β-Tubulin C354 Mutations that Severely Decrease Microtubule Dynamics Do Not Prevent Nuclear Migration in Yeast

Gupta, Mohan L.; Bode, Claudia J.; Thrower, Douglas; Pearson, Chad G.; Suprenant, Kathy A.; Bloom, Kerry; Himes, Richard H.

doi:10.1091/mbc.e02-01-0003

Cited by 65 publications

(85 citation statements)

References 55 publications

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“…The rate at which Kip1 nls--3GFP tracked cMTs during shrinkage was 2.860.2 mm/minute (n514) and during growth 1.260.1 mm/ minute (n515). These rates are comparable to those previously reported for shrinkage and growth rates of cMTs (Gupta et al, 2002;Gupta et al, 2006;Kosco et al, 2001;Tirnauer et al, 1999), indicating that nuclear factors are not required for the plus-end tracking activity of Kip1. We occasionally observed detachment of Kip1 nls--3GFP from the cMTs (data not shown) and found that, in general, Kip1 nls--3GFP tracking of the cMTs was less processive than that of endogenously expressed Kip1-3GFP (Fig.…”

Section: Journal Of Cell Sciencesupporting

confidence: 89%

Kinesin-5 Kip1 is a bi-directional motor that stabilizes microtubules and tracks their plus-ends in vivo

Fridman

Gerson-Gurwitz

Shapira

et al. 2013

Journal of Cell Science

122

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SummaryIn this study, we examined the anaphase functions of the S. cerevisiae kinesin-5 homolog Kip1. We show that Kip1 is attached to the mitotic spindle midzone during late anaphase. This attachment is essential to stabilize interpolar microtubule (iMTs) plus-ends. By detailed examination of iMT dynamics we show that at the end of anaphase, iMTs depolymerize in two stages: during the first stage, one pair of anti-parallel iMTs depolymerizes at a velocity of 7.7 mm/minute; during the second stage, ,90 seconds later, the remaining pair of iMTs depolymerizes at a slower velocity of 5.4 mm/minute. We show that upon the second depolymerization stage, which coincides with spindle breakdown, Kip1 follows the plus-ends of depolymerizing iMTs and translocates toward the spindle poles. This movement is independent of mitotic microtubule motor proteins or the major plus-end binding or tracking proteins. In addition, we show that Kip1 processively tracks the plus-ends of growing and shrinking MTs, both inside and outside the nucleus. The plus-end tracking activity of Kip1 requires its catalytic motor function, because a rigor mutant of Kip1 does not exhibit this activity. Finally, we show that Kip1 is a bi-directional motor: in vitro, at high ionic strength conditions, single Kip1 molecules move processively in the minus-end direction of the MTs, whereas in a multi-motor gliding assay, Kip1 is plus-end directed. The bi-directionality and plus-end tracking activity of Kip1, properties revealed here for the first time, allow Kip1 to perform its multiple functions in mitotic spindle dynamics and to partition the 2-micron plasmid.

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Section: Journal Of Cell Sciencesupporting

confidence: 89%

Kinesin-5 Kip1 is a bi-directional motor that stabilizes microtubules and tracks their plus-ends in vivo

Fridman

Gerson-Gurwitz

Shapira

et al. 2013

Journal of Cell Science

122

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“…Yeast tubulin was purified from the haploid strains MGY1 and MGY1-tax, both of which contain a His 6 tag on the C terminus of ␤-tubulin, to apparent homogeneity by using a His 6 -tag-based affinity purification procedure (26). Our published procedure has been modified slightly to include 100 mM NaCl in the DE52 absorption and washing steps instead of 160 mM, and 250 mM imidazole instead of 300 mM to elute the protein from the Ni-affinity column.…”

Section: Methodsmentioning

confidence: 99%

Understanding tubulin–Taxol interactions: Mutations that impart Taxol binding to yeast tubulin

Gupta

Bode

Georg

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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115

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We have successfully used mutagenesis to engineer Taxol (paclitaxel) binding activity in Saccharomyces cerevisiae tubulin. Taxol, a successful antitumor agent, acts by promoting tubulin assembly and stabilizing microtubules. Several structurally diverse antimitotic compounds, including the epothilones, compete with Taxol for binding to mammalian microtubules, suggesting that Taxol and these compounds share an overlapping binding site. However, Taxol has no effect on tubulin or microtubules from S. cerevisiae, whereas epothilone does. After considering data on Taxol binding to mammalian tubulin and recent modeling studies, we have hypothesized that differences in five key amino acids are responsible for the lack of Taxol binding to yeast tubulin. After changing these amino acids to those found in mammalian brain tubulin, we observed Taxol-related activity in yeast tubulin comparable to that in mammalian tubulin. Importantly, this experimental system can be used to reveal tubulin interactions with Taxol, the epothilones, and other Taxol-like compounds.

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“…This is an attractive possibility that appears to occur in yeast. Differences in tubulins cannot easily explain why microtubules in yeast cells are much more dynamic than those formed from purified yeast tubulin (18).…”

mentioning

confidence: 99%

Intrinsically Slow Dynamic Instability of HeLa Cell Microtubules in Vitro

Newton

DeLuca

Himes

et al. 2002

Journal of Biological Chemistry

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The dynamic behavior of mammalian microtubules has been extensively studied, both in living cells and with microtubules assembled from purified brain tubulin. To understand the intrinsic dynamic behavior of mammalian nonneural microtubules, we purified tubulin from cultured HeLa cells. We find that HeLa cell microtubules exhibit remarkably slow dynamic instability, spending most of their time in an attenuated state. The tempered dynamics contrast sharply with the dynamics of microtubules prepared from purified bovine brain tubulin under similar conditions. In accord with their minimal dynamic instability, assembled HeLa cell microtubules displayed a slow treadmilling rate and a low guanosine-5-triphosphate hydrolysis rate at steady state. We find that unlike brain tubulin, which consists of a heterogeneous mixture of ␤-tubulin isotypes (␤ II , ␤ III , and ␤ IV and a low level of ␤ I ), HeLa cell tubulin consists of ␤ I tubulin (ϳ80%) and a minor amount of ␤ IV tubulin (ϳ20%). The slow dynamic behavior of HeLa cell microtubules in vitro differs strikingly from the dynamic behavior of microtubules in living cultured mammalian cells, supporting the idea that accessory factors create the robust dynamics that occur in cells.

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β-Tubulin C354 Mutations that Severely Decrease Microtubule Dynamics Do Not Prevent Nuclear Migration in Yeast

Cited by 65 publications

References 55 publications

Kinesin-5 Kip1 is a bi-directional motor that stabilizes microtubules and tracks their plus-ends in vivo

Kinesin-5 Kip1 is a bi-directional motor that stabilizes microtubules and tracks their plus-ends in vivo

Understanding tubulin–Taxol interactions: Mutations that impart Taxol binding to yeast tubulin

Intrinsically Slow Dynamic Instability of HeLa Cell Microtubules in Vitro

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