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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 promotes CPP MT depolymerization at MT plus ends. (A) XMAP215 promotes end-dependent CPP MT depolymerization. Shown here are images from a time-lapse series of dim-bright CPP MTs (see Materials and methods) treated with buffer alone or buffer plus 19 nM XMAP215 (interval between still images is 40 s). In each sample, kinesin motility was used to determine MT polarity; translocation of the MT from left to right represents minus end leading and plus end lagging. In the XMAP215-treated sample, the plus end shortens while the minus end remains stable (see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200211095/DC1). (B) Depolymerization by XMAP215 is specific to MT plus ends. Depolymerization rates were quantitated for each MT end from experiments as in A. Error bars denote 10th and 90th percentile; 75th, 50th, and 25th percentiles are represented by the top, middle, and bottom of each box.
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fig5: XMAP215 promotes CPP MT depolymerization at MT plus ends. (A) XMAP215 promotes end-dependent CPP MT depolymerization. Shown here are images from a time-lapse series of dim-bright CPP MTs (see Materials and methods) treated with buffer alone or buffer plus 19 nM XMAP215 (interval between still images is 40 s). In each sample, kinesin motility was used to determine MT polarity; translocation of the MT from left to right represents minus end leading and plus end lagging. In the XMAP215-treated sample, the plus end shortens while the minus end remains stable (see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200211095/DC1). (B) Depolymerization by XMAP215 is specific to MT plus ends. Depolymerization rates were quantitated for each MT end from experiments as in A. Error bars denote 10th and 90th percentile; 75th, 50th, and 25th percentiles are represented by the top, middle, and bottom of each box.

Mentions: To test if XMAP215 promotes CPP MT depolymerization by an end-dependent mechanism, we recorded depolymerization live in glass flow-cells using time-lapse fluorescence microscopy. Rhodamine-labeled CPP MTs were bound to glass using kinesin and then treated with buffer or buffer containing XMAP215. In buffer alone, MTs were relatively stable for 30 min; in the presence of 19 nM XMAP215, they depolymerized over several minutes in an endwise fashion (Fig. 5 A). There was a strong polarity bias to depolymerization. We used dim-bright CPP MTs and kinesin motility to determine that XMAP215 depolymerized the MT plus end at a rate 5–10 times faster than buffer alone, whereas minus end depolymerization was not measurably affected (Fig. 5 B). In the presence of XMAP215, 92 out of 95 MTs (96.8%) had faster rates of depolymerization on their lagging (plus) ends than on their leading (minus) ends. Thus, XMAP215 specifically promotes CPP MT depolymerization at plus ends (see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200211095/DC1). Its polymerization-promoting activity is also plus end specific (Gard and Kirschner, 1987; Vasquez et al., 1994).


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 promotes CPP MT depolymerization at MT plus ends. (A) XMAP215 promotes end-dependent CPP MT depolymerization. Shown here are images from a time-lapse series of dim-bright CPP MTs (see Materials and methods) treated with buffer alone or buffer plus 19 nM XMAP215 (interval between still images is 40 s). In each sample, kinesin motility was used to determine MT polarity; translocation of the MT from left to right represents minus end leading and plus end lagging. In the XMAP215-treated sample, the plus end shortens while the minus end remains stable (see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200211095/DC1). (B) Depolymerization by XMAP215 is specific to MT plus ends. Depolymerization rates were quantitated for each MT end from experiments as in A. Error bars denote 10th and 90th percentile; 75th, 50th, and 25th percentiles are represented by the top, middle, and bottom of each box.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172913&req=5

fig5: XMAP215 promotes CPP MT depolymerization at MT plus ends. (A) XMAP215 promotes end-dependent CPP MT depolymerization. Shown here are images from a time-lapse series of dim-bright CPP MTs (see Materials and methods) treated with buffer alone or buffer plus 19 nM XMAP215 (interval between still images is 40 s). In each sample, kinesin motility was used to determine MT polarity; translocation of the MT from left to right represents minus end leading and plus end lagging. In the XMAP215-treated sample, the plus end shortens while the minus end remains stable (see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200211095/DC1). (B) Depolymerization by XMAP215 is specific to MT plus ends. Depolymerization rates were quantitated for each MT end from experiments as in A. Error bars denote 10th and 90th percentile; 75th, 50th, and 25th percentiles are represented by the top, middle, and bottom of each box.
Mentions: To test if XMAP215 promotes CPP MT depolymerization by an end-dependent mechanism, we recorded depolymerization live in glass flow-cells using time-lapse fluorescence microscopy. Rhodamine-labeled CPP MTs were bound to glass using kinesin and then treated with buffer or buffer containing XMAP215. In buffer alone, MTs were relatively stable for 30 min; in the presence of 19 nM XMAP215, they depolymerized over several minutes in an endwise fashion (Fig. 5 A). There was a strong polarity bias to depolymerization. We used dim-bright CPP MTs and kinesin motility to determine that XMAP215 depolymerized the MT plus end at a rate 5–10 times faster than buffer alone, whereas minus end depolymerization was not measurably affected (Fig. 5 B). In the presence of XMAP215, 92 out of 95 MTs (96.8%) had faster rates of depolymerization on their lagging (plus) ends than on their leading (minus) ends. Thus, XMAP215 specifically promotes CPP MT depolymerization at plus ends (see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200211095/DC1). Its polymerization-promoting activity is also plus end specific (Gard and Kirschner, 1987; Vasquez et al., 1994).

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