Supplementary Materials

The nucleus acts as a ruler tailoring cell responses to spatial constraints

A. J. Lomakin, C. J. Cattin, D. Cuvelier, Z. Alraies, M. Molina, G. P. F. Nader N. Srivastava, P. J. Saez, J. M. Garcia-Arcos, I. Y. Zhitnyak, A. Bhargava, M. K. Driscoll, E. S. Welf, R. Fiolka, R. J. Petrie, N. S. De Silva, J. M. González-Granado, N. Manel, A. M. Lennon-Duménil, D. J. Müller, M. Piel

Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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  • Figs. S1 to S11
  • Tables S1 to S3
  • Caption for Movies S1 to S7
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Images, Video, and Other Media

Movie S1
Rounded nonadherent HeLa-Kyoto cells sense the difference between 10 and 5 μm confinement — spatially confining cells to 5 but not 10 μm height stimulates rapid recruitment of myosin II from the cytosol to the cortex. Left, a representative cell expressing MYH9-GFP (pseudocolored using the thermal LUT mode with hot and cold tones indicating high and low levels of MYH9-GFP fluorescence signal respectively) immediately after the 10 μm height is reached. Right, the same cell after 5 μm confinement. Time interval between consecutive images, 5 s. Scale bar, 5 μm.
Movie S2
Rounded nonadherent HeLa-Kyoto cells undergo a discrete morphodynamic switch from a noncontractile cell cortex and filopodia formation to a highly contractile cortex and vigorous plasma membrane blebbing in response to 10-to-5 μm confinement. Left, a representative cell expressing MYH9-GFP (pseudocolored in magenta) and LifeAct-mCherry (pseudocolored in cyan) during 20-to-10 μm confinement. Right, the same cell during 10-to-5 μm confinement. A differential-interference-contrast (DIC) image of the confining flat microcantilever is shown in the grey channel. Time interval between consecutive images, 5 s. Scale bar, 5 μm.
Movie S3
Rounded nonadherent HeLa-Kyoto cells expand their nuclei and unfold the nuclear envelope prior to the onset of the contractile cell response manifested in active plasma membrane blebbing during 10-to-5 μm confinement. The representative cell expresses LAP2-GFP as a marker of the nuclear envelope (green) and is stained with the SiR-actin probe to reveal F-actin (pseudocolored in magenta). The cell was confined from 20-to-10-to-5 μm. The video is artificially paused several times during the 10-to-5 μm transition to accentuate nuclear expansion-unfolding taking place before the cell retracts the very first rounded blebs. These structures represent passive bleb-like protrusions appearing upon physical squeezing of the cell (i.e. when intracellular fluid is pushed against the plasma membrane through pre-existing cracks in the cell cortex). Our additional observations (data not shown) reveal that such "passive" blebs almost instantaneously accumulate submembranous cortical F-actin. The cortex of the "passive" blebs gets retracted by active myosin II motors initiating a continuous active blebbing. "Active" blebs are more numerous, often of a larger length, and significantly smaller in width compared to their "passive" counterparts. Time interval between consecutive images, 5 s. Scale bar, 5 μm.
Movie S4
Rounded nonadherent, enucleated HeLa-Kyoto cells (cytoplasts) do not sense the difference between confinement heights relevant to the contractile response in nucleated cells. Left, a representative nucleated cell expressing MYH9-GFP (pseudocolored in magenta) and LifeActmCherry (pseudocolored in cyan) that survived the treatments and centrifugation protocol required for enucleated cytoplast generation. Right, a representative enucleated cytoplast. The video starts from depicting a nonconfined state of these cells followed by 20-to-10-to-5 μm confinement. Note, despite its smaller size (height), the nucleated cell that survived the cytoplast generation protocol initiates the sustained, active contractile response (cortical accumulation of myosin II and vigorous plasma membrane blebbing) right before the confining cantilever stops at 5 μm during the 27 movement from 10 to 5 μm. However, the enucleated cytoplast displays only a passive response in the form of transient bleb-like protrusions generated due to physical squeezing of the cytoplast during the 10-to-5 μm confinement. Time interval between consecutive images, 5 s. Scale bar, 5 μm.
Movie S5
Compression of the cell cortex down to 0.5 μm confinement height yields only local and highly transient bleb-like protrusions without any global effect on the levels of actomyosin contractility. Left, a representative spread adherent HeLa-Kyoto cell expressing MYH9-GFP (pseudocolored in magenta), LifeAct-mCherry (pseudocolored in cyan) and additionally stained with DAPI (pseudocolored in yellow). A differential-interference-contrast (DIC) image of the confining flat microcantilever is shown in the grey channel. The precise position of the cantilever was adjusted such that only the lamellar but not organelle-rich compartment is engaged in the local cell compression. Right, a time-lapse video of the cell responding to strong local cortical compression (500 nm confinement). Time interval between consecutive images, 5 s. Scale bar, 10 μm.
Movie S6
Induction of the global sustained contractile cell response to local nuclear but not cortex/cytoplasm confinement. The video depicts representative spread adherent HeLa-Kyoto cells expressing MYH9-GFP (pseudocolored in magenta), LifeAct-mCherry (pseudocolored in cyan) and additionally stained with DAPI (pseudocolored in yellow) to reveal the nuclear compartment. A differential-interference-contrast (DIC) image of the confining flat microcantilever is shown in the grey channel. The precise position of the cantilever was adjusted such that only the nuclear compartment of the lower cell and the lamellar region of the upper cell are engaged in the local confinement. Confinement height, 2 μm. Note, while local cortex/cytoplasm confinement produces local, short-lived bleb-like protrusions (passive response to physical compression), local nuclear-specific confinement results in a global, sustained increase in cell contractility even in the non-confined cell region that switches to this state with a delay (activation signal propagation). Time interval between consecutive images, 5 s. Scale bar, 10 μm.
Movie S7
Populations of LifeAct-GFP-expressing immature dendritic cells (iDCs) subjected to confinement in microfabricated devices. Primary bone marrow-derived iDCs from LifeAct-GFP mice were treated with control siRNA or siRNA targeting cPLA2a. The cells were separately confined to 4 and 3 μm heights and imaged using fluorescence videomicroscopy. Time interval between consecutive images, 2 min. Scale bar, 25 μm.