Abstract
Microtubules (MTs) are important targets for imaging in living cells because of their vital roles in cellular processes. The dynamics (polymerization/depolymerization) of MTs has been imaged in living cells by utilizing MT-targeted drugs as scaffolds. We previously developed a unique MT-binding motif derived from a MT-associated protein, Tau. The Tau-derived peptide (TP) binds to the inner surface of MTs without inhibiting the dynamics of MTs. We introduce a new protocol for live-cell imaging of MTs by using fluorescently labeled TP. We exemplify that tetramethylrhodamine (TMR)-labeled TP (TP-TMR) is spontaneously internalized into HepG2 cells and binds to intracellular MTs, enabling visualization of MTs in living cells. TP-TMR shows no apparent effects on polymerization/depolymerization of MTs and no cytotoxicity. Thus, the peptide-based approach is useful for long-term imaging of MTs.
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References
Hess H, Ross JL (2017) Non-equilibrium assembly of microtubules: from molecules to autonomous chemical robots. Chem Soc Rev 46:5570–5587
Sahl SJ, Hell SW, Jakobs S (2017) Fluorescence nanoscopy in cell biology. Nat Rev Mol Cell Biol 18:685–701
Zwetsloot AJ, Tut G, Straube A (2018) Measuring microtubule dynamics. Essays Biochem 62:725–735
Wang L, Frei MS, Salim A, Johnsson K (2019) Small-molecule fluorescent probes for live-cell super-resolution microscopy. J Am Chem Soc 141:2770–2781
Souto AA, Acuña AU, Andreu JM et al (1996) New fluorescent water-soluble taxol derivatives. Angew Chem Int Ed Engl 34:2710–2712
Guy R, Scott Z, Sloboda R, Nicolaou K (1996) Fluorescent taxoids. Chem Biol 3:1021–1031
Evangelio JA, Abal M, Barasoain I et al (1998) Fluorescent taxoids as probes of the microtubule cytoskeleton. Cell Motil Cytoskeleton 39:73–90
Devaraj NK, Hilderbrand S, Upadhyay R et al (2010) Bioorthogonal turn-on probes for imaging small molecules inside living cells. Angew Chem Int Ed Engl 49:2869–2872
Lukinavičius G, Reymond L, D’Este E et al (2014) Fluorogenic probes for live-cell imaging of the cytoskeleton. Nat Methods 11:731–733
Lukinavičius G, Reymond L, Umezawa K et al (2016) Fluorogenic probes for multicolor imaging in living cells. J Am Chem Soc 138:9365–9368
Lee MM, Gao Z, Peterson BR (2017) Synthesis of a fluorescent analogue of paclitaxel that selectively binds microtubules and sensitively detects efflux by P-glycoprotein. Angew Chem Int Ed Engl 56:6927–6931
Butkevich AN, Ta H, Ratz M et al (2018) Two-color 810 nm STED nanoscopy of living cells with endogenous SNAP-tagged fusion proteins. ACS Chem Biol 13:475–480
Lukinavičius G, Mitronova GY, Schnorrenberg S et al (2018) Fluorescent dyes and probes for super-resolution microscopy of microtubules and tracheoles in living cells and tissues. Chem Sci 9:3324–3334
Zhang H, Wang C, Jiang T et al (2015) Microtubule-targetable fluorescent probe: site-specific detection and super-resolution imaging of ultratrace tubulin in microtubules of living cancer cells. Anal Chem 87:5216–5222
Hamley IW (2017) Small bioactive peptides for biomaterials design and therapeutics. Chem Rev 117:14015–14041
Zhang P, Cui Y, Anderson CF et al (2018) Peptide-based nanoprobes for molecular imaging and disease diagnostics. Chem Soc Rev 47:3490–3529
Qi G-B, Gao Y-J, Wang L, Wang H (2018) Self-assembled peptide-based nanomaterials for biomedical imaging and therapy. Adv Mater 30:1703444
Tsutsumi H, Mihara H (2013) Soft materials based on designed self-assembling peptides: from design to application. Mol BioSyst 9:609–617
Inaba H, Matsuura K (2019) Peptide nanomaterials designed from natural supramolecular systems. Chem Rec 19:843–858
Inaba H, Yamamoto T, Kabir AMR et al (2018) Molecular encapsulation inside microtubules based on Tau-derived peptides. Chem Eur J 24:14958–14967
Inaba H, Yamamoto T, Iwasaki T et al (2019) Stabilization of microtubules by encapsulation of the GFP using a Tau-derived peptide. Chem Commun 55:9072–9075
Inaba H, Yamamoto T, Iwasaki T et al (2019) Fluorescent Tau-derived peptide for monitoring microtubules in living cells. ACS Omega 4:11245–11250
Inaba H, Nagata M, Miyake KJ et al (2020) Cyclic Tau-derived peptides for stabilization of microtubules. Polym J 52:1143–1151. https://doi.org/10.1038/s41428-020-0356-3
Dai Y, Whittal RM, Li L (1999) Two-layer sample preparation: a method for MALDI-MS analysis of complex peptide and protein mixtures. Anal Chem 71:1087–1091
Acknowledgments
This work was supported by KAKENHI (No. 17K14517 and 19K15699 for H.I.) from the Japan Society for the Promotion of Science (JSPS), the Inamori Foundation, and Konica Minolta Science and Technology Foundation for Konica Minolta Imaging Science Encouragement Award.
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Inaba, H., Matsuura, K. (2021). Live-Cell Fluorescence Imaging of Microtubules by Using a Tau-Derived Peptide. In: Kim, SB. (eds) Live Cell Imaging. Methods in Molecular Biology, vol 2274. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1258-3_15
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DOI: https://doi.org/10.1007/978-1-0716-1258-3_15
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