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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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MCAK exhibits tip tracking behavior in living cells. (A) GFP-MCAK can be detected at the ends of MTs in an interphase HeLa cell. (B) Kymograph of tips traveling from the centrosomal region to the edge of the cell. (C) RFP-MCAK roughly colocalizes with GFP-EB1 in live HeLa cells cotransfected with both constructs.
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fig1: MCAK exhibits tip tracking behavior in living cells. (A) GFP-MCAK can be detected at the ends of MTs in an interphase HeLa cell. (B) Kymograph of tips traveling from the centrosomal region to the edge of the cell. (C) RFP-MCAK roughly colocalizes with GFP-EB1 in live HeLa cells cotransfected with both constructs.

Mentions: When fluorescent MCAK is expressed in live cultured cells at levels that do not significantly alter MT polymer levels, MCAK can be detected on MT lattice and in obvious densities on MT tips (Fig. 1 A). Time-lapse imaging shows tips polymerizing toward the cell edge (tracking) in a manner similar to that which has been previously reported for GFP-EB1 (Fig. 1 B; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200411089/DC1). In living HeLa cells the MCAK tip densities are coincident with GFP-EB1 (Fig. 1 C). Tracking of MCAK to MT tips is not dependent on a functional motor domain, as it can be seen clearly in cells transfected with GFP-ML-MCAK (Fig. 2 B; Video 3), a construct in which the 50-kD motor domain has been removed and the NH2-terminal domain of MCAK is fused directly to the COOH-terminal domain (Maney et al., 1998). Tracking tips of GFP-ML-MCAK are consistently longer than those seen with GFP-wt-MCAK (Fig. 2, A′ and B′), suggesting that the motor may influence the off-rate from the lattice. Another mutant of MCAK that contains three point mutations in the motor domain rendering the protein inactive with respect to depolymerizing activity (GFP-hypir-MCAK) also exhibits tip tracking (Fig. 2 C, Table I; Video 4). Tip localization and tracking is seen in both interphase and mitotic cells. This is a striking observation because MCAK has been previously shown to be a potent depolymerizer of MTs (Desai et al., 1999; Hunter et al., 2003). Therefore, the presence of MCAK on the tips of polymerizing MTs in interphase and mitotic cells suggests that MCAK's depolymerizing activity is transiently inhibited on tips.


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)

MCAK exhibits tip tracking behavior in living cells. (A) GFP-MCAK can be detected at the ends of MTs in an interphase HeLa cell. (B) Kymograph of tips traveling from the centrosomal region to the edge of the cell. (C) RFP-MCAK roughly colocalizes with GFP-EB1 in live HeLa cells cotransfected with both constructs.
© Copyright Policy
Related In: Results  -  Collection

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fig1: MCAK exhibits tip tracking behavior in living cells. (A) GFP-MCAK can be detected at the ends of MTs in an interphase HeLa cell. (B) Kymograph of tips traveling from the centrosomal region to the edge of the cell. (C) RFP-MCAK roughly colocalizes with GFP-EB1 in live HeLa cells cotransfected with both constructs.
Mentions: When fluorescent MCAK is expressed in live cultured cells at levels that do not significantly alter MT polymer levels, MCAK can be detected on MT lattice and in obvious densities on MT tips (Fig. 1 A). Time-lapse imaging shows tips polymerizing toward the cell edge (tracking) in a manner similar to that which has been previously reported for GFP-EB1 (Fig. 1 B; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200411089/DC1). In living HeLa cells the MCAK tip densities are coincident with GFP-EB1 (Fig. 1 C). Tracking of MCAK to MT tips is not dependent on a functional motor domain, as it can be seen clearly in cells transfected with GFP-ML-MCAK (Fig. 2 B; Video 3), a construct in which the 50-kD motor domain has been removed and the NH2-terminal domain of MCAK is fused directly to the COOH-terminal domain (Maney et al., 1998). Tracking tips of GFP-ML-MCAK are consistently longer than those seen with GFP-wt-MCAK (Fig. 2, A′ and B′), suggesting that the motor may influence the off-rate from the lattice. Another mutant of MCAK that contains three point mutations in the motor domain rendering the protein inactive with respect to depolymerizing activity (GFP-hypir-MCAK) also exhibits tip tracking (Fig. 2 C, Table I; Video 4). Tip localization and tracking is seen in both interphase and mitotic cells. This is a striking observation because MCAK has been previously shown to be a potent depolymerizer of MTs (Desai et al., 1999; Hunter et al., 2003). Therefore, the presence of MCAK on the tips of polymerizing MTs in interphase and mitotic cells suggests that MCAK's depolymerizing activity is transiently inhibited on tips.

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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