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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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Model for the potential mechanism of XMAP215 as an antipause factor. Depicted here are the growing MT end (top) as a sheet-like structure of protofilament extensions and the shrinking MT end (bottom) with curled protofilaments (adapted from Miyamoto et al., 2002). The hypothetical paused MT end structure (middle) is positioned as an obligate intermediate between the two, drawn here with a blunt-ended, closed tube structure. We propose that XMAP215 destabilizes this pause state by weakening interprotofilament bonds and/or preventing tube closure, increasing transition to either the growing or shrinking state (arrows). Based on work by Cassimeris et al. (2001), we depict XMAP215 here as a long curved molecule that can bind protofilaments along their long axis.
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fig7: Model for the potential mechanism of XMAP215 as an antipause factor. Depicted here are the growing MT end (top) as a sheet-like structure of protofilament extensions and the shrinking MT end (bottom) with curled protofilaments (adapted from Miyamoto et al., 2002). The hypothetical paused MT end structure (middle) is positioned as an obligate intermediate between the two, drawn here with a blunt-ended, closed tube structure. We propose that XMAP215 destabilizes this pause state by weakening interprotofilament bonds and/or preventing tube closure, increasing transition to either the growing or shrinking state (arrows). Based on work by Cassimeris et al. (2001), we depict XMAP215 here as a long curved molecule that can bind protofilaments along their long axis.

Mentions: This interpretation of what the CPP lattice mimics prompts us to propose that XMAP215 destabilizes the pause state, acting as an antipause factor (Fig. 7) . MTs frequently pause in vivo, spending prolonged time neither growing nor shrinking at the resolution level of the light microscope (Shelden and Wadsworth, 1993; Tirnauer et al., 1999; Rusan et al., 2001). MTs also pause during phases of polymerization and depolymerization in Xenopus extracts (Tirnauer et al., 2002). Pauses are infrequent in reports of pure tubulin dynamics (Walker et al., 1988), but it is possible that pure MTs undergo micropauses too short to be detected by conventional imaging. In this pause state, MTs can theoretically transition into either growth or shrinkage, and a factor that destabilizes the pause state would increase MT dynamicity. Whether the MT transits to growth or shrinkage may depend on its environmental cues (tubulin concentration, other proteins, nucleotides, salt, or buffer); this would explain the apparently contradictory behavior of XMAP215 in different contexts. An antipause factor would also increase both apparent polymerization and depolymerization rates if polymerization and depolymerization were rate limited by micropauses. Higher resolution tracking of growing ends with pure tubulin could test this assumption. The antipause hypothesis could also account for the plus end specificity of XMAP215 if, for example, micropauses, corresponding to loss of protofilament extensions (Chretien et al., 1995), limit plus end growth and shrinkage more than minus end growth and shrinkage. Indeed, the pause model was introduced to account for different stabilities of the plus and minus ends (Tran et al., 1997).


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)

Model for the potential mechanism of XMAP215 as an antipause factor. Depicted here are the growing MT end (top) as a sheet-like structure of protofilament extensions and the shrinking MT end (bottom) with curled protofilaments (adapted from Miyamoto et al., 2002). The hypothetical paused MT end structure (middle) is positioned as an obligate intermediate between the two, drawn here with a blunt-ended, closed tube structure. We propose that XMAP215 destabilizes this pause state by weakening interprotofilament bonds and/or preventing tube closure, increasing transition to either the growing or shrinking state (arrows). Based on work by Cassimeris et al. (2001), we depict XMAP215 here as a long curved molecule that can bind protofilaments along their long axis.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172913&req=5

fig7: Model for the potential mechanism of XMAP215 as an antipause factor. Depicted here are the growing MT end (top) as a sheet-like structure of protofilament extensions and the shrinking MT end (bottom) with curled protofilaments (adapted from Miyamoto et al., 2002). The hypothetical paused MT end structure (middle) is positioned as an obligate intermediate between the two, drawn here with a blunt-ended, closed tube structure. We propose that XMAP215 destabilizes this pause state by weakening interprotofilament bonds and/or preventing tube closure, increasing transition to either the growing or shrinking state (arrows). Based on work by Cassimeris et al. (2001), we depict XMAP215 here as a long curved molecule that can bind protofilaments along their long axis.
Mentions: This interpretation of what the CPP lattice mimics prompts us to propose that XMAP215 destabilizes the pause state, acting as an antipause factor (Fig. 7) . MTs frequently pause in vivo, spending prolonged time neither growing nor shrinking at the resolution level of the light microscope (Shelden and Wadsworth, 1993; Tirnauer et al., 1999; Rusan et al., 2001). MTs also pause during phases of polymerization and depolymerization in Xenopus extracts (Tirnauer et al., 2002). Pauses are infrequent in reports of pure tubulin dynamics (Walker et al., 1988), but it is possible that pure MTs undergo micropauses too short to be detected by conventional imaging. In this pause state, MTs can theoretically transition into either growth or shrinkage, and a factor that destabilizes the pause state would increase MT dynamicity. Whether the MT transits to growth or shrinkage may depend on its environmental cues (tubulin concentration, other proteins, nucleotides, salt, or buffer); this would explain the apparently contradictory behavior of XMAP215 in different contexts. An antipause factor would also increase both apparent polymerization and depolymerization rates if polymerization and depolymerization were rate limited by micropauses. Higher resolution tracking of growing ends with pure tubulin could test this assumption. The antipause hypothesis could also account for the plus end specificity of XMAP215 if, for example, micropauses, corresponding to loss of protofilament extensions (Chretien et al., 1995), limit plus end growth and shrinkage more than minus end growth and shrinkage. Indeed, the pause model was introduced to account for different stabilities of the plus and minus ends (Tran et al., 1997).

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