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The actin cortex acts as a mechanical memory of morphology in confined migrating cells

Nature Physics volume 21, pages 1451–1461 (2025)Cite this article

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Abstract

Cell migration in narrow microenvironments occurs in numerous physiological processes. It involves successive cycles of confinement and release that drive important morphological changes. However, it remains unclear whether migrating cells can retain a memory of their past morphological states that could potentially facilitate their navigation through confined spaces. We demonstrate that local geometry governs a switch between two cell morphologies, thereby facilitating cell passage through long and narrow gaps. We combined cell migration assays on standardized microsystems with biophysical modelling and biochemical perturbations to show that migrating cells have a long-term memory of past confinement events. The morphological cell states correlate across transitions through actin cortex remodelling. These findings indicate that mechanical memory in migrating cells plays an active role in their migratory potential in confined environments.

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Fig. 1: A morphological switch ensures a successful crossing on long bridges.
Fig. 2: Impact of symmetry-breaking and polarization states on cell dynamics.
Fig. 3: Morphological switch dynamics and mechanical memory.
Fig. 4: Pharmacological perturbation of mechanical memory dynamics.
Fig. 5: Geometrical perturbation of the mechanical memory dynamics.

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Data availability

All data are available upon request to the corresponding authors. Source data are provided with this paper.

Code availability

Codes are available upon request to D.B.B.

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Acknowledgements

We are grateful to members of S.G.’s laboratory for feedback and suggestions. We thank E. Hannezo, J. O. Rädler, M. Piel, O. du Roure and J. Heuvingh for inspiring discussions. Y.K. and S.G. acknowledge J. B. Braquenier from Nikon Instruments Belux and the Nikon BioImaging Lab in Leiden (the Netherlands) for their support with the Nikon Spatial Array Confocal enhanced-resolution confocal microscopy. We thank D. S. Herrador and M. Balland for their help in improving the microprinting method. D.B.B. was supported by the NOMIS Foundation as a NOMIS Fellow and by an EMBO Postdoctoral Fellowship (ALTF 343-2022). Y.K., M.L. and S.G. acknowledge funding from the University of Mons (FEDER Prostem Research Project no. 1510614, Wallonia DG06), the F.R.S.-FNRS (Epiforce Project no. T.0092.21, Cellsqueezer Project no. J.0061.23 and Optopattern Project no. U.NO26.22) and the Interreg projects ANTIRESI and MICROPLAITE, which are financially supported by Interreg France-Wallonie-Vlaanderen (Fonds Européen de Développement Régional). Y.K. and M.L. are financially supported by F.R.S.-FNRS as FRIA Grantee FNRS and Postdoctoral Fellow (Chargé de Recherches), respectively. Y.K. and S.G. acknowledge le Fonds pour la Recherche Médicale dans le Hainaut (FRMH). G.C. was supported by a grant from the Biotechnology and Biological Sciences Research Council (grant no. BB/V007483/1).

Author information

Authors and Affiliations

  1. SYMBIOSE Lab, Research Institute for Biosciences, CIRMAP, University of Mons – UMONS, Mons, Belgium

    Yohalie Kalukula, Marine Luciano & Sylvain Gabriele

  2. London Centre for Nanotechnology, University College London, London, UK

    Gleb Simanov & Guillaume Charras

  3. Institute for the Physics of Living Systems, University College London, London, UK

    Gleb Simanov & Guillaume Charras

  4. Department of Cell and Developmental Biology, University College London, London, UK

    Gleb Simanov & Guillaume Charras

  5. Biozentrum, University of Basel, Basel, Switzerland

    David B. Brückner

  6. Department of Physics, University of Basel, Basel, Switzerland

    David B. Brückner

  7. Institute for Science and Technology Austria, Klosterneuburg, Austria

    David B. Brückner

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  1. Yohalie Kalukula
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  2. Marine Luciano
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  4. Guillaume Charras
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  5. David B. Brückner
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Contributions

S.G. and Y.K. conceived of the project, and S.G. supervised the project. Y.K. developed the micropatterns and performed the time-lapse cell experiments, cell tracking and confocal imaging. D.B.B. developed the theoretical model and performed the simulations. M.L. contributed to the experiments and commented on the paper. G.C. provided cell lines and commented on the paper. G.S. conceived of the Arpc1b MCF-10A cell line. Y.K., D.B.B. and S.G. analysed the data and prepared the figures. The article was written by Y.K., D.B.B. and S.G. and read and corrected by all authors, who each contributed to the interpretation of the results.

Corresponding authors

Correspondence to David B. Brückner or Sylvain Gabriele.

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Nature Physics thanks Falko Ziebert and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Examples of morphological switch on glass fiber and in 3D collagen fiber matrix.

(a) Time-lapse sequence of a single MDCK epithelial cell migrating on a smooth glass wire (diameter = 5.3 µm). After detaching from the tissue, the epithelial cell rounds up and migrate on the glass fiber. The temporal evolution of the long cell axis and the migration speed indicates that the cell morphological switch from an elongated to a compacted morphology is associated with an increase in migration speed. Scale bar, 50 µm. Adapted with permission from 17. (b) Time-lapse sequence of a single fibroblast (NIH3T3) migrating in a 3D matrix composed of aligned collagen fibers. Scale bar, 50 µm. The temporal evolution of the long cell axis and the migration speed indicates that the cell morphological switch from an elongated to a compacted morphology is associated with an increase in migration speed with a maximum speed around 80 µm/min. Adapted with permission from 18.

Source data

Extended Data Fig. 2 Semi-infinite narrow segments.

(a) Theoretical expectation of the percentage of compacted cell on the bridge of dumbbells (W = 6 µm and L = 160 µm) with square area of 1600 µm2 versus 1D semi-infinite line of W = 6 µm and L = 500 µm. On 6 µm line, percentage of compacted cell is expressed as the percentage of time spent under the compacted morphology over a 20-hour time-lapse. (b) Time-lapse sequence of a compacted cell morphology migrating on a one-dimensional (1D) micropatterned line of W = 6 µm and L = 500 µm. Scale bar, 20 µm. (c) Representative color-coded trajectories with elongated (CSI < 0.4) and compacted (CSI > 0.4) morphologies. (d) Percentage of compacted cells on the bridge of dumbbells (W = 6 µm and L = 160 µm) with square area of 1600 µm2 (n = 56, N = 16) versus 1D semi-infinite line of W = 6 µm and L = 500 µm (n = 10, N = 2). On 6 µm line, percentage of compacted cell is expressed as the percentage of time spent under the compacted morphology over a 20-hour time-lapse.

Source data

Extended Data Fig. 3 Analysis of memory dynamics from cell trajectories.

(a) Selection of n = 55 color-coded single cell trajectories of individual MCF-10A cells migrating on FN dumbbell micropatterns for 20 hours. Trajectories on the bridge are color-coded by state: elongated (CSI < 0.4) and compacted (CSI > 0.4) morphologies. (b) State trajectories computed from position trajectories in (A). A morphological state is assigned to each transition on the bridge, based on the morphological state adopted for most time-points during each transition. (c) Memory analysis tree diagram for events C (compacted transition) and E (elongated transition) obtained from state trajectories in (b). Numbers of the branches indicate probabilities. (d) Trajectories of successful and unsuccessful transitions, defined as follows: in successful transitions, the cell (nucleus) enters the bridge and transmigrates to the other island. In unsuccessful transitions, the nucleus enters the bridge and returns to the same island. Fraction of unsuccessful, successful and total transition, which are repartitioned into two categories, elongated and compacted.

Extended Data Fig. 4 Morphological switching dynamics and mechanical memory on 320 µm-long bridges.

(a) Representative sequence of a back-and-forth motion of an individual MCF-10A cell migrating on an FN dumbbell micropattern with a 320 µm-long bridge. (b) Selection of n = 50 color-coded cell trajectories of individual MCF-10A cells migrating on FN dumbbell micropatterns for 30 hours. Trajectories on the bridge are color-coded with elongated (CSI < 0.4) and compacted (CSI > 0.4) morphologies, and square zones. (c) Statistical tree representation of the experimental probabilities for elongated and compacted states over three generation of successive crossings (N ≥ 3 replicates for each condition). (d) Histogram representation of the probabilities for elongated, P(E), and compacted, P(C), states for various combinations of morphological switches: elongated to elongated P(E | E), compacted to compacted P(C | C), elongated to compacted P(E | C) and compacted to elongated P(C | E). Data are presented as mean values ± SD.

Source data

Extended Data Fig. 5 Physico-chemical footprints.

(a) Schematic representation of the two experimental procedures used to assess pattern conditioning by migrating cells. Cells were seeded on human plasma (hp) fibronectin (FN) micropattern, then detached and fixed either (i) after 5 hours (before the time-lapse) or (ii) 24 hours (after the time-lapse) of culture on micropatterns. (b) Epifluorescence images showing human plasma fibronectin, cellular laminin and cellular FN in control conditions after t = 5 h and t = 24 h of cell culture (top to bottom). Scale bar, 50 μm. (c) Normalized laminin intensity for control (n = 62 patterns, N = 3), 5-hour conditioning (n = 67 patterns, N = 3), and 24-hour conditioning (n = 46 patterns, N = 3). (d) Normalized fibronectin intensity from control (n = 73 patterns, N = 3), 5-hour conditioning (n = 125 patterns, N = 3), and 24 hour-conditioning (n = 74 patterns, N = 3). Boxplots range from the first quartile (Q1) to the third quartile (Q3), with the median (50th percentile) indicated by a line. Whiskers extend from the box to the minimum and maximum data points within 1.5 times the interquartile range.**p < 0.01, ns = not significant (Kruskal-Wallis test).

Source data

Extended Data Fig. 6 Spatial distribution of microtubule filaments.

Confocal microscopy images in enhanced-resolution mode of the basal planes of (a) elongated (left) and (b) compacted (right) cell morphologies. β-tubulin staining (inverted image) and the cell body outline is represented with a black dashed line. Scale bar, 20 µm. In bottom images, β-tubulin filaments in elongated and compacted cells were color-coded according to their spatial orientation. Scale bar, 20 µm. Distribution of the normalized intensity of microtubules (c) at the front and (d) at the rear of elongated (n = 3, N = 2) and compacted (n = 2, N = 2) cell morphologies. (e) Front-to-rear tubulin intensity ratio of elongated (n = 48, N = 3) and compacted (n = 54, N = 3) morphologies during crossing events. Boxplots range from the first quartile (Q1) to the third quartile (Q3), with the median (50th percentile) indicated by a line. Whiskers extend from the box to the minimum and maximum data points within 1.5 times the interquartile range. ****p < 0.0001 (Student’s t test).

Source data

Extended Data Fig. 7 Myosin cortex in elongated and compacted cells.

Typical equatorial focal plane obtained in enhanced-resolution microscopy showing the spatial distribution of phosphorylated myosin light chain (p-MLC) at the actin cortex in (a-b) elongated and (c-d) compacted cells. Plot profiles in (b) and (c) shows the normalized intensity of actin and p-MLC as a function of the distance from the membrane. (e) Full width at half maximum (FWHM) for myosin cortex in elongated (orange, n = 29) and compacted (purple, n = 29) cells. Boxplots range from the first quartile (Q1) to the third quartile (Q3), with the median (50th percentile) indicated by a line. Whiskers extend from the box to the minimum and maximum data points within 1.5 times the interquartile range. ****p < 0.0001 (Student’s t test). Scale bars, 10 µm.

Source data

Extended Data Fig. 8 Effect of a nocodazole treatment on confined migration.

(a) Time-lapse sequence of a compacted cell entering a bridge before (control, CTRL) and after treatment with nocodazole (orange). CTRL is DMSO for nocodazole experiments. Scale bar, 10 µm. White arrows show the direction of migration. (b) Cell shape index on bridge for CTRL (n = 21, N = 5) and nocodazole-treated cells (n = 23, N = 6). (c) Cell area on square for CTRL (n = 18, N = 6) and nocodazole-treated cells (n = 22, N = 7). (d) Successful crossing percentage for CTRL (n = 146, N = 18) and nocodazole-treated cells (n = 33, N = 8) and (e) dwell time on square for CTRL (n = 22, grey) and nocodazole-treated cells (n = 12, light orange). Boxplots range from the first quartile (Q1) to the third quartile (Q3), with the median (50th percentile) indicated by a line. Whiskers extend from the box to the minimum and maximum data points within 1.5 times the interquartile range.****p < 0.0001 and n.s. = not significant (Student’s t test).

Source data

Extended Data Fig. 9 Thickness of the actin cortex in cells spread on squares.

Enhanced-resolution confocal images of (a) a partial spreading and (b) a full spreading of epithelial cells on FN square of 1600 µm2. Cells are immunostained for F-actin with phalloidin (in green) and DNA with DAPI (in bue). Inverted images show normal and side views of the actin cytoskeleton at the apical side. The spreading rate is 50.1% for partial spreading (a) and 87.2% for full spreading (b). Scale bars, 20 µm. (c) Full width at half maximum (FWHM) represents the actin cortex thickness for partial (n = 14, N = 4) and full (n = 18, N = 4) spreading conditions. Boxplots range from the first quartile (Q1) to the third quartile (Q3), with the median (50th percentile) indicated by a line. Whiskers extend from the box to the minimum and maximum data points within 1.5 times the interquartile range. ****p < 0.0001 (Student’s t test).

Source data

Extended Data Fig. 10 Experimentally measured and simulated percentages of successful crossing on interconnected dumbbell micropattern.

Percentages of successful crossing with (a) 900 µm2 squares for elongated (E, n = 34, N = 3) and compacted (C, n = 34, N = 3) and (b) 2500 µm2 squares for elongated (E, n = 50, N = 3) and compacted (C, n = 50, N = 3). Experimentally measured and model-estimated percentages of repolarization during successful crossing on interconnected dumbbell micropattern with (c) 900 µm2 squares for elongated (E, n = 21, N = 3) and compacted (C, n = 30 cells, N = 3)cells and (d) 2500 µm2 squares for elongated (E, n = 41, N = 3) and compacted (C, n = 27 cells, N = 3) cells. Data are presented as mean values ± SD. ****p < 0.0001 (Student’s t test).

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–8, Video captions 1–18 and Theory note.

Reporting Summary

41567_2025_2980_MOESM3_ESM.mp4

Supplementary Video 1. Time-lapse sequences showing the back-and-forth motion of an individual MCF-10A epithelial cell on dumbbell-shaped fibronectin micropatterns with bridge lengths of 40 µm, 80 µm, 120 µm, 160 µm and 320 µm and square ends measuring 40 µm × 40 µm. The cell is imaged in differential interference contrast (DIC) mode, with the micropattern labelled with rhodamine-conjugated fibronectin and the nucleus stained with Hoechst. The scale bar represents 20 µm and 50 µm for 320 µm bridge length, the frame rate is 3 min per frame.

41567_2025_2980_MOESM4_ESM.mp4

Supplementary Video 2. Typical sequence of automatic tracking of an individual MCF-10A cell observed in DIC mode and analysed using the Fiji plugin Cellpose 2.0, which provides the cell outline for each frame. This enables the determination of the cell’s spreading area, perimeter and the coordinates of the cell front, rear and centre of mass.

41567_2025_2980_MOESM5_ESM.mp4

Supplementary Video 3. Representative time-lapse sequences (t = 20 h) showing the elongated and compacted morphologies adopted by an individual MCF-10A epithelial cell during its back-and-forth motion on a dumbbell-shaped fibronectin micropattern with a bridge length of 160 µm. The cell is imaged using differential interference contrast (DIC) microscopy, with the micropattern labelled with rhodamine-conjugated fibronectin and the nucleus stained with Hoechst. The scale bar represents 20 µm, and images were captured at 3-min intervals.

41567_2025_2980_MOESM6_ESM.mp4

Supplementary Video 4. Representative time-lapse sequences showing both a failed (t = 24 h) and a successful (t = 3 h and 51 min) crossing event during the back-and-forth motion of an individual MCF-10A epithelial cell on a dumbbell-shaped fibronectin micropattern with a bridge length of 160 µm. The cell is imaged using differential interference contrast (DIC) microscopy, with the micropattern labelled with rhodamine-conjugated fibronectin and the nucleus stained with Hoechst. The scale bar represents 20 µm, and images were captured at 3-min intervals.

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Supplementary Video 5. Time-lapse sequences of actin retrograde flow in both elongated and compacted lamellipodia, observed in confocal mode using a ×100 silicone objective (N.A. = 1.35). Images were acquired at a rate of one frame every 1.25 s for a total duration of 5 min.

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Supplementary Video 6. High-resolution confocal Z-stack sequence (Nikon Spatial Array Confocal, NSPARC) of an elongated cell morphology, obtained usinga galvano scanner at × 60 magnification (Plan Apo oil immersion objective, N.A. 1.42) with a step size of 0.10 μm for three channels (DAPI, TRITC and FITC).Actin filaments are shown in black, and DAPI is shown in blue.

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Supplementary Video 7. High-resolution confocal Z-stack images (Nikon Spatial Array Confocal, NSPARC) of a compacted cell morphology, obtained using agalvano scanner at × 60 magnification (Plan Apo oil immersion objective, N.A. 1.42) with a step size of 0.17 μm for three channels (DAPI, TRITC and FITC).Actin filaments are shown in black, and DAPI is shown in blue.

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Supplementary Video 8. Schematic movies of simulations of elongated (top), compacted (middle) and switching (bottom) cells moving through the confiningdumbbell pattern. Colour of the dot indicates state of polarization (orange: elongated, a = 1; purple: compacted, a = –2), and arrow indicates the polarity of thecell. Plots on the right indicate simultaneous migration trajectories along the x direction. The dumbbell-shaped micropattern is composed of a bridge of 160 μmin length and 6 μm in width connected at each extremity to a square of 40 μm × 40 μm.

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Supplementary Video 9. Series of time-lapse sequences in DIC mode of an epithelial cell (MCF-10A) migrating on a dumbbell-shaped fibronectinmicropattern, featuring a bridge 160 μm in length and 6 μm in width, with square ends measuring 40 μm × 40 μm. The first and second sequences depict,respectively, an elongated and a compacted cell both expressing Golgi targeted CellLight Golgi-GFP BacMam 2.0 (red) and the nucleus is stained blue withHoechst. The third sequence depicts a migrating MCF-10A cell expressing ArpC1B–mScarlet. Arp2/3 complex localization is shown in black with the nucleusin blue stained with Hoechst. Scale bar, 50 μm.

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Supplementary Video 10. Time-lapse sequences of a morphological switch from an elongated to a compacted state, captured in epifluorescence mode with actinfilaments labelled using SPY555-FastAct™ (t = 24 h) and in DIC mode (t = 24 h). The epithelial cell (MCF-10A) migrates on a dumbbell-shaped fibronectinmicropattern, featuring a bridge 160 μm in length and 6 μm in width, with square ends measuring 40 μm × 40 μm. The nucleus is stained blue with Hoechst.Scale bar, 40 μm.

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Supplementary Video 11. Time-lapse sequence in DIC mode of an epithelial cell (MCF-10A) migrating on fibronectin microstripe measuring 500 µm in length and 6 µm in width (t = 20 h). The nucleus is stained blue with Hoechst. Scale bar, 10 µm.

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Supplementary Video 12. Successive time-lapse sequences in DIC mode of an individual epithelial cell (MCF-10A) migrating on a dumbbell-shapedfibronectin micropattern, featuring a bridge 160 μm in length and 6 μm in width, with square ends measuring 40 μm × 40 μm. Nocodazole-treated cells exhibita loss of polarity, characterized by oscillations in the first sequence. In the second sequence, the cells fail to enter the bridge, and in the third sequence, theyremain compacted while also failing to enter the bridge. In the first sequence, the cell is treated with 2.5 μM nocodazole at t = 1 h and 30 min (t = 30 h). In thesecond and third sequences, the cell is treated with 2.5 μM nocodazole at t = 2 h and 30 min (t = 30 h).

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Supplementary Video 13. Time-lapse sequence in DIC mode of an epithelial cell (MCF-10A) migrating on a dumbbell-shaped fibronectin micropattern,featuring a bridge 160 μm in length and 6 μm in width, with square ends measuring 40 μm × 40 μm (t = 20 h and 55 min). The cell is treated with 20 nMLatrunculin B at t = 03 h and 50 min. Scale bar, 10 μm.

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Supplementary Video 14. Time-lapse sequence in DIC mode of an epithelial cell (MCF-10A) migrating on a dumbbell-shaped fibronectin micropattern,featuring a bridge 160 μm in length and 6 μm in width, with square ends measuring 40 μm × 40 μm (t = 21 h and 40 min). The cell is treated with 10 μMY27632 at t = 03 h and 15 min. Scale bar, 50 μm.

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Supplementary Video 15. Time-lapse sequence in DIC mode of an epithelial cell (MCF-10A) migrating on a dumbbell-shaped fibronectin micropattern,featuring a bridge 160 μm in length and 6 μm in width, with square ends measuring 30 μm × 30 μm (t = 20 h). The fibronectin micropattern is red and thenucleus in blue. Scale bar, 20 μm.

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Supplementary Video 16. Time-lapse sequence in DIC mode of an epithelial cell (MCF-10A) migrating on a dumbbell-shaped fibronectin micropattern, featuring a bridge 160 µm in length and 6 µm in width, with square ends measuring 50 µm × 50 µm (t = 20 h). The fibronectin micropattern is depicted in red, and the nucleus is stained blue. Scale bar, 20 µm.

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Supplementary Video 17. Time-lapse sequence in DIC mode of an epithelial cell (MCF-10A) migrating on a dumbbell-shaped fibronectin micropattern, featuring a bridge 160 µm in length and 6 µm in width, with square ends measuring 50 µm × 50 µm (t = 20 h). The cell is treated with 1 nM of Jasplakinolide 30 min before the beginning of the time-lapse experiment. The fibronectin micropattern is depicted in red, and the nucleus is stained blue. Scale bar, 20 µm.

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Supplementary Video 18. Time-lapse sequences in DIC mode of an MCF-10A cell migrating on interconnected dumbbell-shaped micropatterns with 160-μmlongbridges and square deconfinement zones of 2500 μm² (first sequence) and 900 μm² (second sequence). The first sequence spans 59 h and 54 min, and thesecond sequence lasts 17 h and 30 min. The nucleus is stained blue with Hoechst. Scale bar, 100 μm.

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Kalukula, Y., Luciano, M., Simanov, G. et al. The actin cortex acts as a mechanical memory of morphology in confined migrating cells. Nat. Phys. 21, 1451–1461 (2025). https://doi.org/10.1038/s41567-025-02980-z

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