowing one to compare the intensity of fluorescence in the different strains. num1Δ and nip100Δ cells showed increased intensity of Pac1–3GFP dots in the bud. See Videos 6–9 (available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). (B) Relative fluorescence intensity of motile Pac1–3GFP dots in wild-type and mutant cells. The average corrected fluorescence per dot is plotted; n = 25 dots for wild type, 75 dots for dyn1Δ, 170 dots for num1Δ, 171 dots for nip100Δ. Error bars represent standard error. Strains: PAC1–3GFP, YJC2770; PAC1–3GFP dyn1Δ, YJC2907; PAC1–3GFP num1Δ, YJC2905; PAC1–3GFP nip100Δ, YJC2904.
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fig5: Localization of Pac1–3GFP in living dyn1Δ, num1Δ, and nip100Δ cells. (A) DIC and a frame from movies of Pac1–3GFP fluorescence in isogenic wild-type and mutant cells. The video camera and microscope settings were the same, allowing one to compare the intensity of fluorescence in the different strains. num1Δ and nip100Δ cells showed increased intensity of Pac1–3GFP dots in the bud. See Videos 6–9 (available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). (B) Relative fluorescence intensity of motile Pac1–3GFP dots in wild-type and mutant cells. The average corrected fluorescence per dot is plotted; n = 25 dots for wild type, 75 dots for dyn1Δ, 170 dots for num1Δ, 171 dots for nip100Δ. Error bars represent standard error. Strains: PAC1–3GFP, YJC2770; PAC1–3GFP dyn1Δ, YJC2907; PAC1–3GFP num1Δ, YJC2905; PAC1–3GFP nip100Δ, YJC2904.

Mentions: If Pac1 functions in the bud to assist dynein to move the spindle into the neck, then its localization to distal ends of microtubules may depend on other components of the dynein pathway. To test for such dependence, we examined Pac1–3GFP localization in isogenic mutants carrying deletions of genes in the dynein pathway. The video camera and computer settings for collecting the fluorescence images were the same in all cases, allowing one to compare the intensity of fluorescence between strains. In cells deleted for the cortical attachment molecule Num1, we observed a twofold increase in the intensity of Pac1–3GFP cytoplasmic dots in the bud (P < 0.0001, based on measurements by a blinded observer). Fig. 5 shows the intensities. Videos 6 and 7 (available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1) are representative of wild-type and num1Δ strains, respectively. Pac1 dots in num1Δ cells moved rapidly and sometimes formed streaks across the bud cytoplasm, indicating that they likely correspond to the distal ends of cytoplasmic microtubules. Cytoplasmic microtubules in num1Δ cells are known to have dynamics consistent with these observations (Geiser et al., 1997; Heil-Chapdelaine et al., 2000; Farkasovsky and Kuntzel, 2001). The fluorescence intensity of Pac1–3GFP dots was also increased in the dynactin mutants nip100Δ (P < 0.0001; Fig. 5; Video 8, available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1) and arp1Δ (unpublished data). In contrast, Pac1–3GFP dot intensity was slightly reduced in dyn1Δ cells, but by an insignificant margin compared with wild-type cells (P = 0.015; Fig. 5; Video 9, 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 Pac1–3GFP in living dyn1Δ, num1Δ, and nip100Δ cells. (A) DIC and a frame from movies of Pac1–3GFP fluorescence in isogenic wild-type and mutant cells. The video camera and microscope settings were the same, allowing one to compare the intensity of fluorescence in the different strains. num1Δ and nip100Δ cells showed increased intensity of Pac1–3GFP dots in the bud. See Videos 6–9 (available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). (B) Relative fluorescence intensity of motile Pac1–3GFP dots in wild-type and mutant cells. The average corrected fluorescence per dot is plotted; n = 25 dots for wild type, 75 dots for dyn1Δ, 170 dots for num1Δ, 171 dots for nip100Δ. Error bars represent standard error. Strains: PAC1–3GFP, YJC2770; PAC1–3GFP dyn1Δ, YJC2907; PAC1–3GFP num1Δ, YJC2905; PAC1–3GFP nip100Δ, YJC2904.
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fig5: Localization of Pac1–3GFP in living dyn1Δ, num1Δ, and nip100Δ cells. (A) DIC and a frame from movies of Pac1–3GFP fluorescence in isogenic wild-type and mutant cells. The video camera and microscope settings were the same, allowing one to compare the intensity of fluorescence in the different strains. num1Δ and nip100Δ cells showed increased intensity of Pac1–3GFP dots in the bud. See Videos 6–9 (available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). (B) Relative fluorescence intensity of motile Pac1–3GFP dots in wild-type and mutant cells. The average corrected fluorescence per dot is plotted; n = 25 dots for wild type, 75 dots for dyn1Δ, 170 dots for num1Δ, 171 dots for nip100Δ. Error bars represent standard error. Strains: PAC1–3GFP, YJC2770; PAC1–3GFP dyn1Δ, YJC2907; PAC1–3GFP num1Δ, YJC2905; PAC1–3GFP nip100Δ, YJC2904.
Mentions: If Pac1 functions in the bud to assist dynein to move the spindle into the neck, then its localization to distal ends of microtubules may depend on other components of the dynein pathway. To test for such dependence, we examined Pac1–3GFP localization in isogenic mutants carrying deletions of genes in the dynein pathway. The video camera and computer settings for collecting the fluorescence images were the same in all cases, allowing one to compare the intensity of fluorescence between strains. In cells deleted for the cortical attachment molecule Num1, we observed a twofold increase in the intensity of Pac1–3GFP cytoplasmic dots in the bud (P < 0.0001, based on measurements by a blinded observer). Fig. 5 shows the intensities. Videos 6 and 7 (available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1) are representative of wild-type and num1Δ strains, respectively. Pac1 dots in num1Δ cells moved rapidly and sometimes formed streaks across the bud cytoplasm, indicating that they likely correspond to the distal ends of cytoplasmic microtubules. Cytoplasmic microtubules in num1Δ cells are known to have dynamics consistent with these observations (Geiser et al., 1997; Heil-Chapdelaine et al., 2000; Farkasovsky and Kuntzel, 2001). The fluorescence intensity of Pac1–3GFP dots was also increased in the dynactin mutants nip100Δ (P < 0.0001; Fig. 5; Video 8, available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1) and arp1Δ (unpublished data). In contrast, Pac1–3GFP dot intensity was slightly reduced in dyn1Δ cells, but by an insignificant margin compared with wild-type cells (P = 0.015; Fig. 5; Video 9, 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