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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 of MCAK protein is dependent on phosphorylation. (A) GFP-AAAAA-MCAK binds to MT tips (left), whereas GFP-EEEEE-MCAK is not found on tips (right). See also Videos 4 and 5 (available at http://www.jcb.org/cgi/content/full/jcb.200411089/DC1). (B) Two fields of CHO cells labeled for endogenous MCAK (B) and MTs (B′). The top field of CHO cells are control cells. The bottom field of CHO cells were cultured for 5 h in 10 μM roscovitine and then fixed. Increased association of endogenous MCAK with distal ends of MTs is evident (B, bottom; arrows). (C) MCAK protein tracks on tips (arrows) in a HeLa cell transfected with RFP-MCAK and cultured subsequently in 10 μM roscovitine (Video 6).
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fig3: Tip tracking of MCAK protein is dependent on phosphorylation. (A) GFP-AAAAA-MCAK binds to MT tips (left), whereas GFP-EEEEE-MCAK is not found on tips (right). See also Videos 4 and 5 (available at http://www.jcb.org/cgi/content/full/jcb.200411089/DC1). (B) Two fields of CHO cells labeled for endogenous MCAK (B) and MTs (B′). The top field of CHO cells are control cells. The bottom field of CHO cells were cultured for 5 h in 10 μM roscovitine and then fixed. Increased association of endogenous MCAK with distal ends of MTs is evident (B, bottom; arrows). (C) MCAK protein tracks on tips (arrows) in a HeLa cell transfected with RFP-MCAK and cultured subsequently in 10 μM roscovitine (Video 6).

Mentions: Because phosphorylation has been invoked as a mechanism for inactivating MCAK's depolymerizing activity (Andrews et al., 2004; Lan et al., 2004; Ohi et al., 2004), we tested whether tip-associated MCAK was inhibited by phosphorylation. We found that our more active (Fig. S1 A) phospho-mutant of MCAK (GFP-AAAAA-MCAK) exhibited robust tip tracking (Table III, Fig. 3 A; Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200411089/DC1). In this mutant all of the sites known to be phosphorylated by Aurora B kinase have been mutated to alanine (Andrews et al., 2004). In contrast, a less active (Fig. 3 A) phospho-mimic mutant of MCAK (GFP-EEEEE-MCAK) showed no tip tracking (Table III, Fig. 3 A; Video 6). The fact that active GFP-AAAAA-MCAK tip tracks suggests that phosphorylation is not the means by which MCAK is inhibited on tips of polymerizing MTs.


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 of MCAK protein is dependent on phosphorylation. (A) GFP-AAAAA-MCAK binds to MT tips (left), whereas GFP-EEEEE-MCAK is not found on tips (right). See also Videos 4 and 5 (available at http://www.jcb.org/cgi/content/full/jcb.200411089/DC1). (B) Two fields of CHO cells labeled for endogenous MCAK (B) and MTs (B′). The top field of CHO cells are control cells. The bottom field of CHO cells were cultured for 5 h in 10 μM roscovitine and then fixed. Increased association of endogenous MCAK with distal ends of MTs is evident (B, bottom; arrows). (C) MCAK protein tracks on tips (arrows) in a HeLa cell transfected with RFP-MCAK and cultured subsequently in 10 μM roscovitine (Video 6).
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fig3: Tip tracking of MCAK protein is dependent on phosphorylation. (A) GFP-AAAAA-MCAK binds to MT tips (left), whereas GFP-EEEEE-MCAK is not found on tips (right). See also Videos 4 and 5 (available at http://www.jcb.org/cgi/content/full/jcb.200411089/DC1). (B) Two fields of CHO cells labeled for endogenous MCAK (B) and MTs (B′). The top field of CHO cells are control cells. The bottom field of CHO cells were cultured for 5 h in 10 μM roscovitine and then fixed. Increased association of endogenous MCAK with distal ends of MTs is evident (B, bottom; arrows). (C) MCAK protein tracks on tips (arrows) in a HeLa cell transfected with RFP-MCAK and cultured subsequently in 10 μM roscovitine (Video 6).
Mentions: Because phosphorylation has been invoked as a mechanism for inactivating MCAK's depolymerizing activity (Andrews et al., 2004; Lan et al., 2004; Ohi et al., 2004), we tested whether tip-associated MCAK was inhibited by phosphorylation. We found that our more active (Fig. S1 A) phospho-mutant of MCAK (GFP-AAAAA-MCAK) exhibited robust tip tracking (Table III, Fig. 3 A; Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200411089/DC1). In this mutant all of the sites known to be phosphorylated by Aurora B kinase have been mutated to alanine (Andrews et al., 2004). In contrast, a less active (Fig. 3 A) phospho-mimic mutant of MCAK (GFP-EEEEE-MCAK) showed no tip tracking (Table III, Fig. 3 A; Video 6). The fact that active GFP-AAAAA-MCAK tip tracks suggests that phosphorylation is not the means by which MCAK is inhibited on tips of polymerizing MTs.

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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