Limits...
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.

Show MeSH

Related in: MedlinePlus

Microtubule and spindle behavior in wild-type and pac1Δ strains. (A and B) Frames from movies of GFP-labeled microtubules during movement of the mitotic spindle into the mother–bud neck. Arrows indicate the position of the neck. The time elapsed in seconds is indicated. See Videos 1, 2, and 3 (available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). Strains: wild-type GFP–TUB1, YJC2350; pac1Δ GFP–TUB1, YJC2501.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172672&req=5

fig1: Microtubule and spindle behavior in wild-type and pac1Δ strains. (A and B) Frames from movies of GFP-labeled microtubules during movement of the mitotic spindle into the mother–bud neck. Arrows indicate the position of the neck. The time elapsed in seconds is indicated. See Videos 1, 2, and 3 (available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). Strains: wild-type GFP–TUB1, YJC2350; pac1Δ GFP–TUB1, YJC2501.

Mentions: To test whether Pac1 is required for microtubule plastering and sliding, we examined cytoplasmic microtubule behavior during movement of the mitotic spindle into the neck in wild type and pac1Δ mutants expressing GFP–tubulin (GFP–Tub1). Movies of living cells were viewed by two independent blinded observers, who evaluated cells in which cytoplasmic microtubules were observed during penetration of the spindle into the neck. In 10 of 30 wild-type cells, microtubules slid along the bud cortex (Fig. 1 A; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). This frequency is consistent with previous published data (Adames and Cooper, 2000). In 27 pac1Δ cells, no cases of microtubule sliding along the bud cortex were observed (Fig. 1 B; Videos 2 and 3, available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). Instead, microtubules in pac1Δ cells swept laterally in the bud, rotating about the SPB. The distal ends of the microtubules occasionally encountered the cortex and appeared attached, but only for a short time (11 ± 6 s; n = 11 events in eight cells). These microtubules then bent, grew, or shrunk, but did not slide, as observed for dynein and dynactin mutants (Adames and Cooper, 2000). Microtubule growth and shrinkage rates were similar in pac1Δ and wild-type mitotic cells. Growth rates were 4.92 μm/min (n = 11) and 4.67 μm/min (n = 13) for pac1Δ and wild-type cells, respectively; and shrinkage rates were 5.59 μm/min (n = 13) and 4.91 μm/min (n = 16). No qualitative differences were observed in the frequency of microtubule catastrophe and rescue for pac1Δ versus wild-type cells. Interestingly, cytoplasmic microtubules laterally plastered along the bud cortex in a few pac1Δ cells. These microtubules did not slide but dissociated from the bud cortex after a short time (10 s; n = 2).


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

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

Microtubule and spindle behavior in wild-type and pac1Δ strains. (A and B) Frames from movies of GFP-labeled microtubules during movement of the mitotic spindle into the mother–bud neck. Arrows indicate the position of the neck. The time elapsed in seconds is indicated. See Videos 1, 2, and 3 (available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). Strains: wild-type GFP–TUB1, YJC2350; pac1Δ GFP–TUB1, YJC2501.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Microtubule and spindle behavior in wild-type and pac1Δ strains. (A and B) Frames from movies of GFP-labeled microtubules during movement of the mitotic spindle into the mother–bud neck. Arrows indicate the position of the neck. The time elapsed in seconds is indicated. See Videos 1, 2, and 3 (available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). Strains: wild-type GFP–TUB1, YJC2350; pac1Δ GFP–TUB1, YJC2501.
Mentions: To test whether Pac1 is required for microtubule plastering and sliding, we examined cytoplasmic microtubule behavior during movement of the mitotic spindle into the neck in wild type and pac1Δ mutants expressing GFP–tubulin (GFP–Tub1). Movies of living cells were viewed by two independent blinded observers, who evaluated cells in which cytoplasmic microtubules were observed during penetration of the spindle into the neck. In 10 of 30 wild-type cells, microtubules slid along the bud cortex (Fig. 1 A; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). This frequency is consistent with previous published data (Adames and Cooper, 2000). In 27 pac1Δ cells, no cases of microtubule sliding along the bud cortex were observed (Fig. 1 B; Videos 2 and 3, available at http://www.jcb.org/cgi/content/full/jcb.200209022/DC1). Instead, microtubules in pac1Δ cells swept laterally in the bud, rotating about the SPB. The distal ends of the microtubules occasionally encountered the cortex and appeared attached, but only for a short time (11 ± 6 s; n = 11 events in eight cells). These microtubules then bent, grew, or shrunk, but did not slide, as observed for dynein and dynactin mutants (Adames and Cooper, 2000). Microtubule growth and shrinkage rates were similar in pac1Δ and wild-type mitotic cells. Growth rates were 4.92 μm/min (n = 11) and 4.67 μm/min (n = 13) for pac1Δ and wild-type cells, respectively; and shrinkage rates were 5.59 μm/min (n = 13) and 4.91 μm/min (n = 16). No qualitative differences were observed in the frequency of microtubule catastrophe and rescue for pac1Δ versus wild-type cells. Interestingly, cytoplasmic microtubules laterally plastered along the bud cortex in a few pac1Δ cells. These microtubules did not slide but dissociated from the bud cortex after a short time (10 s; n = 2).

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