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MCAK associates with the tips of polymerizing microtubules.

Moore AT, Rankin KE, von Dassow G, Peris L, Wagenbach M, Ovechkina Y, Andrieux A, Job D, Wordeman L - J. Cell Biol. (2005)

Bottom Line: Tip tracking of MCAK is inhibited by phosphorylation and is dependent on the extreme COOH-terminal tail of MCAK.Tip tracking is not essential for MCAK's MT-depolymerizing activity.We propose that tip tracking is a mechanism by which MCAK is preferentially localized to regions of the cell that modulate the plus ends of MTs.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195, USA.

ABSTRACT
MCAK is a member of the kinesin-13 family of microtubule (MT)-depolymerizing kinesins. We show that the potent MT depolymerizer MCAK tracks (treadmills) with the tips of polymerizing MTs in living cells. Tip tracking of MCAK is inhibited by phosphorylation and is dependent on the extreme COOH-terminal tail of MCAK. Tip tracking is not essential for MCAK's MT-depolymerizing activity. We propose that tip tracking is a mechanism by which MCAK is preferentially localized to regions of the cell that modulate the plus ends of MTs.

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Tip tracking is positively correlated with apparent lattice affinity rather than tubulin affinity. (A) Wild-type MCAK binds MT lattice in the absence of ATP (left, lane 1). Excess free tubulin dimer can compete off 13–15% of lattice-associated wtMCAK (left, lanes 2 and 3). Phosphorylation of MCAK significantly decreases the apparent affinity of wtMCAK for stabilized MTs (right, lane 1). Excess free tubulin competes off a further 2–9% of lattice-bound phosphorylated MCAK (right lanes 2 and 3). (B) MCAK-Q710 binds MT lattice in the absence of ATP (left, lane 1). Excess tubulin dimer can compete off 30–45% of lattice-bound MCAK-Q710 (left, lanes 2 and 3). Phosphorylation of MCAK-Q710 decreases the apparent affinity of MCAK-Q710 for MT lattice to a similar extent as is seen with wtMCAK (right, lane 1). Excess free tubulin competes off a further 9–11% of lattice-bound phosphorylated MCAK-Q710 (right lanes 2 and 3).
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fig4: Tip tracking is positively correlated with apparent lattice affinity rather than tubulin affinity. (A) Wild-type MCAK binds MT lattice in the absence of ATP (left, lane 1). Excess free tubulin dimer can compete off 13–15% of lattice-associated wtMCAK (left, lanes 2 and 3). Phosphorylation of MCAK significantly decreases the apparent affinity of wtMCAK for stabilized MTs (right, lane 1). Excess free tubulin competes off a further 2–9% of lattice-bound phosphorylated MCAK (right lanes 2 and 3). (B) MCAK-Q710 binds MT lattice in the absence of ATP (left, lane 1). Excess tubulin dimer can compete off 30–45% of lattice-bound MCAK-Q710 (left, lanes 2 and 3). Phosphorylation of MCAK-Q710 decreases the apparent affinity of MCAK-Q710 for MT lattice to a similar extent as is seen with wtMCAK (right, lane 1). Excess free tubulin competes off a further 9–11% of lattice-bound phosphorylated MCAK-Q710 (right lanes 2 and 3).

Mentions: Presently, it is not known how proteins track with MT ends. Both copolymerization with tubulin and preferential affinity for MT end structures have been proposed. MCAK has been previously shown to possess an affinity for both tubulin and MT ends. To distinguish between these two mechanisms, we compared tip-tracking (wt-unphosphorylated) and nontip-tracking (wt-phosphorylated and Q710) MCAK proteins for their ability to be competed off MT lattice by free tubulin. If tubulin copolymerization were the mechanism by which MCAK preferentially associates with MT ends, then one might expect versions of MCAK that tip track would interact with free tubulin to a greater degree than versions that do not. We have previously shown that tubulin inhibits lattice association of MCAK (Moore and Wordeman, 2004a). We compared the extent to which unphosphorylated and Ipl1/Sli15-phosphorylated wild-type MCAK bind to assembled MTs, and the extent to which that binding is limited by excess tubulin monomer. The former tip tracks, whereas the latter presumably does not. Under conditions in which 100% of nonphosphorylated MCAK is bound to lattice, only about two thirds of Ipl1-phosphorylated MCAK is found in the pellet (Fig. 4 A, lanes 1). However, both unphosphorylated and phosphorylated MCAK can be competed off lattice to a similar extent by addition of excess free tubulin: 19% in the former case, and 13% (9% out of 68%) of the bound fraction in the latter case (Fig. 4 A, lanes 2). Therefore phosphorylation, which is expected to reduce tip tracking, changes the affinity of MCAK for lattice but not for free tubulin.


MCAK associates with the tips of polymerizing microtubules.

Moore AT, Rankin KE, von Dassow G, Peris L, Wagenbach M, Ovechkina Y, Andrieux A, Job D, Wordeman L - J. Cell Biol. (2005)

Tip tracking is positively correlated with apparent lattice affinity rather than tubulin affinity. (A) Wild-type MCAK binds MT lattice in the absence of ATP (left, lane 1). Excess free tubulin dimer can compete off 13–15% of lattice-associated wtMCAK (left, lanes 2 and 3). Phosphorylation of MCAK significantly decreases the apparent affinity of wtMCAK for stabilized MTs (right, lane 1). Excess free tubulin competes off a further 2–9% of lattice-bound phosphorylated MCAK (right lanes 2 and 3). (B) MCAK-Q710 binds MT lattice in the absence of ATP (left, lane 1). Excess tubulin dimer can compete off 30–45% of lattice-bound MCAK-Q710 (left, lanes 2 and 3). Phosphorylation of MCAK-Q710 decreases the apparent affinity of MCAK-Q710 for MT lattice to a similar extent as is seen with wtMCAK (right, lane 1). Excess free tubulin competes off a further 9–11% of lattice-bound phosphorylated MCAK-Q710 (right lanes 2 and 3).
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Related In: Results  -  Collection

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fig4: Tip tracking is positively correlated with apparent lattice affinity rather than tubulin affinity. (A) Wild-type MCAK binds MT lattice in the absence of ATP (left, lane 1). Excess free tubulin dimer can compete off 13–15% of lattice-associated wtMCAK (left, lanes 2 and 3). Phosphorylation of MCAK significantly decreases the apparent affinity of wtMCAK for stabilized MTs (right, lane 1). Excess free tubulin competes off a further 2–9% of lattice-bound phosphorylated MCAK (right lanes 2 and 3). (B) MCAK-Q710 binds MT lattice in the absence of ATP (left, lane 1). Excess tubulin dimer can compete off 30–45% of lattice-bound MCAK-Q710 (left, lanes 2 and 3). Phosphorylation of MCAK-Q710 decreases the apparent affinity of MCAK-Q710 for MT lattice to a similar extent as is seen with wtMCAK (right, lane 1). Excess free tubulin competes off a further 9–11% of lattice-bound phosphorylated MCAK-Q710 (right lanes 2 and 3).
Mentions: Presently, it is not known how proteins track with MT ends. Both copolymerization with tubulin and preferential affinity for MT end structures have been proposed. MCAK has been previously shown to possess an affinity for both tubulin and MT ends. To distinguish between these two mechanisms, we compared tip-tracking (wt-unphosphorylated) and nontip-tracking (wt-phosphorylated and Q710) MCAK proteins for their ability to be competed off MT lattice by free tubulin. If tubulin copolymerization were the mechanism by which MCAK preferentially associates with MT ends, then one might expect versions of MCAK that tip track would interact with free tubulin to a greater degree than versions that do not. We have previously shown that tubulin inhibits lattice association of MCAK (Moore and Wordeman, 2004a). We compared the extent to which unphosphorylated and Ipl1/Sli15-phosphorylated wild-type MCAK bind to assembled MTs, and the extent to which that binding is limited by excess tubulin monomer. The former tip tracks, whereas the latter presumably does not. Under conditions in which 100% of nonphosphorylated MCAK is bound to lattice, only about two thirds of Ipl1-phosphorylated MCAK is found in the pellet (Fig. 4 A, lanes 1). However, both unphosphorylated and phosphorylated MCAK can be competed off lattice to a similar extent by addition of excess free tubulin: 19% in the former case, and 13% (9% out of 68%) of the bound fraction in the latter case (Fig. 4 A, lanes 2). Therefore phosphorylation, which is expected to reduce tip tracking, changes the affinity of MCAK for lattice but not for free tubulin.

Bottom Line: Tip tracking of MCAK is inhibited by phosphorylation and is dependent on the extreme COOH-terminal tail of MCAK.Tip tracking is not essential for MCAK's MT-depolymerizing activity.We propose that tip tracking is a mechanism by which MCAK is preferentially localized to regions of the cell that modulate the plus ends of MTs.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195, USA.

ABSTRACT
MCAK is a member of the kinesin-13 family of microtubule (MT)-depolymerizing kinesins. We show that the potent MT depolymerizer MCAK tracks (treadmills) with the tips of polymerizing MTs in living cells. Tip tracking of MCAK is inhibited by phosphorylation and is dependent on the extreme COOH-terminal tail of MCAK. Tip tracking is not essential for MCAK's MT-depolymerizing activity. We propose that tip tracking is a mechanism by which MCAK is preferentially localized to regions of the cell that modulate the plus ends of MTs.

Show MeSH
Related in: MedlinePlus