Abstract
Three genes that encode MAP65-1 family proteins have been identified in the Nicotiana tabacum genome. In this study, NtMAP65-1c fusion protein was shown to bind and bundle microtubules (MTs). Further in vitro investigations demonstrated that NtMAP65-1c not only alters MT assembly and nucleation, but also exhibits high MT stabilizing activity against cold or katanin-induced destabilization. Analysis of NtMAP65-1c-GFP expressing BY-2 cells clearly demonstrated that NtMAP65-1c was able to bind to MTs during specific stages of the cell cycle. Furthermore, in vivo, NtMAP65-1c-GFP-bound cortical MTs displayed an increase in resistance against the MT-disrupting drug, propyzamide, as well as against cold temperatures. Taken together, these results strongly suggest that NtMAP65-1c stabilizes MTs and is involved in the regulation of MT organization and cellular dynamics.
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Abbreviations
- MAPs:
-
Microtubule-associated proteins
- PIPES:
-
Piperazine-N,N′-bis (2-ethanesulfonic acid 1,4-piperazinediethanesulfonic acid)
- EGTA:
-
Ethylene glycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
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Acknowledgments
We thank Prof. Ying Fu at China Agricultural University for generously providing the katanin construct. This research was supported by grants from the National Basic Research Program of China (2006CB100101 to M.Y.), the 111 Project (B06003), the National Natural Science Foundation of China (30770186 and 31070258 to T. M.; 30830058 and 30721062 to M.Y.) and Chinese Universities Scientific Fund (2009JS72 to T.M.).
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Qiutao Meng and Jizhou Du have contributed equally to this paper and are considered as joint first authors.
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11103_2010_9694_MOESM1_ESM.jpg
NtMAP65-1c decreased the critical concentration (Cc) for tubulin polymerization. (a) Tubulins at concentration of 5 or 10 μM underwent polymerization in presence or absence of 2 μM NtMAP65-1c at 35°C for 30 min. After centrifuging at 100,000 g for 20 min, the supernatant and pellet were subjected to SDS–PAGE. Tubulins assembled without GTP were used a control. (b) The amount of MTs in the pellet of (a) was estimated following density scanning of gels. There was a notable increase in the amount of tubulin in the pellet in the presence of NtMAP65-1c, as compared to control. (JPEG 574 kb)
11103_2010_9694_MOESM2_ESM.jpg
NtMAP65-1a is capable of stabilizing MTs against in vitro cold treatment. Tubulins (20 μM) were assembled with NtMAP65-1a fusion protein at 35°C for 20 min and then incubated at 4°C for 20 min. The pellets were subjected to SDS–PAGE. As a positive control, paclitaxel (10 μM) was used in place of NtMAP65-1a. (JPEG 586 kb)
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Meng, Q., Du, J., Li, J. et al. Tobacco microtubule-associated protein, MAP65-1c, bundles and stabilizes microtubules. Plant Mol Biol 74, 537–547 (2010). https://doi.org/10.1007/s11103-010-9694-4
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DOI: https://doi.org/10.1007/s11103-010-9694-4