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Microtubules orient the mitotic spindle in yeast through dynein-dependent interactions with the cell cortex.

Carminati JL, Stearns T - J. Cell Biol. (1997)

Bottom Line: The behavior of cytoplasmic microtubules revealed distinct interactions with the cell cortex that result in associated spindle movement and orientation.Dynein-mutant cells had defects in these cortical interactions, resulting in misoriented spindles.These results indicate that microtubules and dynein interact to produce dynamic cortical interactions, and that these interactions result in the force driving spindle orientation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Stanford University, Stanford, California 94305-5020, USA.

ABSTRACT
Proper orientation of the mitotic spindle is critical for successful cell division in budding yeast. To investigate the mechanism of spindle orientation, we used a green fluorescent protein (GFP)-tubulin fusion protein to observe microtubules in living yeast cells. GFP-tubulin is incorporated into microtubules, allowing visualization of both cytoplasmic and spindle microtubules, and does not interfere with normal microtubule function. Microtubules in yeast cells exhibit dynamic instability, although they grow and shrink more slowly than microtubules in animal cells. The dynamic properties of yeast microtubules are modulated during the cell cycle. The behavior of cytoplasmic microtubules revealed distinct interactions with the cell cortex that result in associated spindle movement and orientation. Dynein-mutant cells had defects in these cortical interactions, resulting in misoriented spindles. In addition, microtubule dynamics were altered in the absence of dynein. These results indicate that microtubules and dynein interact to produce dynamic cortical interactions, and that these interactions result in the force driving spindle orientation.

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Linear regression analysis of microtubule polymerization and depolymerization rates. (A) Two examples of life-history graphs of wild-type cytoplasmic microtubules, plotting the  microtubule length vs time. In the first example, a microtubule in  a budded cell shrinks and then undergoes a rescue event. After  the next growth phase, a catastrophe event occurs, and the microtubule continues shrinking until the end of the time sequence analyzed. In the second example, one catastrophe occurs between  the growing and shrinking phases of a microtubule in a budded  cell. The microtubule analyzed in the second graph is shown in  Fig. 3 C and is present in the bud from t = 210 to t = 560 s. Rates,  as determined by linear regression, are shown over the corresponding growth or shrinkage phases. (B) Examples of life-history graphs of cytoplasmic microtubules in dynein-mutant cells.  Note the overall longer length of the microtubules as compared  with wild-type microtubules. Both examples are microtubules  from budded cells.
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Figure 5: Linear regression analysis of microtubule polymerization and depolymerization rates. (A) Two examples of life-history graphs of wild-type cytoplasmic microtubules, plotting the microtubule length vs time. In the first example, a microtubule in a budded cell shrinks and then undergoes a rescue event. After the next growth phase, a catastrophe event occurs, and the microtubule continues shrinking until the end of the time sequence analyzed. In the second example, one catastrophe occurs between the growing and shrinking phases of a microtubule in a budded cell. The microtubule analyzed in the second graph is shown in Fig. 3 C and is present in the bud from t = 210 to t = 560 s. Rates, as determined by linear regression, are shown over the corresponding growth or shrinkage phases. (B) Examples of life-history graphs of cytoplasmic microtubules in dynein-mutant cells. Note the overall longer length of the microtubules as compared with wild-type microtubules. Both examples are microtubules from budded cells.

Mentions: Rates of microtubule growth and shrinkage in living yeast cells were determined by examining changes in microtubule length that occurred during time-lapse recordings (Fig. 5 A and Materials and Methods). Transitions between growth and shrinkage of microtubules were also examined, and catastrophe and rescue frequencies were determined (Materials and Methods). Cytoplasmic microtubules grew on average at a rate of 0.491 μm/min and shrank on average at a rate of 1.350 μm/min (Table III). Interestingly, a significant difference was seen in shrinking rates between unbudded cells and budded mitotic cells; shrinking occurred in unbudded cells at a rate of 1.784 μm/ min, as compared with a rate of 0.820 μm/min in budded cells (Table III). In contrast, no significant difference was seen in microtubule growth rates, indicating that more microtubule turnover occurs in unbudded cells. Small-budded cells with duplicated spindle pole bodies but no spindle exhibited microtubule dynamics similar to that of unbudded cells (data not shown).


Microtubules orient the mitotic spindle in yeast through dynein-dependent interactions with the cell cortex.

Carminati JL, Stearns T - J. Cell Biol. (1997)

Linear regression analysis of microtubule polymerization and depolymerization rates. (A) Two examples of life-history graphs of wild-type cytoplasmic microtubules, plotting the  microtubule length vs time. In the first example, a microtubule in  a budded cell shrinks and then undergoes a rescue event. After  the next growth phase, a catastrophe event occurs, and the microtubule continues shrinking until the end of the time sequence analyzed. In the second example, one catastrophe occurs between  the growing and shrinking phases of a microtubule in a budded  cell. The microtubule analyzed in the second graph is shown in  Fig. 3 C and is present in the bud from t = 210 to t = 560 s. Rates,  as determined by linear regression, are shown over the corresponding growth or shrinkage phases. (B) Examples of life-history graphs of cytoplasmic microtubules in dynein-mutant cells.  Note the overall longer length of the microtubules as compared  with wild-type microtubules. Both examples are microtubules  from budded cells.
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Related In: Results  -  Collection

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Figure 5: Linear regression analysis of microtubule polymerization and depolymerization rates. (A) Two examples of life-history graphs of wild-type cytoplasmic microtubules, plotting the microtubule length vs time. In the first example, a microtubule in a budded cell shrinks and then undergoes a rescue event. After the next growth phase, a catastrophe event occurs, and the microtubule continues shrinking until the end of the time sequence analyzed. In the second example, one catastrophe occurs between the growing and shrinking phases of a microtubule in a budded cell. The microtubule analyzed in the second graph is shown in Fig. 3 C and is present in the bud from t = 210 to t = 560 s. Rates, as determined by linear regression, are shown over the corresponding growth or shrinkage phases. (B) Examples of life-history graphs of cytoplasmic microtubules in dynein-mutant cells. Note the overall longer length of the microtubules as compared with wild-type microtubules. Both examples are microtubules from budded cells.
Mentions: Rates of microtubule growth and shrinkage in living yeast cells were determined by examining changes in microtubule length that occurred during time-lapse recordings (Fig. 5 A and Materials and Methods). Transitions between growth and shrinkage of microtubules were also examined, and catastrophe and rescue frequencies were determined (Materials and Methods). Cytoplasmic microtubules grew on average at a rate of 0.491 μm/min and shrank on average at a rate of 1.350 μm/min (Table III). Interestingly, a significant difference was seen in shrinking rates between unbudded cells and budded mitotic cells; shrinking occurred in unbudded cells at a rate of 1.784 μm/ min, as compared with a rate of 0.820 μm/min in budded cells (Table III). In contrast, no significant difference was seen in microtubule growth rates, indicating that more microtubule turnover occurs in unbudded cells. Small-budded cells with duplicated spindle pole bodies but no spindle exhibited microtubule dynamics similar to that of unbudded cells (data not shown).

Bottom Line: The behavior of cytoplasmic microtubules revealed distinct interactions with the cell cortex that result in associated spindle movement and orientation.Dynein-mutant cells had defects in these cortical interactions, resulting in misoriented spindles.These results indicate that microtubules and dynein interact to produce dynamic cortical interactions, and that these interactions result in the force driving spindle orientation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Stanford University, Stanford, California 94305-5020, USA.

ABSTRACT
Proper orientation of the mitotic spindle is critical for successful cell division in budding yeast. To investigate the mechanism of spindle orientation, we used a green fluorescent protein (GFP)-tubulin fusion protein to observe microtubules in living yeast cells. GFP-tubulin is incorporated into microtubules, allowing visualization of both cytoplasmic and spindle microtubules, and does not interfere with normal microtubule function. Microtubules in yeast cells exhibit dynamic instability, although they grow and shrink more slowly than microtubules in animal cells. The dynamic properties of yeast microtubules are modulated during the cell cycle. The behavior of cytoplasmic microtubules revealed distinct interactions with the cell cortex that result in associated spindle movement and orientation. Dynein-mutant cells had defects in these cortical interactions, resulting in misoriented spindles. In addition, microtubule dynamics were altered in the absence of dynein. These results indicate that microtubules and dynein interact to produce dynamic cortical interactions, and that these interactions result in the force driving spindle orientation.

Show MeSH
Related in: MedlinePlus