E10-b - OptoMT and OptoTIP: Reversible Optogenetic Labeling of Microtubules and Plus Ends in Live Cells

Saturday, October 11, 2025

10:00 AM - 12:00 PM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

MC

Megan Chiu

Student
The Bryn Mawr School, United States

Primary Investigator(s)

Xl

Xiaoxuan liu

Graduate Research Assistant
Texas A&M University
Houston, Texas, United States

Co-Author(s)

GM

Guolin Ma

Research Assistant Professor
Texas A&M University Institute of Biosciences & Technology, United States

Last Author(s)

Yubin Zhou

Professor
Texas A&M University
Houton, Texas, United States

Introduction: : Microtubules (MTs) form a dynamic, polarized cytoskeletal network essential for a wide range of cellular processes, including intracellular transport, cell division, signal transduction, and spatial organization of organelles (1). Visualizing the MT cytoskeleton and tracking its dynamics are essential for understanding its function. However, current approaches, including fluorescent tubulin overexpression, antibody-based immunostaining, and fluorophore-conjugated tubulin probes, are limited by perturbations to MT polymerization or incompatibility with live-cell imaging (2,3). In this study, we developed two optogenetic tools, OptoMT and OptoTIP, by fusing the light-sensitive CRY2 to native domains that weakly bind tubulin or the plus-end tracking protein EB1. These single-component probes allow for reversible labeling of MTs and track their plus ends in live cells, respectively, with tunable speed and low background activity.

Materials and

Methods: : Plasmid construction: To generate OptoMT, the PHR domain (aa 1-498) of Arabidopsis thaliana CRY2 (Addgene # 70159) was PCR amplified and inserted into pmCherry-C1 and pEGFP-C1 (Clontech), followed by the insertion of the MT binding domain from CLIP170 (aa 129-350) at the BspEI and EcoRI sites in the downstream of CRY2-PHR. To generate OptoTIP, the EB1-binding SxIP motif of dystonin (aa 5469-5485) was synthesized and inserted to replace the MT binding domain.
Live-cell imaging, photoactivation and image analysis: One day after transfection in HeLa cells, live-cell imaging was performed on a Nikon Eclipse Ti-E microscope equipped with an A1R-A1 confocal module with LU-N4 laser sources (argon-ion: 405 and 488 nm; diode: 561 nm). Photoactivation with persistent illumination or pulses of repeated dark-light cycles was achieved by an external blue light source (470 nm, 40 µW/mm2, ThorLabs Inc., Newton, NJ, USA). The acquired confocal images were analyzed by using MATLAB. The defined regions of interest such as MT or MT plus ends (comets) and the nearby cytosolic areas were separated by using thresholding. Next, the fluorescence intensity values of the MT region versus the neighboring cytosolic mean intensity were determined to obtain the MT-to-cytosol ratio (FMT/Fcytosol or Fcomet/Fcytosol). Images were saved in tiff format and further processed using MATLAB. The apparent half-life time of fluorescent signal was calculated by using a single component exponential decay function. Quantitative data are presented as the mean ± SEM. Sample sizes (n) were listed for each experiment.

Results, Conclusions, and Discussions:: We designed OptoMT to harness the light-induced oligomerization of optogenetic modules (4) to cluster a weak tubulin-binding domain, thereby shifting its equilibrium toward MT association and enabling real-time, light-controllable cytoskeleton labeling (Fig. 1A). To implement this strategy, we made a chimeric construct by fusing the light-sensitive CRY2-PHR domain with the tubulin-binding domain of CLIP170 (5). We transfected mCherry-tagged OptoMT into HeLa cells and performed live cell imaging. In the absence of light, OptoMT was diffusely localized in the cytoplasm, consistent with its weak basal affinity for tubulin. Upon blue light illumination, we observed rapid and reversible formation of MT structure patterns (Fig. 1B). OptoMT displayed rapid kinetics, with a light-induced activation half-life (t1/2, ON) of 10 seconds and a decay half-life (t1/2, OFF) of 240 seconds upon withdrawal of light, indicating fast response times suitable for live-cell imaging (Fig. 1C). The specificity of OptoMT was validated through colocalization with endogenous tubulin based on immunostaining (Fig. 2A). In addition, light-controlled labeling enabled clear visualization of MT dynamics across distinct mitotic stages (Fig. 2B), without detectable effects on cell cycle progression.
To enable reversible tracking of growing microtubule (MT) plus ends, we extended a similar optogenetic engineering strategy to create OptoTIP by fusing CRY2-PHR to the MT tip localization SxIP motif from dystonin (6), a known binder to MT end-binding protein 1 (EB1) (Fig. 3A). Without light, OptoTIP was diffusely localized in the cytoplasm, consistent with its weak basal affinity for EB1. When exposed to blue light, mCherry-OptoTIP formed dynamic, reversible "comet-like" patterns at MT ends (Fig. 3B). Under repeated dark–light cycles, OptoTIP exhibited reversible comet-like localization, with an activation half-life (t₁/₂, ON) of 11 seconds and a decay half-life (t₁/₂, OFF) of 204 seconds (Fig. 3C).
This work presents OptoMT and OptoTIP for reversible and light-controlled labeling and tracking of the MT dynamics with high spatial and temporal precision. In future studies, we will evaluate their utility for in vivo imaging in whole organisms. This work establishes a foundation for versatile optogenetic platforms capable of controlling microtubule motor activity, cargo interactions, and severing, providing powerful tools to dissect microtubule regulation.

Acknowledgements and/or References (Optional): : 1. Akhmanova, A., and Steinmetz, M. O. (2015) Control of microtubule organization and dynamics: two ends in the limelight, Nature reviews. Molecular cell biology 16, 711-726.
2. Xue, L., Karpenko, I. A., Hiblot, J., and Johnsson, K. (2015) Imaging and manipulating proteins in live cells through covalent labeling, Nature chemical biology 11, 917-923.
3. Dumontet, C., and Jordan, M. A. (2010) Microtubule-binding agents: a dynamic field of cancer therapeutics, Nature reviews. Drug discovery 9, 790-803.
4. Tan, P., He, L., Han, G., and Zhou, Y. (2017) Optogenetic Immunomodulation: Shedding Light on Antitumor Immunity, Trends Biotechnol 35, 215-226.
5. Mishima, M., Maesaki, R., Kasa, M., Watanabe, T., Fukata, M., Kaibuchi, K., and Hakoshima, T. (2007) Structural basis for tubulin recognition by cytoplasmic linker protein 170 and its autoinhibition, Proc Natl Acad Sci U S A 104, 10346-10351.
6. Kumar, P., and Wittmann, T. (2012) +TIPs: SxIPping along microtubule ends, Trends Cell Biol 22, 418-428.