Non-equilibration of hydrostatic pressure in blebbing cells

Abstract

Current models for protrusive motility in animal cells focus on cytoskeleton-based mechanisms, where localized protrusion is driven by local regulation of actin biochemistry1,2,3. In plants and fungi, protrusion is driven primarily by hydrostatic pressure4,5,6. For hydrostatic pressure to drive localized protrusion in animal cells7,8, it would have to be locally regulated, but current models treating cytoplasm as an incompressible viscoelastic continuum9 or viscous liquid10 require that hydrostatic pressure equilibrates essentially instantaneously over the whole cell. Here, we use cell blebs as reporters of local pressure in the cytoplasm. When we locally perfuse blebbing cells with cortex-relaxing drugs to dissipate pressure on one side, blebbing continues on the untreated side, implying non-equilibration of pressure on scales of approximately 10 µm and 10 s. We can account for localization of pressure by considering the cytoplasm as a contractile, elastic network infiltrated by cytosol. Motion of the fluid relative to the network generates spatially heterogeneous transients in the pressure field, and can be described in the framework of poroelasticity11,12.

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Figure 1: Localization of GFP–actin and GFP–MRLC in blebs shows that expansion is a passive process and retraction an active process necessitating actin and MRLC.
Figure 2: Local perfusion can locally inhibit blebbing.
Figure 3: Compounds with dual or global effects on blebbing.
Figure 4: Poroelastic description of blebbing.

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Acknowledgements

The authors would like to acknowledge the Nikon Imaging Centre at Harvard Medical School and, in particular, J. Waters. The authors would also like to acknowledge J. Horn at the HMS machine shop for manufacturing the perfusion chamber. G.T.C. was in receipt of a Wellcome Trust Overseas Fellowship. M.A.H. is supported by a Programme Grant from the Wellcome Trust. L.M. was supported by NSF-MRSEC at Harvard University. This work was supported by a grant from the NIH to T.J.M.

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Correspondence to Guillaume T. Charras.

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Supplementary information

Supplementary Notes

This contains Supplementary Discussion, Supplementary Methods, Supplementary Table 1 and Legends to accompany the Supplementary Figures S1-S4 and Supplementary Videos S1-S12. (DOC 202 kb)

Supplementary Figure S1

Dose response of bleb diameter and frequency with increasing osmotic pressure (PDF 164 kb)

Supplementary Figure S2

Distribution of maximum bleb expansion velocities and minimum bleb retraction velocities. (PDF 168 kb)

Supplementary Figure S3

Dissassembly of the actin cortex posterior to bleb expansion (PDF 150 kb)

Supplementary Figure S4

Volume fluctuations in a blebbing microplast (PDF 121 kb)

Supplementary Video S1

Bath treatment of cells with high concentration sucrose (MOV 4519 kb)

Supplementary Video S2

Bath treatment of cells with WGA (MOV 4872 kb)

Supplementary Video S3

Local perfusion of WGA (MOV 3031 kb)

Supplementary Video S4

Local perfusion of Sucrose (MOV 3697 kb)

Supplementary Video S5

Local perfusion of Blebbistatin (MOV 4290 kb)

Supplementary Video S6

Local perfusion of 3-(4-pyridyl)indole (MOV 4789 kb)

Supplementary Video S7

Local perfusion of Latrunculin (MOV 4233 kb)

Supplementary Video S8

Local perfusion of Staurosporine (MOV 4858 kb)

Supplementary Video S9

Local perfusion of HA 1077 (MOV 5119 kb)

Supplementary Video S10

Colocalisation of actin and myosin II at the cell cortex (MOV 3470 kb)

Supplementary Video S11

MRLC localisation during blebbistatin treatment (MOV 4861 kb)

Supplementary Video S12

Dose response of bleb diameter and frequency with increasing osmotic pressure (MOV 401 kb)

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Charras, G., Yarrow, J., Horton, M. et al. Non-equilibration of hydrostatic pressure in blebbing cells. Nature 435, 365–369 (2005). https://doi.org/10.1038/nature03550

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