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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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Subcellular distribution of GFP-Num1p in diploid strains lacking Bni1p (A, FMY795), Kar9p (B, FMY838), or Dyn1p (C, FMY872). (D and E) show diploid cells expressing galactose-induced yEGFP3-NUM1 (FMY519) in the absence (D) or presence (E) of 20 μg/ml nocodazole. Microtubules were visualized by indirect immunofluorescence using antitubulin antibodies (middle panel of rows D and E), and nuclear regions were stained with DAPI (rows A–E, right).
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Figure 8: Subcellular distribution of GFP-Num1p in diploid strains lacking Bni1p (A, FMY795), Kar9p (B, FMY838), or Dyn1p (C, FMY872). (D and E) show diploid cells expressing galactose-induced yEGFP3-NUM1 (FMY519) in the absence (D) or presence (E) of 20 μg/ml nocodazole. Microtubules were visualized by indirect immunofluorescence using antitubulin antibodies (middle panel of rows D and E), and nuclear regions were stained with DAPI (rows A–E, right).

Mentions: Num1p may transiently coexist or even cooperate with a Bni1p–Kar9p complex at the bud tip, although the two cortical cMT capture sites appear to operate at different stages of the cell cycle (see Discussion). To test this possibility, we have studied the influence of Bni1p, Kar9p, and Dyn1p on the bud tip localization of GFP-Num1p. The two Bni1p-deficient diploid cells (FMY795) shown in Fig. 8 A contain a prominent GFP-Num1p dot at a lateral region of the bud cortex, in addition to several dots at the mother cortex, whereas a Num1p dot at the bud tip is clearly missing in virtually all observed budded cells. In contrast, a prominent GFP-Num1p dot is seen at the bud tip of cells lacking either Kar9p (Fig. 8 B) or Dyn1p (Fig. 8 C).


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

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

Subcellular distribution of GFP-Num1p in diploid strains lacking Bni1p (A, FMY795), Kar9p (B, FMY838), or Dyn1p (C, FMY872). (D and E) show diploid cells expressing galactose-induced yEGFP3-NUM1 (FMY519) in the absence (D) or presence (E) of 20 μg/ml nocodazole. Microtubules were visualized by indirect immunofluorescence using antitubulin antibodies (middle panel of rows D and E), and nuclear regions were stained with DAPI (rows A–E, right).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199608&req=5

Figure 8: Subcellular distribution of GFP-Num1p in diploid strains lacking Bni1p (A, FMY795), Kar9p (B, FMY838), or Dyn1p (C, FMY872). (D and E) show diploid cells expressing galactose-induced yEGFP3-NUM1 (FMY519) in the absence (D) or presence (E) of 20 μg/ml nocodazole. Microtubules were visualized by indirect immunofluorescence using antitubulin antibodies (middle panel of rows D and E), and nuclear regions were stained with DAPI (rows A–E, right).
Mentions: Num1p may transiently coexist or even cooperate with a Bni1p–Kar9p complex at the bud tip, although the two cortical cMT capture sites appear to operate at different stages of the cell cycle (see Discussion). To test this possibility, we have studied the influence of Bni1p, Kar9p, and Dyn1p on the bud tip localization of GFP-Num1p. The two Bni1p-deficient diploid cells (FMY795) shown in Fig. 8 A contain a prominent GFP-Num1p dot at a lateral region of the bud cortex, in addition to several dots at the mother cortex, whereas a Num1p dot at the bud tip is clearly missing in virtually all observed budded cells. In contrast, a prominent GFP-Num1p dot is seen at the bud tip of cells lacking either Kar9p (Fig. 8 B) or Dyn1p (Fig. 8 C).

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