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Local control of intracellular microtubule dynamics by EB1 photodissociation

Nature Cell Biology volume 20, pages 252–261 (2018)Cite this article

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Abstract

End-binding proteins (EBs) are adaptors that recruit functionally diverse microtubule plus-end-tracking proteins (+TIPs) to growing microtubule plus ends. To test with high spatial and temporal accuracy how, when and where +TIP complexes contribute to dynamic cell biology, we developed a photo-inactivated EB1 variant (π-EB1) by inserting a blue-light-sensitive protein–protein interaction module between the microtubule-binding and +TIP-binding domains of EB1. π-EB1 replaces endogenous EB1 function in the absence of blue light. By contrast, blue-light-mediated π-EB1 photodissociation results in rapid +TIP complex disassembly, and acutely and reversibly attenuates microtubule growth independent of microtubule end association of the microtubule polymerase CKAP5 (also known as ch-TOG and XMAP215). Local π-EB1 photodissociation allows subcellular control of microtubule dynamics at the second and micrometre scale, and elicits aversive turning of migrating cancer cells. Importantly, light-mediated domain splitting can serve as a template to optically control other intracellular protein activities.

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Fig. 1: Design of a light-sensitive EB1 variant that can replace endogenous EB1 function.
Fig. 2: Spatially and temporally reversible photodissociation of +TIP complexes.
Fig. 3: Attenuation of MT growth by π-EB1 photodissociation.
Fig. 4: π-EB1 photodissociation-induced MT cytoskeleton reorganization.
Fig. 5: EB1-independent MT plus-end localization of the MT polymerase CKAP5.
Fig. 6: Aversive cell turning in response to local π-EB1 photodissociation.

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Acknowledgements

This work was supported by the NIH grants R01 GM079139, R01 GM094819 and S10 RR26758 to T.W., and P41 EB002025 and R35 GM122596 to K.M.H. We thank all members of the Cell and Tissue Biology community for discussions and comments on the manuscript.

Author information

Author notes

  1. Andreas Ettinger

    Present address: Institute of Epigenetics and Stem Cells, Helmholtz Center Munich, München, Germany

Authors and Affiliations

  1. Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA

    Jeffrey van Haren, Rabab A. Charafeddine, Andreas Ettinger & Torsten Wittmann

  2. University of North Carolina, Chapel Hill, NC, USA

    Hui Wang & Klaus M. Hahn

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Contributions

J.v.H. and T.W. designed the experiments, analysed the data and wrote the manuscript. J.v.H. performed most of the experiments and generated most of the reagents. A.E. and R.A.C. contributed to reagent generation and experimental work. H.W. and K.M.H. contributed unpublished reagents.

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Correspondence to Torsten Wittmann.

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Supplementary information

Supplementary Information

Supplementary Figures 1–5, and Supplementary Table and Supplementary Video descriptions.

In the version of the Supplementary Information originally published with this Article, Supplementary Tables 2 and 3 were incorrectly described as ‘List of antibodies’ and ‘List of PCR primer and gRNA sequences’, respectively. The correct descriptions are ‘List of PCR primers and gRNA sequences’ for Supplementary Table 2, and ‘Statistics source data’ for Supplementary Table 3. In addition, Supplementary Video 7 was incorrectly described as ‘Local control of MT growth. CKAP5 association with MT ends is independent of π-EB1 photo-dissociation’. The correct description is ‘CKAP5 association with MT ends is independent of π-EB1 photo-dissociation’. These errors have now been corrected. Further, Supplementary Figures 1–5 of the Supplementary Information file published were of low quality. The file has now been replaced to rectify this.

Life Sciences Reporting Summary

Supplementary Table 1

List of antibodies.

Supplementary Table 2

List of PCR primers and gRNA sequences.

Supplementary Table 3

Statistics source data.

Videos

Supplementary Video 1

Light-mediated SLAIN2 dissociation from MT ends in a π-EB1/EB1 shRNA cell.

Supplementary Video 2

Spatiotemporal control of π-EB1 photo-dissociation.

Supplementary Video 3

Reversible inhibition of MT growth by π-EB1 photo-dissociation.

Supplementary Video 4

Local control of MT growth

Supplementary Video 5

Light-induced MT depolymerization by π-EB1 photo-dissociation.

Supplementary Video 6

Light-induced MT reorganization by π-EB1 photo-dissociation.

Supplementary Video 7

CKAP5 association with MT ends is independent of π-EB1 photo-dissociation.

Supplementary Video 8

CKAP5 dynamics in EB1/3–/– cells.

Supplementary Video 9

Light-induced MT growth inhibition in π-EB1-expressing EB1/3–/– cells.

Supplementary Video 10

π-EB1-expressing EB1/3–/– cell trapped in a virtual blue light box.

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van Haren, J., Charafeddine, R.A., Ettinger, A. et al. Local control of intracellular microtubule dynamics by EB1 photodissociation. Nat Cell Biol 20, 252–261 (2018). https://doi.org/10.1038/s41556-017-0028-5

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