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Astral microtubule dynamics in yeast: a microtubule-based searching mechanism for spindle orientation and nuclear migration into the bud.

Shaw SL, Yeh E, Maddox P, Salmon ED, Bloom K - J. Cell Biol. (1997)

Bottom Line: These forays were evident in unbudded G1 cells, as well as in late telophase cells after spindle disassembly.Stable microtubule interactions with the cell cortex were rarely observed during anaphase, and did not appear to contribute significantly to spindle alignment or elongation into the bud.These studies provide the first mechanistic basis for understanding how spindle orientation and nuclear positioning are established and are indicative of a microtubule-based searching mechanism that requires dynamic microtubules for nuclear migration into the bud.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3280, USA.

ABSTRACT
Localization of dynein-green fluorescent protein (GFP) to cytoplasmic microtubules allowed us to obtain one of the first views of the dynamic properties of astral microtubules in live budding yeast. Several novel aspects of microtubule function were revealed by time-lapse, three-dimensional fluorescence microscopy. Astral microtubules, about four to six in number for each pole, exhibited asynchronous dynamic instability throughout the cell cycle, growing at approximately 0.3-1.5 micron/min toward the cell surface then switching to shortening at similar velocities back to the spindle pole body (SPB). During interphase, a conical array of microtubules trailed the SPB as the nucleus traversed the cytoplasm. Microtubule disassembly by nocodozole inhibited these movements, indicating that the nucleus was pushed around the interior of the cell via dynamic astral microtubules. These forays were evident in unbudded G1 cells, as well as in late telophase cells after spindle disassembly. Nuclear movement and orientation to the bud neck in S/G2 or G2/M was dependent on dynamic astral microtubules growing into the bud. The SPB and nucleus were then pulled toward the bud neck, and further microtubule growth from that SPB was mainly oriented toward the bud. After SPB separation and central spindle formation, a temporal delay in the acquisition of cytoplasmic dynein at one of the spindle poles was evident. Stable microtubule interactions with the cell cortex were rarely observed during anaphase, and did not appear to contribute significantly to spindle alignment or elongation into the bud. Alterations of microtubule dynamics, as observed in cells overexpressing dynein-GFP, resulted in eventual spindle misalignment. These studies provide the first mechanistic basis for understanding how spindle orientation and nuclear positioning are established and are indicative of a microtubule-based searching mechanism that requires dynamic microtubules for nuclear migration into the bud.

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Related in: MedlinePlus

Construction of the  dynein-GFP–containing plasmid, pKBY701. The parent plasmid is based on a high copy, galactose-inducible shuttle vector previously described (Baldari et  al., 1987) that contains the 2-μm origin of replication, URA3, and  Leu2-d. Mitchell et al. (1993) modified the vector for the conditional expression of GST fusions in S. cerevisiae. We inserted the  dynein heavy chain gene (11,333-bp NheI fragment, amino acids  303–4,081) into the XbaI site of pEGKG. The resulting plasmid,  pJU1 contains a galactose-inducible (black), GST (blue)/dynein  (gray) fusion protein. The S65T derivative of GFP (green) (716  bp) was fused to the COOH terminus of dynein, at the unique  SalI site generating the plasmid pKBY701 (22.3 kbp).
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Figure 1: Construction of the dynein-GFP–containing plasmid, pKBY701. The parent plasmid is based on a high copy, galactose-inducible shuttle vector previously described (Baldari et al., 1987) that contains the 2-μm origin of replication, URA3, and Leu2-d. Mitchell et al. (1993) modified the vector for the conditional expression of GST fusions in S. cerevisiae. We inserted the dynein heavy chain gene (11,333-bp NheI fragment, amino acids 303–4,081) into the XbaI site of pEGKG. The resulting plasmid, pJU1 contains a galactose-inducible (black), GST (blue)/dynein (gray) fusion protein. The S65T derivative of GFP (green) (716 bp) was fused to the COOH terminus of dynein, at the unique SalI site generating the plasmid pKBY701 (22.3 kbp).

Mentions: All strains used in this study were derived from strain 15C (Mata, leu2-3, 112, ura3-52, his4-580, trp1Δ, pep4-3) and MAY591 (Matα, leu2-3,112, lys2-801, his3-200, ura3-52) (Saunders and Hoyt, 1992). Haploid derivatives 8d (Mata, lys2-801, trp1Δ, ura3-52, leu2-3,112) and 9d (Matα, lys2-801, his3-200, ura3-52, leu2-3,112) were mated to generate the diploid strain (8d×9d) (Mata/α, lys2/lys2, ura3/ura3, leu2/leu2, his3/+, +/trp1). A complete deletion of the 12,276-bp dynein heavy chain was obtained by fragment-mediated transformation into 9dΔdhc (dhc1::LEU2). The GAL1–glutathione-S-transferase (GST)–dynein-GFP–containing plasmid (pKBY701; see Fig. 1) was introduced into 9d, 9dΔdhc, or the diploid, 8d×9d by standard transformation.


Astral microtubule dynamics in yeast: a microtubule-based searching mechanism for spindle orientation and nuclear migration into the bud.

Shaw SL, Yeh E, Maddox P, Salmon ED, Bloom K - J. Cell Biol. (1997)

Construction of the  dynein-GFP–containing plasmid, pKBY701. The parent plasmid is based on a high copy, galactose-inducible shuttle vector previously described (Baldari et  al., 1987) that contains the 2-μm origin of replication, URA3, and  Leu2-d. Mitchell et al. (1993) modified the vector for the conditional expression of GST fusions in S. cerevisiae. We inserted the  dynein heavy chain gene (11,333-bp NheI fragment, amino acids  303–4,081) into the XbaI site of pEGKG. The resulting plasmid,  pJU1 contains a galactose-inducible (black), GST (blue)/dynein  (gray) fusion protein. The S65T derivative of GFP (green) (716  bp) was fused to the COOH terminus of dynein, at the unique  SalI site generating the plasmid pKBY701 (22.3 kbp).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139970&req=5

Figure 1: Construction of the dynein-GFP–containing plasmid, pKBY701. The parent plasmid is based on a high copy, galactose-inducible shuttle vector previously described (Baldari et al., 1987) that contains the 2-μm origin of replication, URA3, and Leu2-d. Mitchell et al. (1993) modified the vector for the conditional expression of GST fusions in S. cerevisiae. We inserted the dynein heavy chain gene (11,333-bp NheI fragment, amino acids 303–4,081) into the XbaI site of pEGKG. The resulting plasmid, pJU1 contains a galactose-inducible (black), GST (blue)/dynein (gray) fusion protein. The S65T derivative of GFP (green) (716 bp) was fused to the COOH terminus of dynein, at the unique SalI site generating the plasmid pKBY701 (22.3 kbp).
Mentions: All strains used in this study were derived from strain 15C (Mata, leu2-3, 112, ura3-52, his4-580, trp1Δ, pep4-3) and MAY591 (Matα, leu2-3,112, lys2-801, his3-200, ura3-52) (Saunders and Hoyt, 1992). Haploid derivatives 8d (Mata, lys2-801, trp1Δ, ura3-52, leu2-3,112) and 9d (Matα, lys2-801, his3-200, ura3-52, leu2-3,112) were mated to generate the diploid strain (8d×9d) (Mata/α, lys2/lys2, ura3/ura3, leu2/leu2, his3/+, +/trp1). A complete deletion of the 12,276-bp dynein heavy chain was obtained by fragment-mediated transformation into 9dΔdhc (dhc1::LEU2). The GAL1–glutathione-S-transferase (GST)–dynein-GFP–containing plasmid (pKBY701; see Fig. 1) was introduced into 9d, 9dΔdhc, or the diploid, 8d×9d by standard transformation.

Bottom Line: These forays were evident in unbudded G1 cells, as well as in late telophase cells after spindle disassembly.Stable microtubule interactions with the cell cortex were rarely observed during anaphase, and did not appear to contribute significantly to spindle alignment or elongation into the bud.These studies provide the first mechanistic basis for understanding how spindle orientation and nuclear positioning are established and are indicative of a microtubule-based searching mechanism that requires dynamic microtubules for nuclear migration into the bud.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3280, USA.

ABSTRACT
Localization of dynein-green fluorescent protein (GFP) to cytoplasmic microtubules allowed us to obtain one of the first views of the dynamic properties of astral microtubules in live budding yeast. Several novel aspects of microtubule function were revealed by time-lapse, three-dimensional fluorescence microscopy. Astral microtubules, about four to six in number for each pole, exhibited asynchronous dynamic instability throughout the cell cycle, growing at approximately 0.3-1.5 micron/min toward the cell surface then switching to shortening at similar velocities back to the spindle pole body (SPB). During interphase, a conical array of microtubules trailed the SPB as the nucleus traversed the cytoplasm. Microtubule disassembly by nocodozole inhibited these movements, indicating that the nucleus was pushed around the interior of the cell via dynamic astral microtubules. These forays were evident in unbudded G1 cells, as well as in late telophase cells after spindle disassembly. Nuclear movement and orientation to the bud neck in S/G2 or G2/M was dependent on dynamic astral microtubules growing into the bud. The SPB and nucleus were then pulled toward the bud neck, and further microtubule growth from that SPB was mainly oriented toward the bud. After SPB separation and central spindle formation, a temporal delay in the acquisition of cytoplasmic dynein at one of the spindle poles was evident. Stable microtubule interactions with the cell cortex were rarely observed during anaphase, and did not appear to contribute significantly to spindle alignment or elongation into the bud. Alterations of microtubule dynamics, as observed in cells overexpressing dynein-GFP, resulted in eventual spindle misalignment. These studies provide the first mechanistic basis for understanding how spindle orientation and nuclear positioning are established and are indicative of a microtubule-based searching mechanism that requires dynamic microtubules for nuclear migration into the bud.

Show MeSH
Related in: MedlinePlus