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Cortical Num1p interacts with the dynein intermediate chain Pac11p and cytoplasmic microtubules in budding yeast.

Farkasovsky M, Küntzel H - J. Cell Biol. (2001)

Bottom Line: The bud tip location of Num1p depends on Bni1p but does not require Kar9p, Dyn1p, or cMTs, whereas cMT contacts with polar Num1p dots are reduced in cells lacking Dyn1p.Num1p also forms a complex with Bni1p and Kar9p, although Num1p is not required for Bni1p- and Kar9p-dependent nuclear migration to the bud neck in preanaphase cells.In addition, Num1p may transiently cooperate with an associated Bni1p-Kar9p complex at the bud tip of early anaphase cells.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck Institute for Experimental Medicine, D-37075 Göttingen, Germany.

ABSTRACT
Num1p, a cortical 313-kD protein, controls cytoplasmic microtubule (cMT) functions and nuclear migration through the bud neck in anaphase cells. A green fluorescent protein (GFP)-Num1p fusion protein localizes at the bud tip and the distal mother pole of living cells, apparently forming cMT capture sites at late anaphase. In addition, galactose-induced GFP-Num1p is seen at the bud neck and in lateral regions of the mother cortex. The bud tip location of Num1p depends on Bni1p but does not require Kar9p, Dyn1p, or cMTs, whereas cMT contacts with polar Num1p dots are reduced in cells lacking Dyn1p. Num1p associates with the dynein intermediate chain Pac11p in the presence of Dyn1p, and with the alpha-tubulin Tub3p, as shown by coimmune precipitation of tagged proteins. Num1p also forms a complex with Bni1p and Kar9p, although Num1p is not required for Bni1p- and Kar9p-dependent nuclear migration to the bud neck in preanaphase cells. Our data suggest that Num1p controls nuclear migration during late anaphase by forming dynein-interacting cortical cMT capture sites at both cellular poles. In addition, Num1p may transiently cooperate with an associated Bni1p-Kar9p complex at the bud tip of early anaphase cells.

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(A) cMT orientation in MATa mutant cells containing two DAPI-stained chromosomal regions in the mother compartment. Strains FMY691 (WT), FMY705 (kar9Δ), FMY859 (dyn1Δ), and FMY693 (num1Δ) were grown to late exponential phase at 30°C and fixed for indirect immunofluorescence using antitubulin antibodies. 120–150 binucleate cells per mutant strain were scored for cMT orientation. (B) Nuclear position in preanaphase MATa wild-type and mutant cells. Strains: FMY691 (WT), FMY693 (num1Δ), FMY705 (kar9Δ), FMY859 (dyn1Δ), FMY799 (num1Δ kar9Δ), FMY789 (num1Δ dyn1Δ). Strains were grown to early exponential phase at 30°C, and 200–250 DAPI-stained preanaphase cells (single nucleus in the mother) were scored for each strain.
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Figure 6: (A) cMT orientation in MATa mutant cells containing two DAPI-stained chromosomal regions in the mother compartment. Strains FMY691 (WT), FMY705 (kar9Δ), FMY859 (dyn1Δ), and FMY693 (num1Δ) were grown to late exponential phase at 30°C and fixed for indirect immunofluorescence using antitubulin antibodies. 120–150 binucleate cells per mutant strain were scored for cMT orientation. (B) Nuclear position in preanaphase MATa wild-type and mutant cells. Strains: FMY691 (WT), FMY693 (num1Δ), FMY705 (kar9Δ), FMY859 (dyn1Δ), FMY799 (num1Δ kar9Δ), FMY789 (num1Δ dyn1Δ). Strains were grown to early exponential phase at 30°C, and 200–250 DAPI-stained preanaphase cells (single nucleus in the mother) were scored for each strain.

Mentions: Fig. 6 A quantifies the distribution of binucleate mother cells with differing cMT morphologies in num1Δ, dyn1Δ, and kar9Δ strains, as revealed by indirect immunofluorescence of cells treated with antitubulin antibodies and DAPI. Approximately 80% of num1Δ and dyn1Δ cells of this nuclear phenotype contain long cMTs traversing the bud neck, as previously noted (Farkasovsky and Küntzel 1995; Carminati and Stearns 1997; Miller et al. 1998), whereas cMTs do not enter the bud in the majority of kar9Δ binucleate mother cells (Miller and Rose 1998). It is interesting to note that bni1Δ mutants are much more related to num1Δ or dyn1Δ mutants than to kar9Δ or bim1Δ mutants according the cMT phenotype of binucleate mother cells: cMTs extend into the bud in 79–90% of binucleate bni1Δ cells (Fujiwara et al. 1999; Miller et al. 1999).


Cortical Num1p interacts with the dynein intermediate chain Pac11p and cytoplasmic microtubules in budding yeast.

Farkasovsky M, Küntzel H - J. Cell Biol. (2001)

(A) cMT orientation in MATa mutant cells containing two DAPI-stained chromosomal regions in the mother compartment. Strains FMY691 (WT), FMY705 (kar9Δ), FMY859 (dyn1Δ), and FMY693 (num1Δ) were grown to late exponential phase at 30°C and fixed for indirect immunofluorescence using antitubulin antibodies. 120–150 binucleate cells per mutant strain were scored for cMT orientation. (B) Nuclear position in preanaphase MATa wild-type and mutant cells. Strains: FMY691 (WT), FMY693 (num1Δ), FMY705 (kar9Δ), FMY859 (dyn1Δ), FMY799 (num1Δ kar9Δ), FMY789 (num1Δ dyn1Δ). Strains were grown to early exponential phase at 30°C, and 200–250 DAPI-stained preanaphase cells (single nucleus in the mother) were scored for each strain.
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Figure 6: (A) cMT orientation in MATa mutant cells containing two DAPI-stained chromosomal regions in the mother compartment. Strains FMY691 (WT), FMY705 (kar9Δ), FMY859 (dyn1Δ), and FMY693 (num1Δ) were grown to late exponential phase at 30°C and fixed for indirect immunofluorescence using antitubulin antibodies. 120–150 binucleate cells per mutant strain were scored for cMT orientation. (B) Nuclear position in preanaphase MATa wild-type and mutant cells. Strains: FMY691 (WT), FMY693 (num1Δ), FMY705 (kar9Δ), FMY859 (dyn1Δ), FMY799 (num1Δ kar9Δ), FMY789 (num1Δ dyn1Δ). Strains were grown to early exponential phase at 30°C, and 200–250 DAPI-stained preanaphase cells (single nucleus in the mother) were scored for each strain.
Mentions: Fig. 6 A quantifies the distribution of binucleate mother cells with differing cMT morphologies in num1Δ, dyn1Δ, and kar9Δ strains, as revealed by indirect immunofluorescence of cells treated with antitubulin antibodies and DAPI. Approximately 80% of num1Δ and dyn1Δ cells of this nuclear phenotype contain long cMTs traversing the bud neck, as previously noted (Farkasovsky and Küntzel 1995; Carminati and Stearns 1997; Miller et al. 1998), whereas cMTs do not enter the bud in the majority of kar9Δ binucleate mother cells (Miller and Rose 1998). It is interesting to note that bni1Δ mutants are much more related to num1Δ or dyn1Δ mutants than to kar9Δ or bim1Δ mutants according the cMT phenotype of binucleate mother cells: cMTs extend into the bud in 79–90% of binucleate bni1Δ cells (Fujiwara et al. 1999; Miller et al. 1999).

Bottom Line: The bud tip location of Num1p depends on Bni1p but does not require Kar9p, Dyn1p, or cMTs, whereas cMT contacts with polar Num1p dots are reduced in cells lacking Dyn1p.Num1p also forms a complex with Bni1p and Kar9p, although Num1p is not required for Bni1p- and Kar9p-dependent nuclear migration to the bud neck in preanaphase cells.In addition, Num1p may transiently cooperate with an associated Bni1p-Kar9p complex at the bud tip of early anaphase cells.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck Institute for Experimental Medicine, D-37075 Göttingen, Germany.

ABSTRACT
Num1p, a cortical 313-kD protein, controls cytoplasmic microtubule (cMT) functions and nuclear migration through the bud neck in anaphase cells. A green fluorescent protein (GFP)-Num1p fusion protein localizes at the bud tip and the distal mother pole of living cells, apparently forming cMT capture sites at late anaphase. In addition, galactose-induced GFP-Num1p is seen at the bud neck and in lateral regions of the mother cortex. The bud tip location of Num1p depends on Bni1p but does not require Kar9p, Dyn1p, or cMTs, whereas cMT contacts with polar Num1p dots are reduced in cells lacking Dyn1p. Num1p associates with the dynein intermediate chain Pac11p in the presence of Dyn1p, and with the alpha-tubulin Tub3p, as shown by coimmune precipitation of tagged proteins. Num1p also forms a complex with Bni1p and Kar9p, although Num1p is not required for Bni1p- and Kar9p-dependent nuclear migration to the bud neck in preanaphase cells. Our data suggest that Num1p controls nuclear migration during late anaphase by forming dynein-interacting cortical cMT capture sites at both cellular poles. In addition, Num1p may transiently cooperate with an associated Bni1p-Kar9p complex at the bud tip of early anaphase cells.

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Related in: MedlinePlus