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XMAP310: a Xenopus rescue-promoting factor localized to the mitotic spindle.

Andersen SS, Karsenti E - J. Cell Biol. (1997)

Bottom Line: Analysis of the effect of this protein on MT dynamics by time-lapse video microscopy has shown that it increases the rescue frequency 5-10-fold and decreases the shrinkage rate twofold.Cell Biol. 127:985-993), XMAP230, and XMAP310 are localized to the mitotic spindle, they have distinct effects on MT dynamics.While XMAP215 promotes rapid MT growth, XMAP230 decreases the catastrophe frequency and XMAP310 increases the rescue frequency.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Cell Biology Programme, D-69117 Heidelberg, Germany. Andersen@EMBL-Heidelberg.DE

ABSTRACT
To understand the role of microtubule-associated proteins (MAPs) in the regulation of microtubule (MT) dynamics we have characterized MAPs prepared from Xenopus laevis eggs (Andersen, S.S.L., B. Buendia, J.E. Domínguez, A. Sawyer, and E. Karsenti. 1994. J. Cell Biol. 127:1289-1299). Here we report on the purification and characterization of a 310-kD MAP (XMAP310) that localizes to the nucleus in interphase and to mitotic spindle MTs in mitosis. XMAP310 is present in eggs, oocytes, a Xenopus tissue culture cell line, testis, and brain. We have purified XMAP310 to homogeneity from egg extracts. The purified protein cross-links pure MTs. Analysis of the effect of this protein on MT dynamics by time-lapse video microscopy has shown that it increases the rescue frequency 5-10-fold and decreases the shrinkage rate twofold. It has no effect on the growth rate or the catastrophe frequency. Microsequencing data suggest that XMAP230 and XMAP310 are novel MAPs. Although the three Xenopus MAPs characterized so far, XMAP215 (Vasquez, R.J., D.L. Gard, and L. Cassimeris. 1994. J. Cell Biol. 127:985-993), XMAP230, and XMAP310 are localized to the mitotic spindle, they have distinct effects on MT dynamics. While XMAP215 promotes rapid MT growth, XMAP230 decreases the catastrophe frequency and XMAP310 increases the rescue frequency. This may have important implications for the regulation of MT dynamics during spindle morphogenesis and chromosome segregation.

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(Top) XMAP310 localization in the mitotic spindle:  MTs stained with a rabbit anti-tublin pAb in the rhodamine channel (Tubulin), XMAP310 in the fluorescein channel (XMAP310)  and in overlay (Overlay). (Bottom) XMAP230 and XMAP310  colocalize (Overlay) to the central region of the mitotic spindle in  XL177 cells. Images were obtained by confocal microscopy. Bar,  2.5 μm.
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Figure 2: (Top) XMAP310 localization in the mitotic spindle: MTs stained with a rabbit anti-tublin pAb in the rhodamine channel (Tubulin), XMAP310 in the fluorescein channel (XMAP310) and in overlay (Overlay). (Bottom) XMAP230 and XMAP310 colocalize (Overlay) to the central region of the mitotic spindle in XL177 cells. Images were obtained by confocal microscopy. Bar, 2.5 μm.

Mentions: Since XMAP310 had been identified biochemically due to its affinity for MTs, we wanted to determine whether it localized to MTs in vivo. The Q4 mAb was used to probe the localization of XMAP310 at different stages during the cell cycle of Xenopus XL177 tissue culture cells (Fig. 1). In interphase cells XMAP310 was present only in the nucleus (Fig. 1, a–c). The analysis of confocal sections showed that XMAP310 was homogeneously localized throughout the nucleus (data not shown). This localization was maintained through prophase, but XMAP310 relocalized to spindle MTs at the exclusion of astral MTs during metaphase (data not shown and Fig. 1, d–f). The metaphase localization was resistant to preextraction of the cells with detergents (Fig. 1, d–f). During anaphase, XMAP310 was concentrated to areas of high MT density (Fig. 1, g–i), but this staining could be removed by preextraction. During cytokinesis XMAP310 began to reaccumulate in the nucleus (Fig. 1, j and k). Some of the protein that remained diffusely localized in the cytoplasm, was completely removed by preextraction (Fig. 1, l and m, note the faint staining of the nucleus after preextraction). The localization of XMAP310 in the nucleus during interphase and to the spindle during mitosis was confirmed by immunostaining of Xenopus embryos at the mid blastulae stage (data not shown). Double immunostaining of XL177 cells with XMAP230 and XMAP310 mAbs and observation by confocal microscopy revealed that the two proteins colocalize in the mitotic spindle (Fig. 2). However, immunoprecipitation and native molecular weight estimation showed that XMAP230 and XMAP310 do not form a complex in the extract, suggesting that the co-localization in the spindle MTs is due to independent regulation of binding for these two proteins (data not shown; hydrodynamic data for XMAP230 and XMAP310: stokes radius (ao), 13.3 nm and 16.5 nm, respectively; sedimentation coefficients, (S20w) of 5.2 × 10−13 s−1 and 10.1 × 10−13 s−1, respectively; native molecular masses, 285 and 685 kD, respectively).


XMAP310: a Xenopus rescue-promoting factor localized to the mitotic spindle.

Andersen SS, Karsenti E - J. Cell Biol. (1997)

(Top) XMAP310 localization in the mitotic spindle:  MTs stained with a rabbit anti-tublin pAb in the rhodamine channel (Tubulin), XMAP310 in the fluorescein channel (XMAP310)  and in overlay (Overlay). (Bottom) XMAP230 and XMAP310  colocalize (Overlay) to the central region of the mitotic spindle in  XL177 cells. Images were obtained by confocal microscopy. Bar,  2.5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139961&req=5

Figure 2: (Top) XMAP310 localization in the mitotic spindle: MTs stained with a rabbit anti-tublin pAb in the rhodamine channel (Tubulin), XMAP310 in the fluorescein channel (XMAP310) and in overlay (Overlay). (Bottom) XMAP230 and XMAP310 colocalize (Overlay) to the central region of the mitotic spindle in XL177 cells. Images were obtained by confocal microscopy. Bar, 2.5 μm.
Mentions: Since XMAP310 had been identified biochemically due to its affinity for MTs, we wanted to determine whether it localized to MTs in vivo. The Q4 mAb was used to probe the localization of XMAP310 at different stages during the cell cycle of Xenopus XL177 tissue culture cells (Fig. 1). In interphase cells XMAP310 was present only in the nucleus (Fig. 1, a–c). The analysis of confocal sections showed that XMAP310 was homogeneously localized throughout the nucleus (data not shown). This localization was maintained through prophase, but XMAP310 relocalized to spindle MTs at the exclusion of astral MTs during metaphase (data not shown and Fig. 1, d–f). The metaphase localization was resistant to preextraction of the cells with detergents (Fig. 1, d–f). During anaphase, XMAP310 was concentrated to areas of high MT density (Fig. 1, g–i), but this staining could be removed by preextraction. During cytokinesis XMAP310 began to reaccumulate in the nucleus (Fig. 1, j and k). Some of the protein that remained diffusely localized in the cytoplasm, was completely removed by preextraction (Fig. 1, l and m, note the faint staining of the nucleus after preextraction). The localization of XMAP310 in the nucleus during interphase and to the spindle during mitosis was confirmed by immunostaining of Xenopus embryos at the mid blastulae stage (data not shown). Double immunostaining of XL177 cells with XMAP230 and XMAP310 mAbs and observation by confocal microscopy revealed that the two proteins colocalize in the mitotic spindle (Fig. 2). However, immunoprecipitation and native molecular weight estimation showed that XMAP230 and XMAP310 do not form a complex in the extract, suggesting that the co-localization in the spindle MTs is due to independent regulation of binding for these two proteins (data not shown; hydrodynamic data for XMAP230 and XMAP310: stokes radius (ao), 13.3 nm and 16.5 nm, respectively; sedimentation coefficients, (S20w) of 5.2 × 10−13 s−1 and 10.1 × 10−13 s−1, respectively; native molecular masses, 285 and 685 kD, respectively).

Bottom Line: Analysis of the effect of this protein on MT dynamics by time-lapse video microscopy has shown that it increases the rescue frequency 5-10-fold and decreases the shrinkage rate twofold.Cell Biol. 127:985-993), XMAP230, and XMAP310 are localized to the mitotic spindle, they have distinct effects on MT dynamics.While XMAP215 promotes rapid MT growth, XMAP230 decreases the catastrophe frequency and XMAP310 increases the rescue frequency.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Cell Biology Programme, D-69117 Heidelberg, Germany. Andersen@EMBL-Heidelberg.DE

ABSTRACT
To understand the role of microtubule-associated proteins (MAPs) in the regulation of microtubule (MT) dynamics we have characterized MAPs prepared from Xenopus laevis eggs (Andersen, S.S.L., B. Buendia, J.E. Domínguez, A. Sawyer, and E. Karsenti. 1994. J. Cell Biol. 127:1289-1299). Here we report on the purification and characterization of a 310-kD MAP (XMAP310) that localizes to the nucleus in interphase and to mitotic spindle MTs in mitosis. XMAP310 is present in eggs, oocytes, a Xenopus tissue culture cell line, testis, and brain. We have purified XMAP310 to homogeneity from egg extracts. The purified protein cross-links pure MTs. Analysis of the effect of this protein on MT dynamics by time-lapse video microscopy has shown that it increases the rescue frequency 5-10-fold and decreases the shrinkage rate twofold. It has no effect on the growth rate or the catastrophe frequency. Microsequencing data suggest that XMAP230 and XMAP310 are novel MAPs. Although the three Xenopus MAPs characterized so far, XMAP215 (Vasquez, R.J., D.L. Gard, and L. Cassimeris. 1994. J. Cell Biol. 127:985-993), XMAP230, and XMAP310 are localized to the mitotic spindle, they have distinct effects on MT dynamics. While XMAP215 promotes rapid MT growth, XMAP230 decreases the catastrophe frequency and XMAP310 increases the rescue frequency. This may have important implications for the regulation of MT dynamics during spindle morphogenesis and chromosome segregation.

Show MeSH
Related in: MedlinePlus