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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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Mechanism of CPP MT depolymerization by XMAP215. (A) Nocodazole does not depolymerize CPP MTs in our assay. Dimer sequestration was tested by incubating rhodamine-labeled CPP MTs with buffer plus DMSO or 20 μM nocodazole. (B) XMAP215 does not accelerate hydrolysis of GMPCPP. TLC was used to detect the hydrolysis of γ-32P–labeled GMPCPP in CPP MTs treated with buffer alone, full-length XMAP215, NH2-terminal fragment of XMAP215, or Na-BRB80/60% glycerol (positive control). Time points were taken from each reaction at 0, 30, and 60 min. [γ-32P]GMPCPP (CPP), [γ-32P]ATP (ATP), and 32Pi (Pi) are loaded as markers. Release of Pi is seen by the appearance of a second spot in Na-BRB80/60% glycerol, but not in XMAP215 or NH2-terminal XMAP215 samples. Microscopy assays run in parallel demonstrated that treatment with XMAP215 and the NH2-terminal construct depolymerized CPP MTs by the final time point (not depicted). (C) XMAP215 causes protofilament curling on MT ends. CPP MTs were incubated in buffer alone or buffer plus 38.5 nM full-length XMAP215 for 2 min and imaged by negative stain EM. Bars: (left) 200 nm; (right) 50 nm.
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fig6: Mechanism of CPP MT depolymerization by XMAP215. (A) Nocodazole does not depolymerize CPP MTs in our assay. Dimer sequestration was tested by incubating rhodamine-labeled CPP MTs with buffer plus DMSO or 20 μM nocodazole. (B) XMAP215 does not accelerate hydrolysis of GMPCPP. TLC was used to detect the hydrolysis of γ-32P–labeled GMPCPP in CPP MTs treated with buffer alone, full-length XMAP215, NH2-terminal fragment of XMAP215, or Na-BRB80/60% glycerol (positive control). Time points were taken from each reaction at 0, 30, and 60 min. [γ-32P]GMPCPP (CPP), [γ-32P]ATP (ATP), and 32Pi (Pi) are loaded as markers. Release of Pi is seen by the appearance of a second spot in Na-BRB80/60% glycerol, but not in XMAP215 or NH2-terminal XMAP215 samples. Microscopy assays run in parallel demonstrated that treatment with XMAP215 and the NH2-terminal construct depolymerized CPP MTs by the final time point (not depicted). (C) XMAP215 causes protofilament curling on MT ends. CPP MTs were incubated in buffer alone or buffer plus 38.5 nM full-length XMAP215 for 2 min and imaged by negative stain EM. Bars: (left) 200 nm; (right) 50 nm.

Mentions: To test if dimer sequestration accelerates apparent CPP MT depolymerization (by inhibiting readdition of subunits to MT ends), we added nocodazole to CPP MTs diluted in buffer alone (Fig. 6 A). The same concentration of nocodazole added before CPP MT polymerization completely inhibited polymerization (unpublished data). However, this potent monomer-sequestering drug did not stimulate depolymerization of CPP MTs in our assay, presumably because the total tubulin concentration is too low to allow significant readdition of dimer to MT ends. To test if GMPCPP was hydrolyzed during XMAP215-promoted depolymerization, we used MTs polymerized with [γ-32P]GMPCPP and separated from unbound nucleotides by sedimentation through a sucrose cushion. No hydrolysis was observed in buffer or XMAP215, though Na-BRB80/60% glycerol (a positive control; Caplow et al., 1994) did stimulate hydrolysis (Fig. 6 B).


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)

Mechanism of CPP MT depolymerization by XMAP215. (A) Nocodazole does not depolymerize CPP MTs in our assay. Dimer sequestration was tested by incubating rhodamine-labeled CPP MTs with buffer plus DMSO or 20 μM nocodazole. (B) XMAP215 does not accelerate hydrolysis of GMPCPP. TLC was used to detect the hydrolysis of γ-32P–labeled GMPCPP in CPP MTs treated with buffer alone, full-length XMAP215, NH2-terminal fragment of XMAP215, or Na-BRB80/60% glycerol (positive control). Time points were taken from each reaction at 0, 30, and 60 min. [γ-32P]GMPCPP (CPP), [γ-32P]ATP (ATP), and 32Pi (Pi) are loaded as markers. Release of Pi is seen by the appearance of a second spot in Na-BRB80/60% glycerol, but not in XMAP215 or NH2-terminal XMAP215 samples. Microscopy assays run in parallel demonstrated that treatment with XMAP215 and the NH2-terminal construct depolymerized CPP MTs by the final time point (not depicted). (C) XMAP215 causes protofilament curling on MT ends. CPP MTs were incubated in buffer alone or buffer plus 38.5 nM full-length XMAP215 for 2 min and imaged by negative stain EM. Bars: (left) 200 nm; (right) 50 nm.
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Related In: Results  -  Collection

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fig6: Mechanism of CPP MT depolymerization by XMAP215. (A) Nocodazole does not depolymerize CPP MTs in our assay. Dimer sequestration was tested by incubating rhodamine-labeled CPP MTs with buffer plus DMSO or 20 μM nocodazole. (B) XMAP215 does not accelerate hydrolysis of GMPCPP. TLC was used to detect the hydrolysis of γ-32P–labeled GMPCPP in CPP MTs treated with buffer alone, full-length XMAP215, NH2-terminal fragment of XMAP215, or Na-BRB80/60% glycerol (positive control). Time points were taken from each reaction at 0, 30, and 60 min. [γ-32P]GMPCPP (CPP), [γ-32P]ATP (ATP), and 32Pi (Pi) are loaded as markers. Release of Pi is seen by the appearance of a second spot in Na-BRB80/60% glycerol, but not in XMAP215 or NH2-terminal XMAP215 samples. Microscopy assays run in parallel demonstrated that treatment with XMAP215 and the NH2-terminal construct depolymerized CPP MTs by the final time point (not depicted). (C) XMAP215 causes protofilament curling on MT ends. CPP MTs were incubated in buffer alone or buffer plus 38.5 nM full-length XMAP215 for 2 min and imaged by negative stain EM. Bars: (left) 200 nm; (right) 50 nm.
Mentions: To test if dimer sequestration accelerates apparent CPP MT depolymerization (by inhibiting readdition of subunits to MT ends), we added nocodazole to CPP MTs diluted in buffer alone (Fig. 6 A). The same concentration of nocodazole added before CPP MT polymerization completely inhibited polymerization (unpublished data). However, this potent monomer-sequestering drug did not stimulate depolymerization of CPP MTs in our assay, presumably because the total tubulin concentration is too low to allow significant readdition of dimer to MT ends. To test if GMPCPP was hydrolyzed during XMAP215-promoted depolymerization, we used MTs polymerized with [γ-32P]GMPCPP and separated from unbound nucleotides by sedimentation through a sucrose cushion. No hydrolysis was observed in buffer or XMAP215, though Na-BRB80/60% glycerol (a positive control; Caplow et al., 1994) did stimulate hydrolysis (Fig. 6 B).

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.

Show MeSH
Related in: MedlinePlus