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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 expression and incorporation into functional microtubules. (A) Western blot of crude yeast protein extracts probed with anti–α-tubulin antibody. Strain TSY425, containing the GFP–TUB3 plasmid, and strain TPS510, containing  the vector plasmid, were grown in the presence of either 2% galactose, or 2% galactose + 0.5% glucose as the carbon source.  Both strains show endogenous total α-tubulin (TUB1p, TUB3p)  running as a doublet at 56 kD, while the GFP–TUB3 fusion protein runs at 83 kD. Similar results were obtained with a COOH-terminal fusion protein, TUB3–GFP (data not shown). (B) GFP– TUB3 expression rescues the benomyl supersensitivity of a tub3:: TRP1 haploid strain (DBY2375). (Top) Growth of tub3::TRP1  cells transformed with plasmids as noted above, and a 1:10 dilution of cells (second row), when grown with 2% galactose. (Bottom) Growth of the same strains, and 1:10 dilutions, in the presence of 5 μg/ml benomyl. All cells were incubated at 30°C. tub3:: TRP1 containing either vector alone, TUB3–GFP, or TUB1–GFP  remain benomyl supersensitive, while both TUB3 and GFP– TUB3 rescue the benomyl supersensitivity.
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Figure 2: GFP–TUB3 expression and incorporation into functional microtubules. (A) Western blot of crude yeast protein extracts probed with anti–α-tubulin antibody. Strain TSY425, containing the GFP–TUB3 plasmid, and strain TPS510, containing the vector plasmid, were grown in the presence of either 2% galactose, or 2% galactose + 0.5% glucose as the carbon source. Both strains show endogenous total α-tubulin (TUB1p, TUB3p) running as a doublet at 56 kD, while the GFP–TUB3 fusion protein runs at 83 kD. Similar results were obtained with a COOH-terminal fusion protein, TUB3–GFP (data not shown). (B) GFP– TUB3 expression rescues the benomyl supersensitivity of a tub3:: TRP1 haploid strain (DBY2375). (Top) Growth of tub3::TRP1 cells transformed with plasmids as noted above, and a 1:10 dilution of cells (second row), when grown with 2% galactose. (Bottom) Growth of the same strains, and 1:10 dilutions, in the presence of 5 μg/ml benomyl. All cells were incubated at 30°C. tub3:: TRP1 containing either vector alone, TUB3–GFP, or TUB1–GFP remain benomyl supersensitive, while both TUB3 and GFP– TUB3 rescue the benomyl supersensitivity.

Mentions: A dyn1::HIS3 disruption deletion vector was generously provided by M. Andrew Hoyt ( Johns Hopkins University, Baltimore, MD) (Geiser et al., 1997). The HIS3 deletion removes 1701 bp from an EcoRV to SacI site within the 3.5-kb R2 fragment as shown in Fig. 2 of Eshel et al., 1993. The EcoRV site is not shown and is upstream of the two HindIII sites used to make the URA3 disruption deletion. The vector was digested with EcoRI and transformed into the diploid strain, TPS507. Heterozygotes containing the dynein disruption integrated at the DYN1 locus were sporulated and dissected to obtain haploids of the opposite mating type. Haploids containing dyn1::HIS3 were mated, and the resulting diploid strain, TSY524, was transformed with GFP-TUB3 plasmid, pTS417, generating strain TSY526. A smaller deletion of sequences contained within the HIS3 disruption showed a similar phenotype to a larger 7-kb deletion allele, and thus probably acts as a allele (Li et al., 1993). Both strains TSY524 and TSY526 showed phenotypes consistent with previously published results.


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 expression and incorporation into functional microtubules. (A) Western blot of crude yeast protein extracts probed with anti–α-tubulin antibody. Strain TSY425, containing the GFP–TUB3 plasmid, and strain TPS510, containing  the vector plasmid, were grown in the presence of either 2% galactose, or 2% galactose + 0.5% glucose as the carbon source.  Both strains show endogenous total α-tubulin (TUB1p, TUB3p)  running as a doublet at 56 kD, while the GFP–TUB3 fusion protein runs at 83 kD. Similar results were obtained with a COOH-terminal fusion protein, TUB3–GFP (data not shown). (B) GFP– TUB3 expression rescues the benomyl supersensitivity of a tub3:: TRP1 haploid strain (DBY2375). (Top) Growth of tub3::TRP1  cells transformed with plasmids as noted above, and a 1:10 dilution of cells (second row), when grown with 2% galactose. (Bottom) Growth of the same strains, and 1:10 dilutions, in the presence of 5 μg/ml benomyl. All cells were incubated at 30°C. tub3:: TRP1 containing either vector alone, TUB3–GFP, or TUB1–GFP  remain benomyl supersensitive, while both TUB3 and GFP– TUB3 rescue the benomyl supersensitivity.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: GFP–TUB3 expression and incorporation into functional microtubules. (A) Western blot of crude yeast protein extracts probed with anti–α-tubulin antibody. Strain TSY425, containing the GFP–TUB3 plasmid, and strain TPS510, containing the vector plasmid, were grown in the presence of either 2% galactose, or 2% galactose + 0.5% glucose as the carbon source. Both strains show endogenous total α-tubulin (TUB1p, TUB3p) running as a doublet at 56 kD, while the GFP–TUB3 fusion protein runs at 83 kD. Similar results were obtained with a COOH-terminal fusion protein, TUB3–GFP (data not shown). (B) GFP– TUB3 expression rescues the benomyl supersensitivity of a tub3:: TRP1 haploid strain (DBY2375). (Top) Growth of tub3::TRP1 cells transformed with plasmids as noted above, and a 1:10 dilution of cells (second row), when grown with 2% galactose. (Bottom) Growth of the same strains, and 1:10 dilutions, in the presence of 5 μg/ml benomyl. All cells were incubated at 30°C. tub3:: TRP1 containing either vector alone, TUB3–GFP, or TUB1–GFP remain benomyl supersensitive, while both TUB3 and GFP– TUB3 rescue the benomyl supersensitivity.
Mentions: A dyn1::HIS3 disruption deletion vector was generously provided by M. Andrew Hoyt ( Johns Hopkins University, Baltimore, MD) (Geiser et al., 1997). The HIS3 deletion removes 1701 bp from an EcoRV to SacI site within the 3.5-kb R2 fragment as shown in Fig. 2 of Eshel et al., 1993. The EcoRV site is not shown and is upstream of the two HindIII sites used to make the URA3 disruption deletion. The vector was digested with EcoRI and transformed into the diploid strain, TPS507. Heterozygotes containing the dynein disruption integrated at the DYN1 locus were sporulated and dissected to obtain haploids of the opposite mating type. Haploids containing dyn1::HIS3 were mated, and the resulting diploid strain, TSY524, was transformed with GFP-TUB3 plasmid, pTS417, generating strain TSY526. A smaller deletion of sequences contained within the HIS3 disruption showed a similar phenotype to a larger 7-kb deletion allele, and thus probably acts as a allele (Li et al., 1993). Both strains TSY524 and TSY526 showed phenotypes consistent with previously published results.

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