Limits...
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.

Show MeSH

Related in: MedlinePlus

There is a CPP MT–depolymerizing activity in Xenopus egg extract independent of XKCM1. (A) XKCM1 overlaps with the peak of depolymerizing activity on sucrose gradients. 50 μl of clarified CSF extract was sedimented over a 5–20% sucrose gradient. Western blot of fractions showed that XKCM1 is present in fractions 10–18. CPP MT–depolymerizing activity peaked in fractions 9–14 (see B). Arrows below the blot indicate sedimentation values for protein standards run on a parallel gradient. Active fractions are labeled with asterisks. (B) Inhibition of XKCM1 did not inhibit depolymerizing activity in sucrose gradient fractions. Fractions from the sucrose gradient shown in A were assayed for depolymerizing activity, using rhodamine-labeled CPP MTs as described in the Materials and methods. Each fraction was assayed in the absence of ATP and in the presence of random IgG or inhibitory amounts of α-XKCM1 antibody and fixed after 10 min. XKCM1 depolymerizing activity is ATP dependent. As shown, neither the absence of ATP nor the presence of αXKCM1 antibody blocked the depolymerizing activity of active fractions. Active fractions are labeled with asterisks. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172913&req=5

fig1: There is a CPP MT–depolymerizing activity in Xenopus egg extract independent of XKCM1. (A) XKCM1 overlaps with the peak of depolymerizing activity on sucrose gradients. 50 μl of clarified CSF extract was sedimented over a 5–20% sucrose gradient. Western blot of fractions showed that XKCM1 is present in fractions 10–18. CPP MT–depolymerizing activity peaked in fractions 9–14 (see B). Arrows below the blot indicate sedimentation values for protein standards run on a parallel gradient. Active fractions are labeled with asterisks. (B) Inhibition of XKCM1 did not inhibit depolymerizing activity in sucrose gradient fractions. Fractions from the sucrose gradient shown in A were assayed for depolymerizing activity, using rhodamine-labeled CPP MTs as described in the Materials and methods. Each fraction was assayed in the absence of ATP and in the presence of random IgG or inhibitory amounts of α-XKCM1 antibody and fixed after 10 min. XKCM1 depolymerizing activity is ATP dependent. As shown, neither the absence of ATP nor the presence of αXKCM1 antibody blocked the depolymerizing activity of active fractions. Active fractions are labeled with asterisks. Bar, 10 μm.

Mentions: To assay for MT-depolymerizing factors, we added rhodamine-labeled CPP MTs to crude or clarified cytostatic factor (CSF)–arrested Xenopus egg extract (CSF extract) and observed their disappearance over time. CPP MTs are stable to dilution in buffer, but when added to extract, they depolymerize in 5–10 min (Caplow, M., personal communication). To characterize this depolymerizing activity, we sedimented clarified CSF extract on a 5–20% sucrose gradient and assayed fractions for depolymerizing activity. A single ATP-independent peak of activity was observed at ∼9.5S (Fig. 1 B). XKCM1 cosedimented with this peak (Fig. 1 A), but katanin and Op18 did not (unpublished data). The activity appeared to be independent of XKCM1 because XKCM1 requires ATP for efficient MT depolymerization (Desai et al., 1999b). To confirm that XKCM1 was not responsible for the depolymerizing activity, we assayed those fractions in the absence of ATP and in the presence of inhibitory α-XKCM1 antibody (Walczak et al., 1996) (Fig. 1 B). Depolymerizing activity was not blocked, suggesting that another factor was responsible.


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)

There is a CPP MT–depolymerizing activity in Xenopus egg extract independent of XKCM1. (A) XKCM1 overlaps with the peak of depolymerizing activity on sucrose gradients. 50 μl of clarified CSF extract was sedimented over a 5–20% sucrose gradient. Western blot of fractions showed that XKCM1 is present in fractions 10–18. CPP MT–depolymerizing activity peaked in fractions 9–14 (see B). Arrows below the blot indicate sedimentation values for protein standards run on a parallel gradient. Active fractions are labeled with asterisks. (B) Inhibition of XKCM1 did not inhibit depolymerizing activity in sucrose gradient fractions. Fractions from the sucrose gradient shown in A were assayed for depolymerizing activity, using rhodamine-labeled CPP MTs as described in the Materials and methods. Each fraction was assayed in the absence of ATP and in the presence of random IgG or inhibitory amounts of α-XKCM1 antibody and fixed after 10 min. XKCM1 depolymerizing activity is ATP dependent. As shown, neither the absence of ATP nor the presence of αXKCM1 antibody blocked the depolymerizing activity of active fractions. Active fractions are labeled with asterisks. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: There is a CPP MT–depolymerizing activity in Xenopus egg extract independent of XKCM1. (A) XKCM1 overlaps with the peak of depolymerizing activity on sucrose gradients. 50 μl of clarified CSF extract was sedimented over a 5–20% sucrose gradient. Western blot of fractions showed that XKCM1 is present in fractions 10–18. CPP MT–depolymerizing activity peaked in fractions 9–14 (see B). Arrows below the blot indicate sedimentation values for protein standards run on a parallel gradient. Active fractions are labeled with asterisks. (B) Inhibition of XKCM1 did not inhibit depolymerizing activity in sucrose gradient fractions. Fractions from the sucrose gradient shown in A were assayed for depolymerizing activity, using rhodamine-labeled CPP MTs as described in the Materials and methods. Each fraction was assayed in the absence of ATP and in the presence of random IgG or inhibitory amounts of α-XKCM1 antibody and fixed after 10 min. XKCM1 depolymerizing activity is ATP dependent. As shown, neither the absence of ATP nor the presence of αXKCM1 antibody blocked the depolymerizing activity of active fractions. Active fractions are labeled with asterisks. Bar, 10 μm.
Mentions: To assay for MT-depolymerizing factors, we added rhodamine-labeled CPP MTs to crude or clarified cytostatic factor (CSF)–arrested Xenopus egg extract (CSF extract) and observed their disappearance over time. CPP MTs are stable to dilution in buffer, but when added to extract, they depolymerize in 5–10 min (Caplow, M., personal communication). To characterize this depolymerizing activity, we sedimented clarified CSF extract on a 5–20% sucrose gradient and assayed fractions for depolymerizing activity. A single ATP-independent peak of activity was observed at ∼9.5S (Fig. 1 B). XKCM1 cosedimented with this peak (Fig. 1 A), but katanin and Op18 did not (unpublished data). The activity appeared to be independent of XKCM1 because XKCM1 requires ATP for efficient MT depolymerization (Desai et al., 1999b). To confirm that XKCM1 was not responsible for the depolymerizing activity, we assayed those fractions in the absence of ATP and in the presence of inhibitory α-XKCM1 antibody (Walczak et al., 1996) (Fig. 1 B). Depolymerizing activity was not blocked, suggesting that another factor was responsible.

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