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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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Covisualization of GFP-Num1p, 3xHA-Kar9p, and chromosomal DNA in (A) pre-anaphase, (B) mid anaphase, and (C) late anaphase/telophase cells. Strain FMY861 was grown in YPGal + 0.3% glucose at 30°C for 3 h. Formaldehyde-fixed cells were treated with anti-HA mAb and visualized by indirect immunofluorescence. Bar, 10 μm.
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Figure 9: Covisualization of GFP-Num1p, 3xHA-Kar9p, and chromosomal DNA in (A) pre-anaphase, (B) mid anaphase, and (C) late anaphase/telophase cells. Strain FMY861 was grown in YPGal + 0.3% glucose at 30°C for 3 h. Formaldehyde-fixed cells were treated with anti-HA mAb and visualized by indirect immunofluorescence. Bar, 10 μm.

Mentions: Fig. 9 depicts cells of a diploid strain (FMY861) coexpressing galactose-inducible yEGFP3-NUM1 and 3xHA-KAR9 fusion genes. Cells were grown in YPGal to early exponential phase and treated with anti-HA mAbs after fixation. DAPI was added to the mounting medium to stain chromosomal regions. Fig. 9, row A, shows a preanaphase cell containing a HA-Kar9p patch at the bud tip (lanes b and d), which apparently represents a cortical cMT/Kar9p capture site visualizing the plus-end of cMTs by microtubule-bound Kar9p (Miller et al. 1999). The same cell contains a GFP-Num1p patch at the bud tip, which is only partially resolved from the HA-Kar9p patch, as seen in the merged image of Fig. 9, lane d. Fig. 9, row B, shows a cell at early anaphase stage with a GFP-Num1p dot (lane a) and a cMT/HA-Kar9p capture site (lane b) at the bud tip. The merged image of Fig. 9, lane d, clearly resolves the two apparently adjacent cortical GFP-Num1p and HA-Kar9p dots and further demonstrates that the Kar9p-decorated cMTs are in contact with cortical HA-Kar9p but not with GFP-Num1p. HA-Kar9p and GFP-Num1p contact or near proximity was observed in 29% of early anaphase and 75% of mid anaphase cells with Kar9p localization at the bud cortex (dot or line). Anti–HA-stained HA-Kar9p has completely disappeared from the bud at the late anaphase/telophase stage (>90%), as seen in Fig. 9, row C, lane b, whereas GFP-Num1p persists at the bud tip (Fig. 9, lanes a and b).


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

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

Covisualization of GFP-Num1p, 3xHA-Kar9p, and chromosomal DNA in (A) pre-anaphase, (B) mid anaphase, and (C) late anaphase/telophase cells. Strain FMY861 was grown in YPGal + 0.3% glucose at 30°C for 3 h. Formaldehyde-fixed cells were treated with anti-HA mAb and visualized by indirect immunofluorescence. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 9: Covisualization of GFP-Num1p, 3xHA-Kar9p, and chromosomal DNA in (A) pre-anaphase, (B) mid anaphase, and (C) late anaphase/telophase cells. Strain FMY861 was grown in YPGal + 0.3% glucose at 30°C for 3 h. Formaldehyde-fixed cells were treated with anti-HA mAb and visualized by indirect immunofluorescence. Bar, 10 μm.
Mentions: Fig. 9 depicts cells of a diploid strain (FMY861) coexpressing galactose-inducible yEGFP3-NUM1 and 3xHA-KAR9 fusion genes. Cells were grown in YPGal to early exponential phase and treated with anti-HA mAbs after fixation. DAPI was added to the mounting medium to stain chromosomal regions. Fig. 9, row A, shows a preanaphase cell containing a HA-Kar9p patch at the bud tip (lanes b and d), which apparently represents a cortical cMT/Kar9p capture site visualizing the plus-end of cMTs by microtubule-bound Kar9p (Miller et al. 1999). The same cell contains a GFP-Num1p patch at the bud tip, which is only partially resolved from the HA-Kar9p patch, as seen in the merged image of Fig. 9, lane d. Fig. 9, row B, shows a cell at early anaphase stage with a GFP-Num1p dot (lane a) and a cMT/HA-Kar9p capture site (lane b) at the bud tip. The merged image of Fig. 9, lane d, clearly resolves the two apparently adjacent cortical GFP-Num1p and HA-Kar9p dots and further demonstrates that the Kar9p-decorated cMTs are in contact with cortical HA-Kar9p but not with GFP-Num1p. HA-Kar9p and GFP-Num1p contact or near proximity was observed in 29% of early anaphase and 75% of mid anaphase cells with Kar9p localization at the bud cortex (dot or line). Anti–HA-stained HA-Kar9p has completely disappeared from the bud at the late anaphase/telophase stage (>90%), as seen in Fig. 9, row C, lane b, whereas GFP-Num1p persists at the bud tip (Fig. 9, lanes a and b).

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