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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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GFP–TUB3 produces fusion protein that incorporates  into microtubules. (A) Schematic showing the process by which  fluorescent microtubules are generated in vivo. The CEN plasmid carrying GFP–TUB3, pTS417, is transformed into the diploid  yeast strain, TPS507, and the resulting strain, TSY425, is then  grown in the presence of 2% galactose as the carbon source. After galactose induction, fluorescent microtubules are formed in  vivo. (B) Colocalization of GFP–TUB3 with α-tubulin in microtubules. After growth of TSY425 in 2% galactose, cells were  fixed and stained for α-tubulin. Fluorescence of rhodamine- labeled α-tubulin was compared with GFP fluorescence; both  α-tubulin and GFP colocalized to microtubule structures in unbudded and budded cells. Fluorescent cytoplasmic microtubules  and the spindle pole body are seen in unbudded cells (left),  whereas a fluorescent mitotic spindle is seen in the budded cell  shown (right). The right panel also contains an unbudded cell in  which GFP fluorescence of the spindle pole body, but not the microtubules, can be seen.
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Figure 1: GFP–TUB3 produces fusion protein that incorporates into microtubules. (A) Schematic showing the process by which fluorescent microtubules are generated in vivo. The CEN plasmid carrying GFP–TUB3, pTS417, is transformed into the diploid yeast strain, TPS507, and the resulting strain, TSY425, is then grown in the presence of 2% galactose as the carbon source. After galactose induction, fluorescent microtubules are formed in vivo. (B) Colocalization of GFP–TUB3 with α-tubulin in microtubules. After growth of TSY425 in 2% galactose, cells were fixed and stained for α-tubulin. Fluorescence of rhodamine- labeled α-tubulin was compared with GFP fluorescence; both α-tubulin and GFP colocalized to microtubule structures in unbudded and budded cells. Fluorescent cytoplasmic microtubules and the spindle pole body are seen in unbudded cells (left), whereas a fluorescent mitotic spindle is seen in the budded cell shown (right). The right panel also contains an unbudded cell in which GFP fluorescence of the spindle pole body, but not the microtubules, can be seen.

Mentions: To visualize microtubules in living yeast cells, fusions were constructed between the yeast α-tubulin genes and the Aequorea victoria GFP (Prasher et al., 1992; Chalfie et al., 1994; Stearns, 1995). Yeast has four tubulin genes: two α-tubulin (TUB1 and TUB3), one β-tubulin (TUB2), and one γ-tubulin (TUB4). The TUB1 and TUB2 genes are essential for viability, whereas strains deleted for TUB3 are viable but supersensitive to the microtubule-destabilizing drug, benomyl (Neff et al., 1983; Schatz et al., 1986). The α-tubulin genes were chosen for fusion to GFP because yeast cells are relatively insensitive to the level of expression of α-tubulin, whereas overexpression of β-tubulin is lethal (Burke et al., 1989; Katz et al., 1990; Weinstein and Solomon, 1990). TUB1 and TUB3 encode functionally identical α-tubulin proteins (Schatz et al., 1986). Fusions between GFP and the amino and carboxy terminus of TUB3 (GFP–TUB3 and TUB3–GFP, respectively) were constructed and placed under the control of the inducible GAL1 promoter (Fig. 1 A and Materials and Methods).


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

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

GFP–TUB3 produces fusion protein that incorporates  into microtubules. (A) Schematic showing the process by which  fluorescent microtubules are generated in vivo. The CEN plasmid carrying GFP–TUB3, pTS417, is transformed into the diploid  yeast strain, TPS507, and the resulting strain, TSY425, is then  grown in the presence of 2% galactose as the carbon source. After galactose induction, fluorescent microtubules are formed in  vivo. (B) Colocalization of GFP–TUB3 with α-tubulin in microtubules. After growth of TSY425 in 2% galactose, cells were  fixed and stained for α-tubulin. Fluorescence of rhodamine- labeled α-tubulin was compared with GFP fluorescence; both  α-tubulin and GFP colocalized to microtubule structures in unbudded and budded cells. Fluorescent cytoplasmic microtubules  and the spindle pole body are seen in unbudded cells (left),  whereas a fluorescent mitotic spindle is seen in the budded cell  shown (right). The right panel also contains an unbudded cell in  which GFP fluorescence of the spindle pole body, but not the microtubules, can be seen.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2141630&req=5

Figure 1: GFP–TUB3 produces fusion protein that incorporates into microtubules. (A) Schematic showing the process by which fluorescent microtubules are generated in vivo. The CEN plasmid carrying GFP–TUB3, pTS417, is transformed into the diploid yeast strain, TPS507, and the resulting strain, TSY425, is then grown in the presence of 2% galactose as the carbon source. After galactose induction, fluorescent microtubules are formed in vivo. (B) Colocalization of GFP–TUB3 with α-tubulin in microtubules. After growth of TSY425 in 2% galactose, cells were fixed and stained for α-tubulin. Fluorescence of rhodamine- labeled α-tubulin was compared with GFP fluorescence; both α-tubulin and GFP colocalized to microtubule structures in unbudded and budded cells. Fluorescent cytoplasmic microtubules and the spindle pole body are seen in unbudded cells (left), whereas a fluorescent mitotic spindle is seen in the budded cell shown (right). The right panel also contains an unbudded cell in which GFP fluorescence of the spindle pole body, but not the microtubules, can be seen.
Mentions: To visualize microtubules in living yeast cells, fusions were constructed between the yeast α-tubulin genes and the Aequorea victoria GFP (Prasher et al., 1992; Chalfie et al., 1994; Stearns, 1995). Yeast has four tubulin genes: two α-tubulin (TUB1 and TUB3), one β-tubulin (TUB2), and one γ-tubulin (TUB4). The TUB1 and TUB2 genes are essential for viability, whereas strains deleted for TUB3 are viable but supersensitive to the microtubule-destabilizing drug, benomyl (Neff et al., 1983; Schatz et al., 1986). The α-tubulin genes were chosen for fusion to GFP because yeast cells are relatively insensitive to the level of expression of α-tubulin, whereas overexpression of β-tubulin is lethal (Burke et al., 1989; Katz et al., 1990; Weinstein and Solomon, 1990). TUB1 and TUB3 encode functionally identical α-tubulin proteins (Schatz et al., 1986). Fusions between GFP and the amino and carboxy terminus of TUB3 (GFP–TUB3 and TUB3–GFP, respectively) were constructed and placed under the control of the inducible GAL1 promoter (Fig. 1 A and Materials and Methods).

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