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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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Nuclear migration defects in  mutant cells. Haploid MATa cells were grown at 30°C to OD600 = 1.2 in YPD, fixed, and stained with DAPI, and 250–300 large-budded cells were scored for the position of nuclear regions. Strains: FMY691 (WT), FMY693 (num1Δ), FMY705 (kar9Δ), FMY711 (bni1Δ), FMY859 (dyn1Δ), FMY856 (arp1Δ), FMY848 (kip2Δ), FMY852 (kip3Δ), FMY799 (num1Δ kar9Δ), FMY821 (num1Δ bni1Δ), FMY789 (num1Δ dyn1Δ), FMY793 (num1Δ arp1Δ), FMY850 (num1Δ kip2Δ), and FMY854 (num1Δ kip3Δ). D includes cells with three or more nuclei in the mother compartment. >80% of the num1Δ kar9Δ, num1Δ bni1Δ, and num1Δ kip3Δ cells listed under D are multinucleate and multibudded.
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Figure 5: Nuclear migration defects in mutant cells. Haploid MATa cells were grown at 30°C to OD600 = 1.2 in YPD, fixed, and stained with DAPI, and 250–300 large-budded cells were scored for the position of nuclear regions. Strains: FMY691 (WT), FMY693 (num1Δ), FMY705 (kar9Δ), FMY711 (bni1Δ), FMY859 (dyn1Δ), FMY856 (arp1Δ), FMY848 (kip2Δ), FMY852 (kip3Δ), FMY799 (num1Δ kar9Δ), FMY821 (num1Δ bni1Δ), FMY789 (num1Δ dyn1Δ), FMY793 (num1Δ arp1Δ), FMY850 (num1Δ kip2Δ), and FMY854 (num1Δ kip3Δ). D includes cells with three or more nuclei in the mother compartment. >80% of the num1Δ kar9Δ, num1Δ bni1Δ, and num1Δ kip3Δ cells listed under D are multinucleate and multibudded.

Mentions: The three double mutants num1Δ kar9Δ, num1Δ bni1Δ, and num1Δ kip3Δ exhibit additive defects in nuclear migration through the bud neck, as summarized in Fig. 5: the percentage of large-budded cells with more than one DAPI spot in the mother compartment (“binucleate mother cells”) is increased in comparison to the single mutants. In contrast, the num1Δ combinations with dyn1Δ, arp1Δ, and kip2Δ are not significantly affected. Furthermore, the double mutants num1Δ kar9Δ and num1Δ kip3Δ grow slowly at 30°C and arrest at 15°C, whereas all other single and double mutants, including num1Δ bni1Δ, grow like wild type at both temperatures.


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

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

Nuclear migration defects in  mutant cells. Haploid MATa cells were grown at 30°C to OD600 = 1.2 in YPD, fixed, and stained with DAPI, and 250–300 large-budded cells were scored for the position of nuclear regions. Strains: FMY691 (WT), FMY693 (num1Δ), FMY705 (kar9Δ), FMY711 (bni1Δ), FMY859 (dyn1Δ), FMY856 (arp1Δ), FMY848 (kip2Δ), FMY852 (kip3Δ), FMY799 (num1Δ kar9Δ), FMY821 (num1Δ bni1Δ), FMY789 (num1Δ dyn1Δ), FMY793 (num1Δ arp1Δ), FMY850 (num1Δ kip2Δ), and FMY854 (num1Δ kip3Δ). D includes cells with three or more nuclei in the mother compartment. >80% of the num1Δ kar9Δ, num1Δ bni1Δ, and num1Δ kip3Δ cells listed under D are multinucleate and multibudded.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199608&req=5

Figure 5: Nuclear migration defects in mutant cells. Haploid MATa cells were grown at 30°C to OD600 = 1.2 in YPD, fixed, and stained with DAPI, and 250–300 large-budded cells were scored for the position of nuclear regions. Strains: FMY691 (WT), FMY693 (num1Δ), FMY705 (kar9Δ), FMY711 (bni1Δ), FMY859 (dyn1Δ), FMY856 (arp1Δ), FMY848 (kip2Δ), FMY852 (kip3Δ), FMY799 (num1Δ kar9Δ), FMY821 (num1Δ bni1Δ), FMY789 (num1Δ dyn1Δ), FMY793 (num1Δ arp1Δ), FMY850 (num1Δ kip2Δ), and FMY854 (num1Δ kip3Δ). D includes cells with three or more nuclei in the mother compartment. >80% of the num1Δ kar9Δ, num1Δ bni1Δ, and num1Δ kip3Δ cells listed under D are multinucleate and multibudded.
Mentions: The three double mutants num1Δ kar9Δ, num1Δ bni1Δ, and num1Δ kip3Δ exhibit additive defects in nuclear migration through the bud neck, as summarized in Fig. 5: the percentage of large-budded cells with more than one DAPI spot in the mother compartment (“binucleate mother cells”) is increased in comparison to the single mutants. In contrast, the num1Δ combinations with dyn1Δ, arp1Δ, and kip2Δ are not significantly affected. Furthermore, the double mutants num1Δ kar9Δ and num1Δ kip3Δ grow slowly at 30°C and arrest at 15°C, whereas all other single and double mutants, including num1Δ bni1Δ, grow like wild type at both temperatures.

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