微管調控了許多細胞活動，包括細胞內運輸、細胞分裂以及細胞的爬行等等。微管若發生缺陷將導致很多人類疾病。微管會經歷不同的轉譯後修飾，例如:谷氨醯化、乙醯化…等。過去已有離體的研究顯示這些轉譯後修飾可以調控純化微管的構造以及功能，但因為缺乏適合的研究工具，這幾十年來還是一直無從得知微管在活細胞中是如何改變其轉譯後修飾來調控細胞的活動。為此，我們實驗室利用了一系列化學生物學的方式藉由累積一個改造過的去谷氨醯酶—CCP5CD到微管上來去除其谷氨醯化，並藉此初步了解微管谷氨醯化在活細胞中所扮演的生理角色。為了要能夠更精準的調控微管蛋白上的轉譯後修飾，我們欲發展出一套光調控系統。為此，一套藍光誘導雙聚體系統Cry2/CIBN被我們利用來聚集CCP5CD到微管上。初步結果顯示利用藍光去轉移CCP5CD到微管上只有稍微減少微管的谷氨醯化。未來我們將找出更佳的光刺激參數以有效地利用光調控系統來去除微管的谷氨醯化。

Microtubules regulate various cellular activities including intracellular transport, cell division, cell signaling, and cell motility. Defects in microtubules lead to various human diseases. Microtubules undergo different post-translational modifications (PTMs) including glutamylation, acetylation, detyrosination, polyglycylation, and so on. Evidence mainly from in vitro studies have demonstrated that PTMs directly modulate the structure and functions of purified microtubules. However, the detailed mechanism of how microtubules spatiotemporally regulate cellular activities via altering their PTMs is still unclear mainly due to the lack of techniques to spatiotemporally perturb tubulin PTMs in living cells. Recently our lab utilized a series of chemical biology approaches to recruit an engineered deglutamylase, CCP5CD, onto microtubules for local deglutamylation within minutes which enable us to uncover the spatiotemporal roles of tubulin glutamylation. In order to manipulate tubulin PTM with better spatial and temporal accuracy, we here aimed to develop a light controllable system to manipulate tubulin PTMs in living cells. To achieve this, a dimerization pair, Cry2/CIBN that can be induced by blue light, was utilized to locally and rapidly translocate CCP5CD onto microtubules. Our preliminary result showed that light-induced recruitment of CCP5CD onto microtubules only slightly deplete tubulin glutamylation. We will optimize the parameters of light illumination to improve the efficiency of tubulin deglutamylation in the near future.

Aillaud, C., Bosc, C., Saoudi, Y., Denariera, E., Peris, L., Sago, L., Taulet, N., Cieren, A., Tort, O., Magiera, M.M., et al. (2016). Evidence for new C-terminally truncated variants of α-and β-tubulins. Mol. Biol. Cell 27, 640–653.
Benedetti, L., Barentine, A.E.S., Messa, M., Wheeler, H., Bewersdorf, J., andDeCamilli, P. (2018). Light-activated protein interaction with high spatial subcellular confinement. Proc. Natl. Acad. Sci. U. S. A. 115, E2238–E2245.
Berezniuk, I., Lyons, P.J., Sironi, J.J., Xiao, H., Setou, M., Angeletti, R.H., Ikegami, K., andFricker, L.D. (2013). Cytosolic carboxypeptidase 5 removes α- and γ-linked glutamates from tubulin. J. Biol. Chem. 288, 30445–30453.
Bobinnec, Y., Moudjou, M., Fouquet, J.P., Desbruyères, E., Eddé, B., andBornens, M. (1998). Glutamylation of centriole and cytoplasmic tubulin in proliferating non-neuronal cells. Cell Motil. Cytoskeleton 39, 223–232.
Bré, M.H., deNéchaud, B., Wolff, A., andFleury, A. (1994). Glutamylated tubulin probed in ciliates with the monoclonal antibody GT335. Cell Motil. Cytoskeleton 27, 337–349.
Brouhard, G.J., andRice, L.M. (2018). Microtubule dynamics: An interplay of biochemistry and mechanics. Nat. Rev. Mol. Cell Biol. 19, 451–463.
DeRose, R., Miyamoto, T., andInoue, T. (2013). Manipulating signaling at will: chemically-inducible dimerization (CID) techniques resolve problems in cell biology. Pfl{ü}gers Arch. Eur. J. Physiol. 465, 409–417.
vanDijk, J., Rogowski, K., Miro, J., Lacroix, B., Eddé, B., andJanke, C. (2007). A Targeted Multienzyme Mechanism for Selective Microtubule Polyglutamylation. Mol. Cell 26, 437–448.
VanDijk, J., Miro, J., Strub, J.M., Lacroix, B., VanDorsselaer, A., Edde, B., andJanke, C. (2008). Polyglutamylation is a post-translational modification with a broad range of substrates. J. Biol. Chem. 283, 3915–3922.
Garnham, C.P., andRoll-Mecak, A. (2012). The chemical complexity of cellular microtubules: Tubulin post-translational modification enzymes and their roles in tuning microtubule functions. Cytoskeleton 69, 442–463.
Gradilone, S. a, Radtke, B.N., Bogert, P.S., Huang, B.Q., Gajdos, G.B., andLaRusso, N.F. (2013). HDAC6 inhibition restores ciliary expression and decreases tumor growth. Cancer Res. 73, 2259–2270.
Greer, K., Maruta, H., L’Hernault, S.W., andRosenbaum, J.L. (1985). α-tubulin acetylase activity in isolated Chlamydomonas flagella. J. Cell Biol. 101, 2081–2084.
Guntas, G., Hallett, R. a., Zimmerman, S.P., Williams, T., Yumerefendi, H., Bear, J.E., andKuhlman, B. (2015). Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins. Proc. Natl. Acad. Sci. 112, 112–117.
Hong, S.-R., Wang, C.-L., Huang, Y.-S., Chang, Y.-C., Chang, Y.-C., Pusapati, G.V, Lin, C.-Y., Hsu, N., Cheng, H.-C., Chiang, Y.-C., et al. (2018a). Spatiotemporal manipulation of ciliary glutamylation reveals its roles in intraciliary trafficking and Hedgehog signaling. Nat. Commun. 9, 1732.
Hong, S.R., Wang, C.L., Huang, Y.S., Chang, Y.C., Chang, Y.C., Pusapati, G.V, Lin, C.Y., Hsu, N., Cheng, H.C., Chiang, Y.C., et al. (2018b). Spatiotemporal manipulation of ciliary glutamylation reveals its roles in intraciliary trafficking and Hedgehog signaling. Nat. Commun. 9, 1–13.
Idevall-hagren, O., Dickson, E.J., Hille, B., Toomre, D.K., DeCamilli, P., andCamilli, P.De (2012). Optogenetic control of phosphoinositide metabolism. Proc. Natl. Acad. Sci. 109, E2316--E2323.
Ikegami, K., Sato, S., Nakamura, K., Ostrowski, L.E., andSetou, M. (2010). Tubulin polyglutamylation is essential for airway ciliary function through the regulation of beating asymmetry. Proc. Natl. Acad. Sci. U. S. A. 107, 10490–10495.
Inobe, T., andNukina, N. (2016). Rapamycin-induced oligomer formation system of FRB-FKBP fusion proteins. J. Biosci. Bioeng. 122, 40–46.
Janke, C. (2014). The tubulin code: Molecular components, readout mechanisms, and functions. J. Cell Biol. 206, 461–472.
Janke, C., andBulinski, J.C. (2011). Post-translational regulation of the microtubule cytoskeleton: mechanisms and functions. Nat. Rev. Mol. Cell Biol. 12, 773–786.
Janke, C., andKneussel, M. (2010). Tubulin post-translational modifications: Encoding functions on the neuronal microtubule cytoskeleton. Trends Neurosci. 33, 362–372.
Kimura, Y., Kurabe, N., Ikegami, K., Tsutsumi, K., Konishi, Y., Kaplan, O.I., Kunitomo, H., Iino, Y., Blacque, O.E., andSetou, M. (2010). Identification of tubulin deglutamylase among Caenorhabditis elegans and mammalian cytosolic carboxypeptidases (CCPs). J. Biol. Chem. 285, 22936–22941.
L’Hernault, S.W. (2004). Chlamydomonas alpha-tubulin is posttranslationally modified in the flagella during flagellar assembly. J. Cell Biol. 97, 258–263.
Lin, Y.-C., Niewiadomski, P., Lin, B., Nakamura, H., Phua, S.C., Jiao, J., Levchenko, A., Inoue, T., Rohatgi, R., andInoue, T. (2013). Chemically inducible diffusion trap at cilia reveals molecular sieve-like barrier. Nat. Chem. Biol. 9, 437–443.
Magiera, M.M., Singh, P., Gadadhar, S., andJanke, C. (2018). Tubulin Posttranslational Modifications and Emerging Links to Human Disease. Cell 173, 1323–1327.
Nieuwenhuis, J., Adamopoulos, A., Bleijerveld, O.B., Mazouzi, A., Stickel, E., Celie, P., Altelaar, M., Knipscheer, P., Perrakis, A., Blomen, V.A., et al. (2017). Vasohibins encode tubulin detyrosinating activity. Science (80-. ). 358, 1453–1456.
O’Hagan, R., Piasecki, B.P., Silva, M., Phirke, P., Nguyen, K.C.Q., Hall, D.H., Swoboda, P., andBarr, M.M. (2011). The tubulin deglutamylase CCPP-1 regulates the function and stability of sensory cilia in C. elegans. Curr. Biol. 21, 1685–1694.
Pan, Y., Yoon, S., Sun, J., Huang, Z., Lee, C., Allen, M., Wu, Y., Chang, Y.-J., Sadelain, M., Shung, K.K., et al. (2018). Mechanogenetics for the remote and noninvasive control of cancer immunotherapy. Proc. Natl. Acad. Sci. 115, 201714900.
Pathak, N., Austin-Tse, C. a, Liu, Y., Vasilyev, A., andDrummond, I. a (2014). Cytoplasmic carboxypeptidase 5 regulates tubulin glutamylation and zebrafish cilia formation and function. Mol. Biol. Cell 25, 1836–1844.
Regnard, C., Desbruyères, E., Denoulet, P., andEddé, B. (1999). Tubulin polyglutamylase: isozymic variants and regulation during the cell cycle in HeLa cells. J. Cell Sci. 112 ( Pt 2, 4281–4289.
Rogowski, K., vanDijk, J., Magiera, M.M., Bosc, C., Deloulme, J.-C., Bosson, A., Peris, L., Gold, N.D., Lacroix, B., Bosch Grau, M., et al. (2010). A family of protein-deglutamylating enzymes associated with neurodegeneration. Cell 143, 564–578.
Roll-Mecak, A. (2019). How cells exploit tubulin diversity to build functional cellular microtubule mosaics. Curr. Opin. Cell Biol. 56, 102–108.
Sloboda, R.D. (2009). Posttranslational protein modifications in cilia and flagella. (Elsevier).
Song, Y., andBrady, S.T. (2014). Post-translational modifications of tubulin : pathways to functional diversity of microtubules. Trends Cell Biol. 25, 1–12.
Song, Y., Kirkpatrick, L.L., Schilling, A.B., Helseth, D.L., Chabot, N., Keillor, J.W., Johnson, G.V.W., andBrady, S.T. (2013). Transglutaminase and Polyamination of Tubulin: Posttranslational Modification for Stabilizing Axonal Microtubules. Neuron 78, 109–123.
Taslimi, A., Vrana, J.D., Chen, D., Borinskaya, S., Mayer, B.J., Kennedy, M.J., andTucker, C.L. (2014). An optimized optogenetic clustering tool for probing protein interaction and function. Nat. Commun. 5, 4925.
Taslimi, A., Zoltowski, B., G., J.M., Pathak, G., Hughes, R.M., andTucker, C.L. (2016). Optimized second generation CRY2/CIB dimerizers and photoactivatable Cre recombinase. 118, 6072–6078.
Tort, O., Tanco, S., Rocha, C., Bièche, I., Seixas, C., Bosc, C., Andrieux, A., Moutin, M.-J., Avilés, F.X., Lorenzo, J., et al. (2014). The cytosolic carboxypeptidases CCP2 and CCP3 catalyze posttranslational removal of acidic amino acids. Mol. Biol. Cell 25, 3017–3027.
Westermann, S., andWeber, K. (2003). Post-translational modifications regulate microtubule function. Nat. Rev. Mol. Cell Biol. 4, 938–947.
Wloga, D., andGaertig, J. (2010). Post-translational modifications of microtubules. J. Cell Sci. 124, 154.
Yu, I., Garnham, C.P., andRoll-Mecak, A. (2015). Writing and Reading the Tubulin Code. J. Biol. Chem. 290, 17163–17172.
Zhang, Y., Gilquin, B., Khochbin, S., andMatthias, P. (2006). Two catalytic domains are required for protein deacetylation. J. Biol. {…} 1–5.
Zhou, X., Fan, L.X., Li, K., Ramchandran, R., Calvet, J.P., andLi, X. (2014). SIRT2 regulates ciliogenesis and contributes to abnormal centrosome amplification caused by loss of polycystin-1. Hum. Mol. Genet. 23, 1644–1655.