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Boundary-sensing mechanism in branched microtubule networks

Nature Chemical Engineering volume 2, pages 498–510 (2025)Cite this article

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Abstract

The self-organization of cytoskeletal biopolymers, such as microtubules (MTs), depends on mechanosensing and adaptation to confined spaces such as cellular protrusions. Understanding how these active biopolymers coordinate their formation under confinement leads to advances in bioengineering. Here we report the self-organization of branched MT networks in channels with narrow junctions and closed ends, mimicking cellular protrusions. We find that branching MT nucleation occurs in the post-narrowing region only if this region exceeds a minimum length, determined by MT dynamic instability at the closed end and the timescale for nucleation at a distant point. We term this feedback ‘boundary sensing’. Increasing the amount of branching factor TPX2 in the system accelerates MT nucleation and adjusts this minimum length, but excess TPX2 stabilizes MTs at the closed end, disrupting network formation. We performed experiments and simulations to study how this tunable feedback, wherein growing MTs navigate confinement and create nucleation sites, shapes MT architecture. Our findings impact the understanding of MT self-organization during axonal growth, dendrite formation, plant development, fungal guidance and the engineering of biomaterials.

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Fig. 1: Branched MT network formation near a rigid planar boundary.
Fig. 2: MT network formation in a channel of varying width.
Fig. 3: Channel-length-dependent onset of branching MT nucleation in the post-narrowing region.
Fig. 4: Tuning the adaptive response.
Fig. 5: Computational model of MT boundary sensing with experimental comparisons.

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All data generated or analyzed during this study are included in this Article and its Supplementary Information files.

Code availability

The MATLAB codes used for simulations performed in this study are included in the Supplementary Information files.

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Acknowledgements

M.Z. acknowledges an Omenn-Darling Postdoctoral Fellowship from the Princeton Bioengineering Institute. This work was supported by the Princeton University Eric and Wendy Schmidt Transformative Technology Fund.

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Author notes

  1. These authors contributed equally: Meisam Zaferani, Ryungeun Song.

  2. These authors jointly supervised this work.

Authors and Affiliations

  1. Department of Molecular Biology, Princeton University, Princeton, NJ, USA

    Meisam Zaferani, Ryungeun Song, Ned S. Wingreen & Sabine Petry

  2. Department of Mechanical and Aerospace Engineering, Princeton University, Princeton, NJ, USA

    Meisam Zaferani, Ryungeun Song & Howard A. Stone

  3. Omenn-Darling Bioengineering Institute, Princeton University, Princeton, NJ, USA

    Meisam Zaferani

  4. School of Mechanical Engineering, Chungbuk National University, Cheongju-si, Republic of Korea

    Ryungeun Song

  5. Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA

    Ned S. Wingreen

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  1. Meisam Zaferani
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  2. Ryungeun Song
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  3. Ned S. Wingreen
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  4. Howard A. Stone
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Contributions

M.Z., N.S.W., H.A.S. and S.P. conceived the project. M.Z. and R.S. conducted the experiments, data analysis and simulations. M.Z., N.S.W. and H.A.S. developed the model. H.A.S. and S.P. supervised the project. All authors contributed to the writing of the paper.

Corresponding authors

Correspondence to Howard A. Stone or Sabine Petry.

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Nature Chemical Engineering thanks Yusuke T. Maeda and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Discussion and Figs. 1–4.

Reporting Summary

Supplementary Video 1

Self-organization of a branched microtubule (MT) network in front of a flat, rigid physical boundary. The average angle between the MT network and the wall is 85 ± 10°. MTs are visualized in magenta, and EB1 comets appear in green. Branching MT nucleation was induced by the addition of 10 µM RanQ69L.

Supplementary Video 2

Self-organization of a branched MT network within a microchannel containing a narrowing region and a closed end. Branching MT nucleation was induced by the addition of 10 µM RanQ69L.

Supplementary Video 3

Self-organization of a branched MT network within microchannels containing narrowing regions and post-narrowing regions of varying lengths. Branching MT nucleation was induced by the addition of 10 µM RanQ69L.

Supplementary Video 4

Growth of individual MTs within the narrowing region when it was empty (top) compared with growth when the narrowing region was pre-filled with 5–7 MTs (bottom). Branching MT nucleation was induced by the addition of 10 µM RanQ69L.

Supplementary Video 5

Self-organization of a branched MT network through a narrowing region approximately 400 nm in width. Branching MT nucleation was induced by the addition of 10 µM RanQ69L.

Supplementary Video 6

Self-organization of branched MT networks within a microchannel containing two narrowing and two post-narrowing regions. Branching MT nucleation was induced by the addition of 10 µM RanQ69L.

Supplementary Video 7

Self-organization of a branched MT network within microchannels containing narrowing regions and post-narrowing regions of length L = 9 µm in the presence of 10 µM RanQ69L and 350 nM TPX2. Branching MT nucleation was induced by the combined addition of RanQ69L and TPX2.

Supplementary Video 8

Self-organization of a branched MT network within microchannels containing narrowing regions and post-narrowing regions of lengths L = 5 µm and L = 15 µm in the presence of 10 µM RanQ69L and 1 µM TPX2. Branching MT nucleation was induced by the combined addition of RanQ69L and TPX2.

Supplementary Video 9

Computational simulations of branched MT network self-organization in a symmetric bifurcation with side channels of lengths L = 5 µm and L = 15 µm (top). Simulations of branched MT network self-organization in a symmetric bifurcation with higher TPX2 concentration in one side channel (bottom).

Supplementary Video 10

Self-organization of a branched MT network in a symmetric bifurcation with side channels of lengths L = 5 µm and L = 15 µm. Branching MT nucleation was induced by the addition of 10 µM RanQ69L.

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Zaferani, M., Song, R., Wingreen, N.S. et al. Boundary-sensing mechanism in branched microtubule networks. Nat Chem Eng 2, 498–510 (2025). https://doi.org/10.1038/s44286-025-00264-0

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