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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 and B) Subcellular distribution of GFP-Num1p in living diploid cells. (A) Strain FMY519 (relevant genotype GAL1p-yEGFP3-NUM1/NUM1) was grown in YPD to early exponential growth and induced in YPGal + 0.3% glucose for 2–3 h. (B, upper row) CEN.PK2 cells containing pFM313 (NUM1p-yEGFP3-NUM1, CEN6/ARSH4) were grown in YPD to early exponential phase. (B, lower row) CEN.PK2 cells containing the pRS416 derivatives pFM314 (a, GFP fused to Num1p residues 1–421), pFM315 (b, GFP fused to Num1p residues 1–1876) and the empty vector pRS416 (c). (C and D) Costaining of microtubules in FMY519 cells by indirect immunofluorescence using antitubulin antibodies. (D, upper row) Merged images of GFP-Num1p and microtubules; lower row, DAPI-stained nuclear regions.
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Figure 2: (A and B) Subcellular distribution of GFP-Num1p in living diploid cells. (A) Strain FMY519 (relevant genotype GAL1p-yEGFP3-NUM1/NUM1) was grown in YPD to early exponential growth and induced in YPGal + 0.3% glucose for 2–3 h. (B, upper row) CEN.PK2 cells containing pFM313 (NUM1p-yEGFP3-NUM1, CEN6/ARSH4) were grown in YPD to early exponential phase. (B, lower row) CEN.PK2 cells containing the pRS416 derivatives pFM314 (a, GFP fused to Num1p residues 1–421), pFM315 (b, GFP fused to Num1p residues 1–1876) and the empty vector pRS416 (c). (C and D) Costaining of microtubules in FMY519 cells by indirect immunofluorescence using antitubulin antibodies. (D, upper row) Merged images of GFP-Num1p and microtubules; lower row, DAPI-stained nuclear regions.

Mentions: Fig. 2 A visualizes the cellular distribution of galactose-induced GFP-Num1p in living diploid cells. In addition to the previously noted dot-like Num1p distribution at the mother cortex, we observe prominent dots at the bud tip, the opposite pole of the mother compartment, and the bud neck, which were not visible in fixed NUM1 cells treated with anti-Num1p antibodies (Farkasovsky and Küntzel 1995). A similar distribution of GFP-Num1p was observed in galactose-grown haploid cells (data not shown). Furthermore, a yEGFP3-NUM1 fusion gene was placed under the control of the NUM1 promoter and introduced into the diploid wt strain CEN.PK2 as a centromeric plasmid (pFM313).


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

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

(A and B) Subcellular distribution of GFP-Num1p in living diploid cells. (A) Strain FMY519 (relevant genotype GAL1p-yEGFP3-NUM1/NUM1) was grown in YPD to early exponential growth and induced in YPGal + 0.3% glucose for 2–3 h. (B, upper row) CEN.PK2 cells containing pFM313 (NUM1p-yEGFP3-NUM1, CEN6/ARSH4) were grown in YPD to early exponential phase. (B, lower row) CEN.PK2 cells containing the pRS416 derivatives pFM314 (a, GFP fused to Num1p residues 1–421), pFM315 (b, GFP fused to Num1p residues 1–1876) and the empty vector pRS416 (c). (C and D) Costaining of microtubules in FMY519 cells by indirect immunofluorescence using antitubulin antibodies. (D, upper row) Merged images of GFP-Num1p and microtubules; lower row, DAPI-stained nuclear regions.
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Related In: Results  -  Collection

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Figure 2: (A and B) Subcellular distribution of GFP-Num1p in living diploid cells. (A) Strain FMY519 (relevant genotype GAL1p-yEGFP3-NUM1/NUM1) was grown in YPD to early exponential growth and induced in YPGal + 0.3% glucose for 2–3 h. (B, upper row) CEN.PK2 cells containing pFM313 (NUM1p-yEGFP3-NUM1, CEN6/ARSH4) were grown in YPD to early exponential phase. (B, lower row) CEN.PK2 cells containing the pRS416 derivatives pFM314 (a, GFP fused to Num1p residues 1–421), pFM315 (b, GFP fused to Num1p residues 1–1876) and the empty vector pRS416 (c). (C and D) Costaining of microtubules in FMY519 cells by indirect immunofluorescence using antitubulin antibodies. (D, upper row) Merged images of GFP-Num1p and microtubules; lower row, DAPI-stained nuclear regions.
Mentions: Fig. 2 A visualizes the cellular distribution of galactose-induced GFP-Num1p in living diploid cells. In addition to the previously noted dot-like Num1p distribution at the mother cortex, we observe prominent dots at the bud tip, the opposite pole of the mother compartment, and the bud neck, which were not visible in fixed NUM1 cells treated with anti-Num1p antibodies (Farkasovsky and Küntzel 1995). A similar distribution of GFP-Num1p was observed in galactose-grown haploid cells (data not shown). Furthermore, a yEGFP3-NUM1 fusion gene was placed under the control of the NUM1 promoter and introduced into the diploid wt strain CEN.PK2 as a centromeric plasmid (pFM313).

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.

Show MeSH
Related in: MedlinePlus