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Identification of XMAP215 as a microtubule-destabilizing factor in Xenopus egg extract by biochemical purification.

Shirasu-Hiza M, Coughlin P, Mitchison T - J. Cell Biol. (2003)

Bottom Line: Consistent with the purification results, we find that XMAP215 is necessary for GMPCPP-MT destabilization in extracts and that recombinant full-length XMAP215 as well as an NH2-terminal fragment have depolymerizing activity in vitro.Stimulation of depolymerization is specific for the MT plus end.These results provide evidence for a robust MT-destabilizing activity intrinsic to this microtubule-associated protein and suggest that destabilization may be part of its essential biochemical functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, 250 Longwood Ave., Boston, MA 02115, USA. mshirasu@hms.harvard.edu

ABSTRACT
Microtubules (MTs) polymerized with GMPCPP, a slowly hydrolyzable GTP analogue, are stable in buffer but are rapidly depolymerized in Xenopus egg extracts. This depolymerization is independent of three previously identified MT destabilizers (Op18, katanin, and XKCM1/KinI). We purified the factor responsible for this novel depolymerizing activity using biochemical fractionation and a visual activity assay and identified it as XMAP215, previously identified as a prominent MT growth-promoting protein in Xenopus extracts. Consistent with the purification results, we find that XMAP215 is necessary for GMPCPP-MT destabilization in extracts and that recombinant full-length XMAP215 as well as an NH2-terminal fragment have depolymerizing activity in vitro. Stimulation of depolymerization is specific for the MT plus end. These results provide evidence for a robust MT-destabilizing activity intrinsic to this microtubule-associated protein and suggest that destabilization may be part of its essential biochemical functions. We propose that the substrate in our assay, GMPCPP-stabilized MTs, serves as a model for the pause state of MT ends and that the multiple activities of XMAP215 are unified by a mechanism of antagonizing MT pauses.

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XMAP215 contributes to CPP MT–depolymerizing activity in CSF extract. (A) Full-length XMAP215 copeaks with depolymerizing activity on a sucrose gradient. Sucrose gradient fractions were analyzed by Western blot with COOH-terminal–specific α-XMAP215 antibody. Active fractions are labeled with asterisks. (B) Crude extract was specifically depleted of full-length XMAP215, XKCM1, or both. Samples were depleted with random IgG, COOH-terminal α-XMAP215 antibody (ΔXMAP215), α-XKCM1 antibody (ΔXKCM1), or both α-XMAP215 and α-XKCM1 antibodies (Δboth). Western blots for the four major depolymerizers (XMAP215, XKCM1, katanin, and Op18) are shown for each condition. (C) Depletion of XMAP215 and XKCM1 from crude extract inhibited CPP MT depolymerization. Rhodamine-labeled CPP MTs were incubated in depleted extracts for 10 min. Representative fluorescence images of each sample are shown. Buffer used for negative control was CSF-XB (extract buffer used, see Materials and methods). Bar, 10 μm.
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fig3: XMAP215 contributes to CPP MT–depolymerizing activity in CSF extract. (A) Full-length XMAP215 copeaks with depolymerizing activity on a sucrose gradient. Sucrose gradient fractions were analyzed by Western blot with COOH-terminal–specific α-XMAP215 antibody. Active fractions are labeled with asterisks. (B) Crude extract was specifically depleted of full-length XMAP215, XKCM1, or both. Samples were depleted with random IgG, COOH-terminal α-XMAP215 antibody (ΔXMAP215), α-XKCM1 antibody (ΔXKCM1), or both α-XMAP215 and α-XKCM1 antibodies (Δboth). Western blots for the four major depolymerizers (XMAP215, XKCM1, katanin, and Op18) are shown for each condition. (C) Depletion of XMAP215 and XKCM1 from crude extract inhibited CPP MT depolymerization. Rhodamine-labeled CPP MTs were incubated in depleted extracts for 10 min. Representative fluorescence images of each sample are shown. Buffer used for negative control was CSF-XB (extract buffer used, see Materials and methods). Bar, 10 μm.

Mentions: We next investigated whether XMAP215 constituted a CPP MT–depolymerizing factor in CSF extracts. Though we had purified a set of NH2-terminal XMAP215 fragments from crude extract, we could not detect those fragments by Western blot in crude or clarified extract. XMAP215 appeared to exist as a full-length 215-kD species. This full-length XMAP215 comigrated with the 9.5S peak of depolymerizing activity we originally observed during sucrose gradient sedimentation of clarified extract (Fig. 3 A). It is not clear if we purified a rare, truncated species of XMAP215 that is highly active in our assay or if endogenous full-length protein was proteolyzed during the purification. The latter explanation seems likely as our depolymerizing factor decreased in sedimentation value from 9.5 to 6S (unpublished data) during the purification and as XMAP215 is known to be labile to a variety of nonspecific proteases in vitro (Gard, D., personal communication).


Identification of XMAP215 as a microtubule-destabilizing factor in Xenopus egg extract by biochemical purification.

Shirasu-Hiza M, Coughlin P, Mitchison T - J. Cell Biol. (2003)

XMAP215 contributes to CPP MT–depolymerizing activity in CSF extract. (A) Full-length XMAP215 copeaks with depolymerizing activity on a sucrose gradient. Sucrose gradient fractions were analyzed by Western blot with COOH-terminal–specific α-XMAP215 antibody. Active fractions are labeled with asterisks. (B) Crude extract was specifically depleted of full-length XMAP215, XKCM1, or both. Samples were depleted with random IgG, COOH-terminal α-XMAP215 antibody (ΔXMAP215), α-XKCM1 antibody (ΔXKCM1), or both α-XMAP215 and α-XKCM1 antibodies (Δboth). Western blots for the four major depolymerizers (XMAP215, XKCM1, katanin, and Op18) are shown for each condition. (C) Depletion of XMAP215 and XKCM1 from crude extract inhibited CPP MT depolymerization. Rhodamine-labeled CPP MTs were incubated in depleted extracts for 10 min. Representative fluorescence images of each sample are shown. Buffer used for negative control was CSF-XB (extract buffer used, see Materials and methods). Bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172913&req=5

fig3: XMAP215 contributes to CPP MT–depolymerizing activity in CSF extract. (A) Full-length XMAP215 copeaks with depolymerizing activity on a sucrose gradient. Sucrose gradient fractions were analyzed by Western blot with COOH-terminal–specific α-XMAP215 antibody. Active fractions are labeled with asterisks. (B) Crude extract was specifically depleted of full-length XMAP215, XKCM1, or both. Samples were depleted with random IgG, COOH-terminal α-XMAP215 antibody (ΔXMAP215), α-XKCM1 antibody (ΔXKCM1), or both α-XMAP215 and α-XKCM1 antibodies (Δboth). Western blots for the four major depolymerizers (XMAP215, XKCM1, katanin, and Op18) are shown for each condition. (C) Depletion of XMAP215 and XKCM1 from crude extract inhibited CPP MT depolymerization. Rhodamine-labeled CPP MTs were incubated in depleted extracts for 10 min. Representative fluorescence images of each sample are shown. Buffer used for negative control was CSF-XB (extract buffer used, see Materials and methods). Bar, 10 μm.
Mentions: We next investigated whether XMAP215 constituted a CPP MT–depolymerizing factor in CSF extracts. Though we had purified a set of NH2-terminal XMAP215 fragments from crude extract, we could not detect those fragments by Western blot in crude or clarified extract. XMAP215 appeared to exist as a full-length 215-kD species. This full-length XMAP215 comigrated with the 9.5S peak of depolymerizing activity we originally observed during sucrose gradient sedimentation of clarified extract (Fig. 3 A). It is not clear if we purified a rare, truncated species of XMAP215 that is highly active in our assay or if endogenous full-length protein was proteolyzed during the purification. The latter explanation seems likely as our depolymerizing factor decreased in sedimentation value from 9.5 to 6S (unpublished data) during the purification and as XMAP215 is known to be labile to a variety of nonspecific proteases in vitro (Gard, D., personal communication).

Bottom Line: Consistent with the purification results, we find that XMAP215 is necessary for GMPCPP-MT destabilization in extracts and that recombinant full-length XMAP215 as well as an NH2-terminal fragment have depolymerizing activity in vitro.Stimulation of depolymerization is specific for the MT plus end.These results provide evidence for a robust MT-destabilizing activity intrinsic to this microtubule-associated protein and suggest that destabilization may be part of its essential biochemical functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, 250 Longwood Ave., Boston, MA 02115, USA. mshirasu@hms.harvard.edu

ABSTRACT
Microtubules (MTs) polymerized with GMPCPP, a slowly hydrolyzable GTP analogue, are stable in buffer but are rapidly depolymerized in Xenopus egg extracts. This depolymerization is independent of three previously identified MT destabilizers (Op18, katanin, and XKCM1/KinI). We purified the factor responsible for this novel depolymerizing activity using biochemical fractionation and a visual activity assay and identified it as XMAP215, previously identified as a prominent MT growth-promoting protein in Xenopus extracts. Consistent with the purification results, we find that XMAP215 is necessary for GMPCPP-MT destabilization in extracts and that recombinant full-length XMAP215 as well as an NH2-terminal fragment have depolymerizing activity in vitro. Stimulation of depolymerization is specific for the MT plus end. These results provide evidence for a robust MT-destabilizing activity intrinsic to this microtubule-associated protein and suggest that destabilization may be part of its essential biochemical functions. We propose that the substrate in our assay, GMPCPP-stabilized MTs, serves as a model for the pause state of MT ends and that the multiple activities of XMAP215 are unified by a mechanism of antagonizing MT pauses.

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