cell microfilament staining  (Cytoskeleton Inc)

Journal: Nature Communications

Article Title: Actin based cell chirality emerging at the boundary of 2D-microtissue directs chiral multicellular pattern formation

doi: 10.1038/s41467-026-76921-6

Figure Lengend Snippet: a (Left) Typical example of chiral organisation of the actin cytoskeleton (magenta) in a single fibroblast-type cell plated on a circular fibronectin island (48 μm diameter). Actin was labelled with phalloidin, and the nucleus with Hoechst-33342 (green). (Right) Phase-contrast image of a multicellular chiral alignment pattern resembling the Cyrillic letter И on a rectangular fibronectin island (600 × 300 µm ) 48 h after seeding. Scale bars, 10 μm (left), 100 μm (right). b Time-lapse imaging of a LifeAct-transfected cell (magenta) interacting with the boundary of the rectangular fibronectin (FN; white) island (Cell 1; dashed semicircular outline). Only selected regions of the rectangular island were visualised by high-magnification live-cell imaging; time points (hh:mm) started 30 min after seeding. Nuclei 1 and 2 (green) were labelled with Hoechst-33342. Note that the stress fibres in these cells are tilted relative to the boundary. Scale bar, 20 μm. See also Supplementary Fig. . c Time-lapse sequence showing the dynamic self-organisation of the actin cytoskeleton (green) and associated nuclear (red) movement in a cell plated on a semicircular fibronectin island (radius 40 μm) at selected time points (hh:mm). Actin and nucleus were visualised using SPY650-FastAct and Hoechst-33342, respectively. See also Supplementary Figs. , and Supplementary Movie . d Histogram showing the distribution of stress fibres (SFs) tilt angles in fixed cells 6 h after plating on semicircular fibronectin island and labelling with phalloidin. Mean ± SEM for n = 204 cells is indicated at the top right; see also Supplementary Figs. and . e , g Time-lapse sequences showing actin dynamics (visualised with SPY650-FastAct) and cell shape changes after release from semicircular ( e ) or elliptical ( g ) confinement. Time points (hh:mm) are referenced to the moment of release, induced by adding streptavidin-fibronectin to PLL-PEG-biotin passivated dishes. See also Supplementary Movies and . Scale bars, 10 μm ( c , e , g ). f , h Quantification of the mean stress fibre tilt angle over time for cells released from semicircular ( f ; n = 26 cells) and elliptical ( h ; n = 28 cells) micropatterns. Confinement was released at t = 1.5 h. Data are presented as mean ± SD.

Article Snippet: To visualise actin cytoskeleton in cells during live imaging, cells were either transfected with LifeAct-GFP DNA plasmid via electroporation (Neon transfection system, Life Technologies) following the manufacturer’s instructions, or labelled using SPY650-FastAct (Cytoskeleton Inc., catalogue no. CY-SC505).

Techniques: Phase Contrast, Imaging, Transfection, Live Cell Imaging, Sequencing