Supplementary Materials

The nucleus measures shape changes for cellular proprioception to control dynamic cell behavior

Valeria Venturini, Fabio Pezzano, Frederic Català Castro, Hanna-Maria Häkkinen, Senda Jiménez-Delgado, Mariona Colomer-Rosell, Monica Marro, Queralt Tolosa-Ramon, Sonia Paz-López, Miguel A. Valverde, Julian Weghuber, Pablo Loza-Alvarez, Michael Krieg, Stefan Wieser, Verena Ruprecht

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

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  • Supplementary Methods
  • Figs. S1 to S7
  • Table S1
  • Captions for Movies S1 to S9
  • References
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Images, Video, and Other Media

Movie S1
Adaptive myosin II dynamics upon cell confinement and role of cPLA2 in cell mechanotransduction (related to Fig. 1 and Fig. 3). Time lapse confocal fluorescence movies of progenitor cells expressing Myl12.1-eGFP (myosin II) on non-adhesive substrates (PLL-PEG) cultured in DMEM media in suspension (left), under 7 μm confinement in control condition (middle) or supplemented with 1 μM cPLA2 inhibitor. Upon confinement, myosin II accumulates at the cortex in control condition and the accumulation is blocked by the inhibition of cPLA2.
Movie S2
Cortex remodeling during amoeboid stable-bleb cell transformation and cortical retrograde flow in stable-bleb polarized cells (related to Fig. 1 and Suppl. Fig. 1). (I) Time lapse confocal fluorescence movie of Myl12.1-eGFP (myosin II) localization during stable-bleb transformation of a progenitor cells under 7 μm mechanical confinement: a non-polarized and nonmotile blebbing cell spontaneously polarizes into stable-bleb cell upon mechanical confinement and initiates cell migration. (II) Time lapse confocal fluorescence movie of the basal cortex of a stable-bleb polarized cell expressing Myl12.1-eGFP (myosin II) under 7 μm mechanical confinement. Myosin II shows a cortical density gradient and retrograde flow (magenta arrow, opposite to the direction of migration indicated by the orange arrow).
Movie S3
Adaptive myosin II morphodynamics under confinement (related to Fig. 1-2 and Suppl. Fig 2). Confocal fluorescence time lapse movie of pluripotent progenitor cells (I, sphere stage) on non-adhesive substrate (PLL-PEG); ectoderm cells (II) on non-adhesive substrate (PLLPEG); and mesoderm cells on fibronectin (III) for 2D substrate (left) and 3D mechanical confinement (7 μm, right). Pluripotent and ectoderm cells are non-motile when plated on 2D substrate and transform into a stable-bleb polarized and motile phenotype upon confinement. Mesoderm cells plated on a 2D fibronectin substrate migrate with a mesenchymal phenotype and transform to stable-bleb amoeboid migration upon mechanical confinement.
Movie S4
Myosin II accumulation is reversible (related to Fig. 1). Confocal fluorescence time lapse movie of progenitor cells expressing Myl12.1-eGFP (myosin II) during confinement (10 μm) and subsequent release (i) and for confined cells squeezing out from underneath a micropillar ("escape reflex", ii). Myosin II is rapidly re-localized from the cortex to the cytoplasm upon confinement release.
Movie S5
Mesenchymal-to-amoeboid transition: mesoderm cells transform to stable-bleb cells upon mechanical confinement (related to Fig. 2). Time lapse confocal fluorescence movie of mesendoderm-induced progenitor cells expressing Myl12.1-eGFP (myosin II) plated on fibronectin substrate showing mesenchymal migration (white asterisk indicates migrating cell, 24 white arrow points at cell protrusions). Upon confinement (7 μm), cells rapidly accumulate myosin II at the cortex and transform to stable-bleb polarized cell (magenta asterisks point at cell front).
Movie S6
The inner nuclear membrane is unfolded under mechanical shape deformation in confinement and the unfolding is stable over time (related to Fig. 3). Time lapse confocal movies of progenitor cells stained with Lap2b-eGFP (i) in suspension, (ii) during confinement and (iii) under 7 μm confinement. The time lapse in (iii) was acquired 1h after mechanical confinement.
Movie S7
Adaptive Myosin II dynamics under hypotonic conditions or ionomycin treatment (related to Fig. 7). Confocal fluorescence time lapse movie of progenitor cells expressing Myl12.1- eGFP (myosin II) on non-adhesive substrate (PLL-PEG) in isotonic media (control, DMEM), upon hypotonic shock (adding milliQ water, 0.5x shock) and upon the addition of 1 μM ionomycin. Hypotonic treatment is followed by a myosin II accumulation at the cortex; further addition of ionomycin leads to a pronounced myosin II accumulation and triggers cell polarization.
Movie S8
Cell dynamics under hypotonic conditions and ionomycin treatment (related to Fig. 7). Bright field time lapse movie of progenitor cells cultured in isotonic condition (top-left), hypotonic media (top-right), hypotonic media supplemented with 1 μM ionomycin in suspension (bottomleft) or under 16 μm confinement (bottom-right). Hypotonic conditions supplemented with 1 μM ionomycin lead to rapid cell polarization and induce a stable-bleb cell transformation (bottomleft), but cells can migrate only under confined conditions (bottom-right).
Movie S9
The endoplasmic reticulum (ER) is immobilized under the nucleus in deformed cells under high confinement (related to Fig. 7 and Suppl. Fig. 6). Time lapse TIRF movie of progenitor cells stained with ER tracker green under increasing mechanical confinement. At low mechanical confinement, the ER is mobile in the nucleus-plasma membrane contact area, while for increasing confinement the ER is immobilized in the region underneath the nucleus. The yellow line marks the nuclear area (visualized from bright field imaging).