Poster: Cytoskeleton: Structure & Function
Abs #
1103: Tobacco high molecular weight microtubule-associated protein (210 kDa MAP) homologous to MOR1/XMAP215 promote tubulin polymerization by forming a complex with tubulin.
|
|
Presenter: |
Hamada, Takahiro , t.hama@sci.himeji-tech.ac.jp |
Authors | Hamada, Takahiro (A) Itoh, Tomohiko J. (B) Shimmen, Teruo (A) Sonobe, Seiji (A) | | Affiliations: |
(A): Department of Life Science, Graduate School of Science, Himeji Institute of Technology
(B): Division of Biological Sciences, Graduate School of Science, Nagoya University
|
|
|
We have succeeded in isolating a high molecular weight microtubule-associated protein (MAP), a 210 kDa MAP, from tobacco BY-2 cells. Analysis of partial amino acid sequence indicated that the 210 kDa MAP was a member of the XMAP215/MOR1 family. Electron microscopy revealed that the 210 kDa MAP was long filamentous protein with average length of 60 nm. The 210 kDa MAP accelerated turbidity increase of tubulin solution in both initial rate and final extent in concentration dependent manner, suggesting that the 210 kDa MAP can promote MT polymerization. Dark-field microscopy indicated that both lengths and numbers of MT increased in the presence of the 210 kDa MAP. When the 210 kDa MAP was mixed with tubulin under a condition in which polymerization of tubulin per se was inhibited by 0.15 M NaCl, complexes were formed. Cosedimentation assay and cross-link experiment suggested that the complex contained the 210 kDa MAP and tubulin. These results strongly suggest that MAP-tubulin complex promote tubulin polymerization. Immunofluorescent microscopy was carried out using monoclonal antibody against the 210 kDa MAP to localize the 210 kDa MAP in BY-2 cells. In interphase, most of the 210 kDa MAP was present in cytoplasm. Fluorescence signals along with cortical microtubules were not observed. As preprophase band (PPB) was converged in late G2 phase, signals of the 210 kDa MAP increased in the PPB. During mitotic phase, spindle and phragmoplast seemed to be covered with cytoplasmic 210 kDa MAP. These observations suggested that the 210 kDa MAP might stabilize MTs by surrounding them, which biases the equilibrium into polymerization.
