Limits...
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.

Show MeSH
Models to explain the presence of MCAK on polymerizing MT tips. (A) EB1 is capable of antagonizing modest levels of MCAK's depolymerizing activity. Cultured cells were transfected with GFP-EB1, RFP-MCAK, or both. Cells expressing low levels of MCAK have a significantly reduced amount of MT polymer relative to control cells (*, T = 0. 0042, P = 0.05). In contrast, there is no significant difference between control levels of MT polymer and low MCAK+EB1 (**, T = 0.4345, P = 0.05). Thus, at low levels of MCAK expression, overexpressed EB1 is capable of restoring bulk MT polymer to control levels. (B) Competition model: EB1 and MCAK compete for high affinity binding sites at the end of the MT. EB1 prevents MCAK from achieving a quorum of occupied protofilaments. (C) The NH2 and COOH terminus interact with MT ends, relieving the MCAK motor domain from inhibition by the COOH terminus and putting it in a state in which the motor becomes receptive to lattice interactions.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171944&req=5

fig5: Models to explain the presence of MCAK on polymerizing MT tips. (A) EB1 is capable of antagonizing modest levels of MCAK's depolymerizing activity. Cultured cells were transfected with GFP-EB1, RFP-MCAK, or both. Cells expressing low levels of MCAK have a significantly reduced amount of MT polymer relative to control cells (*, T = 0. 0042, P = 0.05). In contrast, there is no significant difference between control levels of MT polymer and low MCAK+EB1 (**, T = 0.4345, P = 0.05). Thus, at low levels of MCAK expression, overexpressed EB1 is capable of restoring bulk MT polymer to control levels. (B) Competition model: EB1 and MCAK compete for high affinity binding sites at the end of the MT. EB1 prevents MCAK from achieving a quorum of occupied protofilaments. (C) The NH2 and COOH terminus interact with MT ends, relieving the MCAK motor domain from inhibition by the COOH terminus and putting it in a state in which the motor becomes receptive to lattice interactions.

Mentions: A remaining question is why a MT tip that is enriched for bound MCAK would polymerize at all. MCAK's MT depolymerizing activity must be inhibited. We propose two models, which are not mutually exclusive, to explain the inhibition of MCAK on MT tips (Fig. 5). In the first case, the MT tip clearly associates with both MT stabilizers (such as EB1) and MT destabilizers (MCAK). MT stabilizers, such as EB1, may be capable of successfully competing with MCAK on MT tips by preventing MCAK from reaching a critical concentration at the end of the MT. We coexpressed EB1 and MCAK in live cells and found that excess EB1 can indeed antagonize the MT-depolymerizing activity of MCAK in living cells as long as the excess MCAK levels are relatively low (Fig. 5 A). We call this the competition model (Fig. 5 B). Another possibility is that MCAK's activity is inhibited during initial encounters with living MT ends. Because this binding site is independent of the motor domain, it presumably does not involve stabilizing a curved tubulin conformation. The COOH-terminal tail and NH2 terminus must prefer a binding site unique to the MT end (relative to lattice), such as the extreme end of a dimer (Fig. 5 C, higher affinity site). A subsequent conformational change would then bring the motor into contact with the lattice (Fig. 5 C, low affinity site). This model is also compatible with recruitment of MCAK to MT ends in an inactive form by EB1 (Mennella et al., 2005), although for simplicity we are assuming a direct association of MCAK with MTs. If this conformational change occurs further down on the lattice it will have a neutral effect on MT depolymerization. Thus, if the polymerization rate of MT ends exceeds that of the conformational change of the motor, depolymerization will not occur. It is important to note that the loss of tip tracking by phosphorylation of the NH2 terminus suggests that the NH2 terminus of MCAK is also necessary but not sufficient for tip tracking.


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)

Models to explain the presence of MCAK on polymerizing MT tips. (A) EB1 is capable of antagonizing modest levels of MCAK's depolymerizing activity. Cultured cells were transfected with GFP-EB1, RFP-MCAK, or both. Cells expressing low levels of MCAK have a significantly reduced amount of MT polymer relative to control cells (*, T = 0. 0042, P = 0.05). In contrast, there is no significant difference between control levels of MT polymer and low MCAK+EB1 (**, T = 0.4345, P = 0.05). Thus, at low levels of MCAK expression, overexpressed EB1 is capable of restoring bulk MT polymer to control levels. (B) Competition model: EB1 and MCAK compete for high affinity binding sites at the end of the MT. EB1 prevents MCAK from achieving a quorum of occupied protofilaments. (C) The NH2 and COOH terminus interact with MT ends, relieving the MCAK motor domain from inhibition by the COOH terminus and putting it in a state in which the motor becomes receptive to lattice interactions.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171944&req=5

fig5: Models to explain the presence of MCAK on polymerizing MT tips. (A) EB1 is capable of antagonizing modest levels of MCAK's depolymerizing activity. Cultured cells were transfected with GFP-EB1, RFP-MCAK, or both. Cells expressing low levels of MCAK have a significantly reduced amount of MT polymer relative to control cells (*, T = 0. 0042, P = 0.05). In contrast, there is no significant difference between control levels of MT polymer and low MCAK+EB1 (**, T = 0.4345, P = 0.05). Thus, at low levels of MCAK expression, overexpressed EB1 is capable of restoring bulk MT polymer to control levels. (B) Competition model: EB1 and MCAK compete for high affinity binding sites at the end of the MT. EB1 prevents MCAK from achieving a quorum of occupied protofilaments. (C) The NH2 and COOH terminus interact with MT ends, relieving the MCAK motor domain from inhibition by the COOH terminus and putting it in a state in which the motor becomes receptive to lattice interactions.
Mentions: A remaining question is why a MT tip that is enriched for bound MCAK would polymerize at all. MCAK's MT depolymerizing activity must be inhibited. We propose two models, which are not mutually exclusive, to explain the inhibition of MCAK on MT tips (Fig. 5). In the first case, the MT tip clearly associates with both MT stabilizers (such as EB1) and MT destabilizers (MCAK). MT stabilizers, such as EB1, may be capable of successfully competing with MCAK on MT tips by preventing MCAK from reaching a critical concentration at the end of the MT. We coexpressed EB1 and MCAK in live cells and found that excess EB1 can indeed antagonize the MT-depolymerizing activity of MCAK in living cells as long as the excess MCAK levels are relatively low (Fig. 5 A). We call this the competition model (Fig. 5 B). Another possibility is that MCAK's activity is inhibited during initial encounters with living MT ends. Because this binding site is independent of the motor domain, it presumably does not involve stabilizing a curved tubulin conformation. The COOH-terminal tail and NH2 terminus must prefer a binding site unique to the MT end (relative to lattice), such as the extreme end of a dimer (Fig. 5 C, higher affinity site). A subsequent conformational change would then bring the motor into contact with the lattice (Fig. 5 C, low affinity site). This model is also compatible with recruitment of MCAK to MT ends in an inactive form by EB1 (Mennella et al., 2005), although for simplicity we are assuming a direct association of MCAK with MTs. If this conformational change occurs further down on the lattice it will have a neutral effect on MT depolymerization. Thus, if the polymerization rate of MT ends exceeds that of the conformational change of the motor, depolymerization will not occur. It is important to note that the loss of tip tracking by phosphorylation of the NH2 terminus suggests that the NH2 terminus of MCAK is also necessary but not sufficient for tip tracking.

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