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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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Pure recombinant XMAP215 depolymerizes CPP MTs in vitro. (A) Full-length XMAP215 and an NH2-terminal fragment of XMAP215 both depolymerize CPP MTs, but a COOH-terminal fragment does not. Rhodamine-labeled CPP MTs were incubated for 15 min in buffer containing different concentrations of full-length XMAP215 (F), an NH2-terminal fragment (N), or a COOH-terminal fragment (C). Shown here are representative fluorescence images for four concentrations of each protein. Bar, 10 μm. (B) Full-length XMAP215 and the NH2-terminal fragment have depolymerizing activity between 6.25 and 200 nM. MT polymer was quantitated for each sample by calculating average fluorescent pixel area per field for each protein concentration of full-length (F), NH2-terminal (N), and COOH-terminal (C) XMAP215. MT polymer is expressed as percent median value of buffer control; error bars denote 10th and 90th percentile; the 75th, 50th, and 25th percentiles are represented by the top, middle, and bottom of each box.
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fig4: Pure recombinant XMAP215 depolymerizes CPP MTs in vitro. (A) Full-length XMAP215 and an NH2-terminal fragment of XMAP215 both depolymerize CPP MTs, but a COOH-terminal fragment does not. Rhodamine-labeled CPP MTs were incubated for 15 min in buffer containing different concentrations of full-length XMAP215 (F), an NH2-terminal fragment (N), or a COOH-terminal fragment (C). Shown here are representative fluorescence images for four concentrations of each protein. Bar, 10 μm. (B) Full-length XMAP215 and the NH2-terminal fragment have depolymerizing activity between 6.25 and 200 nM. MT polymer was quantitated for each sample by calculating average fluorescent pixel area per field for each protein concentration of full-length (F), NH2-terminal (N), and COOH-terminal (C) XMAP215. MT polymer is expressed as percent median value of buffer control; error bars denote 10th and 90th percentile; the 75th, 50th, and 25th percentiles are represented by the top, middle, and bottom of each box.

Mentions: We next assayed pure, baculovirus-expressed XMAP215 for CPP MT–depolymerizing activity in vitro, using both full-length and truncated XMAP215 constructs previously characterized by Popov et al. (2001)(see Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200211095/DC1). Both full-length protein and an NH2-terminal fragment (aa 1–560) were able to depolymerize rhodamine-labeled CPP MTs in vitro (Fig. 4 A). In serial titrations, activity for both polypeptides was similar and measurable, beginning between 6.25 and 12.5 nM (Fig. 4 B). The full-length protein sample does contain a small amount of cleaved protein, so we cannot definitively rule out that this is not the active species in our assay; however, the majority of the protein is full-length. The NH2-terminal fragment does not appear to be significantly more potent than the full-length protein. A COOH-terminal fragment of XMAP215 (aa 1168–2065), on the other hand, was completely inactive in the depolymerization assay (Fig. 4, A and B). We measured depolymerizing activity in the visual assay by using fluorescent pixel area per visual field to quantitate MT polymer. Sedimentation assays and quantitation of tubulin in supernatants and pellets gave similar results (unpublished data). Samples with high concentrations of full-length XMAP215 (stoichiometric with tubulin, ∼200 nM) showed less depolymerization and highly bundled MTs (Fig. 4 A). This was also seen, to a lesser extent, in samples with very high concentrations of NH2-terminal fragment (unpublished data). The COOH-terminal fragment did not cause bundling at any concentration.


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)

Pure recombinant XMAP215 depolymerizes CPP MTs in vitro. (A) Full-length XMAP215 and an NH2-terminal fragment of XMAP215 both depolymerize CPP MTs, but a COOH-terminal fragment does not. Rhodamine-labeled CPP MTs were incubated for 15 min in buffer containing different concentrations of full-length XMAP215 (F), an NH2-terminal fragment (N), or a COOH-terminal fragment (C). Shown here are representative fluorescence images for four concentrations of each protein. Bar, 10 μm. (B) Full-length XMAP215 and the NH2-terminal fragment have depolymerizing activity between 6.25 and 200 nM. MT polymer was quantitated for each sample by calculating average fluorescent pixel area per field for each protein concentration of full-length (F), NH2-terminal (N), and COOH-terminal (C) XMAP215. MT polymer is expressed as percent median value of buffer control; error bars denote 10th and 90th percentile; the 75th, 50th, and 25th percentiles are represented by the top, middle, and bottom of each box.
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fig4: Pure recombinant XMAP215 depolymerizes CPP MTs in vitro. (A) Full-length XMAP215 and an NH2-terminal fragment of XMAP215 both depolymerize CPP MTs, but a COOH-terminal fragment does not. Rhodamine-labeled CPP MTs were incubated for 15 min in buffer containing different concentrations of full-length XMAP215 (F), an NH2-terminal fragment (N), or a COOH-terminal fragment (C). Shown here are representative fluorescence images for four concentrations of each protein. Bar, 10 μm. (B) Full-length XMAP215 and the NH2-terminal fragment have depolymerizing activity between 6.25 and 200 nM. MT polymer was quantitated for each sample by calculating average fluorescent pixel area per field for each protein concentration of full-length (F), NH2-terminal (N), and COOH-terminal (C) XMAP215. MT polymer is expressed as percent median value of buffer control; error bars denote 10th and 90th percentile; the 75th, 50th, and 25th percentiles are represented by the top, middle, and bottom of each box.
Mentions: We next assayed pure, baculovirus-expressed XMAP215 for CPP MT–depolymerizing activity in vitro, using both full-length and truncated XMAP215 constructs previously characterized by Popov et al. (2001)(see Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200211095/DC1). Both full-length protein and an NH2-terminal fragment (aa 1–560) were able to depolymerize rhodamine-labeled CPP MTs in vitro (Fig. 4 A). In serial titrations, activity for both polypeptides was similar and measurable, beginning between 6.25 and 12.5 nM (Fig. 4 B). The full-length protein sample does contain a small amount of cleaved protein, so we cannot definitively rule out that this is not the active species in our assay; however, the majority of the protein is full-length. The NH2-terminal fragment does not appear to be significantly more potent than the full-length protein. A COOH-terminal fragment of XMAP215 (aa 1168–2065), on the other hand, was completely inactive in the depolymerization assay (Fig. 4, A and B). We measured depolymerizing activity in the visual assay by using fluorescent pixel area per visual field to quantitate MT polymer. Sedimentation assays and quantitation of tubulin in supernatants and pellets gave similar results (unpublished data). Samples with high concentrations of full-length XMAP215 (stoichiometric with tubulin, ∼200 nM) showed less depolymerization and highly bundled MTs (Fig. 4 A). This was also seen, to a lesser extent, in samples with very high concentrations of NH2-terminal fragment (unpublished data). The COOH-terminal fragment did not cause bundling at any concentration.

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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