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Updated: Jan 20, 2026
Microtubule Binding Assay in CRC Cells: A Method to Examine Microtubule Binding Tau Protein in Colorectal Cancer Cells
Published on: April 30, 2023
Fluorescent Tau-derived Peptide for Monitoring Microtubules in Living Cells
Hiroshi Inaba1,1, Takahisa Yamamoto1, Takashi Iwasaki2
1Department of Chemistry and Biotechnology, Graduate School of Engineering and Centre for Research on Green Sustainable Chemistry, Tottori University, Koyama-Minami 4-101, Tottori 680-8552, Japan.
Abstract:
Microtubules (MTs) are key cytoskeletal components that modulate various cellular activities with their dynamic structural changes, including polymerization and depolymerization. To monitor the dynamics of MTs in living cells, many drug-based fluorescent probes have been developed; however, these also potentially disturb the polymerization/depolymerization of MTs. Here, we report nondrug, peptide-based fluorescent probes to monitor MTs in living cells. We employed a Tau-derived peptide (TP) that has been shown to bind MTs without inhibiting polymerization/depolymerization in vitro. We show that a tetramethylrhodamine (TMR)-labeled TP (TP-TMR) is internalized into HepG2 cells and binds to intracellular MTs, enabling visualization of MTs as clear, fibrous structures. The binding of TP-TMR shows no apparent effects on polymerization/depolymerization of MTs induced by MT-targeted drugs and temperature change. The main uptake mechanism of TP-TMR was elucidated as endocytosis, and partial endosomal escape resulted in the binding of TP-TMR to MTs. TP-TMR exhibited no cytotoxicity compared with MT-targeted drug scaffolds. These results indicate that TP scaffolds can be exploited as useful MT-targeted tools in living cells, such as in long-term imaging of MTs.
Insights
New peptide-based fluorescent probes visualize microtubules (MTs) in living cells without disrupting their dynamics. This offers a non-drug alternative for studying cellular activities and long-term MT imaging.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Imaging
Background:
- Microtubules (MTs) are crucial cytoskeletal elements involved in cellular processes, exhibiting dynamic polymerization and depolymerization.
- Existing drug-based fluorescent probes for monitoring MT dynamics can interfere with MT polymerization and depolymerization.
- There is a need for non-disruptive tools to visualize MTs in living cells.
Purpose of the Study:
- To develop and evaluate novel, non-drug peptide-based fluorescent probes for monitoring microtubule dynamics in living cells.
- To assess the probe's ability to visualize MTs without affecting their polymerization/depolymerization.
- To investigate the probe's cellular uptake mechanism and cytotoxicity.
Main Methods:
- A Tau-derived peptide (TP) known to bind MTs without inhibiting polymerization was labeled with tetramethylrhodamine (TMR) to create TP-TMR.
- TP-TMR was introduced into HepG2 cells to visualize intracellular MTs.
- The effect of TP-TMR on MT polymerization/depolymerization was assessed under drug treatment and temperature changes.
- Cellular uptake mechanisms and cytotoxicity were evaluated.
Main Results:
- TP-TMR was successfully internalized into HepG2 cells and localized to intracellular MTs, appearing as distinct fibrous structures.
- TP-TMR binding did not affect MT polymerization/depolymerization dynamics, even when induced by drugs or temperature shifts.
- Endocytosis was identified as the primary uptake pathway, with partial endosomal escape facilitating MT binding.
- TP-TMR demonstrated no significant cytotoxicity compared to conventional MT-targeted drug scaffolds.
Conclusions:
- Tau-derived peptide (TP) scaffolds, such as TP-TMR, are effective non-drug fluorescent probes for visualizing microtubules in living cells.
- These probes enable accurate monitoring of MTs without perturbing their essential dynamic behaviors.
- TP-TMR represents a promising tool for applications like long-term imaging of microtubule dynamics and cellular studies.
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