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Membrane ruffling is a mechanosensor of extracellular fluid viscosity

Nature Physics volume 18, pages 1112–1121 (2022)Cite this article

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Abstract

Cell behaviour is affected by the physical forces and mechanical properties of cells and their microenvironment. The viscosity of extracellular fluid—a component of the cellular microenvironment—can vary by orders of magnitude, but its effect on cell behaviour remains largely unexplored. Using biocompatible polymers to increase the viscosity of the culture medium, we characterize how viscosity affects cell behaviour. We find that multiple types of adherent cell respond in an unexpected but similar manner to elevated viscosity. In a highly viscous medium, cells double their spread area, exhibit increased focal adhesion formation and turnover, generate significantly greater traction forces and migrate nearly two times faster. We observe that when cells are immersed in a regular medium, these viscosity-dependent responses require an actively ruffling lamellipodium—a dynamic membrane structure at the front of the cell. We present evidence that cells utilize membrane ruffling to sense changes in extracellular fluid viscosity and to trigger adaptive responses.

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Fig. 1: Elevated viscosity enhances single-cell motility and induces graded, reversible cell spreading.
Fig. 2: Membrane ruffling is critical for the cellular responses to viscosity changes and is inversely correlated with viscosity-dependent cell spreading.
Fig. 3: Viscosity-dependent spreading is mediated by integrin.
Fig. 4: Actin polymerization is required for cellular response to elevated viscosity, but myosin II contractility is not.
Fig. 5: Cells generate stronger forces at high viscosity independent of myosin II activity.

Data availability

Source data are available for this paper. All other data that support the plots within this paper and other findings of this study are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank K. M. Fish and A. Doyle for providing scientific insight. Funding: National Heart, Lung, and Blood Institute F31 HL154709 (M.P.); National Institute of Biomedical Imaging and Bioengineering S10 OD025193 and National Institute of Biomedical Imaging and Bioengineering R21 EB029677 (Y.C. and J.C.); Air Force Office of Scientific Research 21RT0264—FA9550-21-1-0284 (Y.C.); National Cancer Institute F99 CA253759 (W.-H.J.); Canadian Institutes of Health Research (PJT-178272) and Natural Sciences and Engineering Research Council of Canada (RGPIN-2020-05881) (S. Plotikov); Ontario Graduate Scholarship and Natural Sciences and Engineering Research Council of Canada PGS-D (E.I.); National Science Foundation 137959 (J.L.).

Author information

Authors and Affiliations

  1. Department of Mechanical Engineering, Johns Hopkins University, Baltimore, MD, USA

    Matthew Pittman, Keva Li, Mingjiu Wang, Junjie Chen, Seungman Park, Wei-Hung Jung, Le Liang, Ishan Barman & Yun Chen

  2. Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD, USA

    Matthew Pittman, Keva Li, Mingjiu Wang, Junjie Chen, Seungman Park, Wei-Hung Jung & Yun Chen

  3. Center for Cell Dynamics, Johns Hopkins University, Baltimore, MD, USA

    Matthew Pittman, Keva Li, Mingjiu Wang, Junjie Chen, Seungman Park, Wei-Hung Jung & Yun Chen

  4. Department of Cell & Systems Biology, University of Toronto, Toronto, Ontario, Canada

    Ernest Iu & Sergey Plotnikov

  5. Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN, USA

    Nilay Taneja & Dylan Burnette

  6. Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA

    Myung Hyun Jo & Taekjip Ha

  7. Department of Civil and Systems Engineering, Johns Hopkins University, Baltimore, MD, USA

    Stavros Gaitanaros

  8. Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Jian Liu

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  1. Matthew Pittman
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Contributions

M.P. and Y.C. devised the experiments and interpreted the results. M.P. performed most of the experiments and data analysis. K.L. performed the motility experiments and analysis. E.I. and S. Plotnikov performed the traction force microscopy and actin retrograde flow experiments and analysed the data. N.T. performed the laser ablation experiments and myosin imaging and analysed the data. M.H.J. performed the TGT experiments. S. Park conducted the simulations. S.G. performed the beam buckling analysis. J.L. performed the computational modelling. M.W., J.C., W.-H.J. and L.L. assisted with the experiments and analysis. M.P. and Y.C. wrote the manuscript; all the other authors provided editorial advice.

Corresponding authors

Correspondence to Jian Liu, Dylan Burnette, Sergey Plotnikov or Yun Chen.

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The authors declare no competing interests.

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Nature Physics thanks Wanda Strychalski 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 Characterization of viscous media and cellular responses to increased viscosity.

a) Viscous solutions were measured using a rheometer; shown are viscosity vs. shear curves for various media used in this study. b) Viscosity of each solution measured at 37o C at 100 Hz shear. c,d) Viscous medium does not significantly affect cell persistence in c) MDA-MB-231 cells or in d) NIH 3T3 fibroblasts. Direction autocorrelation was calculated using DiPer3; in c) N = 305, 370, and 176 for DMEM, 1% MC, and 1% PEO, respectively; in d) N = 120, 120, and 102 for DMEM, 1% MC, and 1% PEO, respectively; error bars: SEM. e,f) Viscous medium induced speed increases across cell types. e) Treating RAW 264.7 macrophages with 1% MC led to an increase in cell speed. Error bars: SD; p < 0.0001 (****); N = 37 (DMEM), 51 (1% MC). f) Treating AG19642 non-wound healing human fibroblasts with 1% MC led to an increase in cell speed. Error bars: SD; p < 0.001 (***); N = 64 (DMEM), 57 (1% MC).

Extended Data Fig. 2 Cells became flatter as they spread in viscous medium.

a) Time lapse xz-slices showed MDA-MB-231 cells, labelled with eGFP-F-tractin, flattening in viscous medium. 1% MC added at 1 minute; scale: 10 μm. b) Cell height was measured 4 μm towards the cell center from the leading edge. Error bars: SD; p ≥ 0.05 (n.s.), < 0.01 (**); N = 4 cells. c) Representative images of an MDA-MB-231 nucleus xz-cross-section stained with Hoescht showing nuclear shape change in viscous medium; 1% MC added at 1 minute; scale: 10 μm. d) Nuclear height decreased, e) nuclear width increased, and f) nuclear volume decreased slightly after MDA-MB-231 cells were treated with 1% MC at 1 minute; error bars: SD; significance reported with respect to DMEM group; p ≥ 0.05 (n.s.), < 0.1 (*), < 0.01 (**), < 0.001 (***), < 0.0001 (****); N = 12 cells.

Extended Data Fig. 3 Characterization of the role of integrin and myosin II in cell spreading and traction force generation in response to increased viscosity.

a) Representative images of an MDA-MB-231 cell on a PLL-coated substrate show no spreading following the addition of 1% MC. Scale: 20 μm. b,c) Both the b) maximum traction stresses and c) total traction forces increased in 1% MC for both untreated and blebbistatin-treated cells. Error bars: SD; p ≥ 0.05 (n.s.), < 0.05 (*); < 0.01 (**); N = 15 cells for each condition. d) Representative SIM images of myosin IIA staining in MDA-MB-231 cells after 30 minutes incubation in 1% MC show the formation of myosin stacks. Scale: 10 μm.

Extended Data Fig. 4 Laser ablation showed decreased cortical tension upon addition of viscous medium followed by a return to baseline.

Both the a) initial cut distance and b) retraction rate showed a decrease and then recovery in 1% MC. Time refers to duration of incubation in 1% MC; error bars: SD; p ≥ 0.05 (n.s.), < 0.05 (*); < 0.001 (***); N = 19 (control), 6 (0–5 min), 9 (5–15 min), 25 (>15 min). c) The time courses of membrane ruffling and cortical tension of cells subjected to 1% MC reveal that ruffle disappearance began instantaneously upon medium change, but a significant reduction in cortical tension occurred only after a 10-minute delay. d) Representative cell images and e) zoomed-in regions show actin arc retraction following laser ablation; white squares highlight ablated regions; ablations performed at 2 seconds; scale: d) 20 μm, e) 10 μm.

Extended Data Fig. 5 Simulations of protruding and ruffling lamellipodia show increased drag in viscous medium.

a) Ruffles in 1% MC experience drag two orders of magnitude greater than ruffles in regular medium. Ruffling was modeled as a periodic behavior with a 20-second period starting with the cell membrane at the leading edge detaching from the substrate to which it was adhered. b,c) Maximal and average drag forces on a protruding lamellipodia in DMEM and in 1% MC were estimated using finite element analysis. d) Force decomposition shows that the force generated by actin to drive protrusion, FProtrusion, and the retrograde forces of contractility and tension that ultimately lead to ruffling, FRetrograde, are oriented almost exclusively in the xy-plane; therefore, FRuffle, the z-oriented component of FRetrograde directly responsible for initiating ruffles, is extremely small. e) In the lamellipodium, the force required to cause buckling increases proportionally with the coefficient of viscous drag. In highly viscous medium, cells are incapable of generating the greatly increased forces required to cause buckling to the same extent as in regular medium; instead, lamellipodial buckling is reduced.

Extended Data Fig. 6 MDA-MB-231 cells spread rapidly in response to increased viscosity despite perturbations to membrane availability and passive membrane reservoirs.

A) Cells treated with 80 μM dynasore to suppress endocytosis underwent rapid spread area increases when exposed to 1% MC. Envelope: SEM; N = 22. B) Cell spread area did not change in response to hypertonic treatment (530 mOsm) and then increased in response to 1% MC following hypertonic treatment. Envelope: SEM; N = 23. C) Following hypotonic treatment (160 mOsm), which led to a small decrease in cell spread area, cell spreading in 1% MC was slowed slightly due to increased membrane tension; spreading increased substantially when cells were exposed to 2% MC. Envelopes: SEM; N = 27 (hypotonic 1% MC), 27 (hypotonic 2% MC).

Extended Data Fig. 7 Methods for ruffle quantification and modelling.

a) To quantify membrane ruffling, confocal z-stacks of fluorescent F-tractin were acquired at 1-minute intervals. The summed projection of these stacks created a strong signal in the tall, actin-rich ruffles, and this signal became weaker as the ruffles changed location or flattened. For each projection, a threshold was manually selected to include membrane ruffling across all frames while excluding other actin structures. The ruffle index (arbitrary unit) was then generated by integrating the thresholded area and normalizing each frame to the mean ruffle area of all the frames captured in DMEM. b) Schematic diagram depicting the interactions between model components.

Supplementary information

Supplementary Information

Supplementary Methods, References, captions for Supplementary Figs. 1–7 and Supplementary Videos 1–15.

Reporting Summary

Supplementary Video 1

On addition of 1% MC, MDA-MB-231 cells begin to migrate more quickly. Two representative cells have been circled (blue/red circles) to highlight the speed increase. The viscous medium was added after 180 min; scale, 200 μm.

Supplementary Video 2

Cells begin to spread immediately on the addition of the viscous medium. 1% MC added at 3:50. Left, eGFP-F-tractin; centre, IRM; right, bright field; time stamp, mm:ss; scale, 20 μm.

Supplementary Video 3

Three-dimensional render of a cell increasing its spread area on the addition of 2% MC. The render was created from a confocal z-stack time lapse of an MDA-MB-231 cell transfected with eGFP-F-tractin. The viscous medium was added at 10:55. Colour coded for height, where cooler colours are higher. The video is displayed at 25 fps; time stamp, mm:ss.

Supplementary Video 4

Three-dimensional render of HEK 293 cells transfected with YFP-EGFR spreading in a viscous medium. The viscous medium was added at 16:30. Colour coded for height, where cooler colours are higher. The video is displayed at 25 fps; time stamp, mm:ss.

Supplementary Video 5

MDA-MB-231 cells increase spread area and membrane ruffles disappear on the addition of 2% MC. The viscous medium is added at 11:45. Left, bright field; right, IRM enhanced with a shadow filter to boost edge contrast. The video is displayed at 70 fps; time stamp, mm:ss; scale, 20 μm.

Supplementary Video 6

Membrane ruffling is suppressed in an MDA-MB-231 cell treated with 2% MC, and then ruffling recovers after dilution to reduce viscosity. The viscous medium is added at 19:50. Between 53:22 and 61:18, the viscous medium is repeatedly diluted with a regular medium to rapidly reduce viscosity. The video is displayed at 25 fps; time stamp, mm:ss; scale, 20 μm.

Supplementary Video 7

An example atypical keratocyte exhibited ruffling and was not motile; following the addition of 1% MC at 07:30, ruffling decreased as the cell increased its spread area and became motile. The video is displayed at 20 fps; time stamp, mm:ss; scale, 20 μm.

Supplementary Video 8

Addition of 1% MC induces explosive remodelling and increased turnover of FAs in MDA-MB-231 cells. The viscous medium is added at 07:10. Left, mEmerald-talin; centre, IRM; right, bright field. The video is displayed at 20 fps; time stamp, mm:ss; scale, 20 μm.

Supplementary Video 9

Viscosity-induced spreading is severely attenuated by inhibiting actin polymerization via latA treatment. DMEM with 100 nM latA was added at 7:30, and 1% MC (also with 100 nM latA) was added at 24:30. Time stamp, mm:ss; scale, 20 μm.

Supplementary Video 10

Laser ablation shows that both retraction rate and initial cut size of ablated actin arcs decrease in MDA-MB-231 cells labelled with eGFP-F-tractin after 5 min incubation in 1% MC and recover after 30 min incubation in 1% MC. Left, 5 min after 1% MC treatment; right, 30 min after 1% MC treatment. Ablation is performed at 2 s. The video is displayed at 8 fps; time stamp, mm:ss; scale, 20 μm.

Supplementary Video 11

Total internal reflection fluorescence microscopy of MDA-MB-231 cells labelled with emiRFP670-paxillin reveals extensive nascent adhesion growth following the addition of 1% MC. The viscous medium is added at 01:15. Left, emiRFP670-paxillin; centre, eGFP-F-tractin; right, combined. The video is displayed at 20 fps; time stamp, mm:ss; scale, 20 μm.

Supplementary Video 12

Simulation of membrane ruffling in a regular medium (DMEM).

Supplementary Video 13

Simulation of membrane ruffling in a viscous medium (1% MC).

Supplementary Video 14

Tutorial video showing the preparation of and imaging with a viscous medium.

Supplementary Video 15

Nominal-case model simulation showing the evolution of FA growth coupled with membrane protrusion and actin retrograde flux. The simulation starts with a nascent FA of 200 nm radius. The grey scale indicates the intensity of FA, which corresponds to the local level of anchored integrin—the sum of [E-I-A] and [E-I]. The membrane position is indicated by the upper boundary. The lengths of the magenta arrows scale with the velocity of the local actin flux. The total length of the simulation is 300 s.

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Statistical source data.

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Statistical source data.

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Statistical source data.

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Statistical source data.

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Pittman, M., Iu, E., Li, K. et al. Membrane ruffling is a mechanosensor of extracellular fluid viscosity. Nat. Phys. 18, 1112–1121 (2022). https://doi.org/10.1038/s41567-022-01676-y

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