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. 2021 Jul 9;134(13):jcs253484. doi: 10.1242/jcs.253484

Fig. 5.

Fig. 5.

Implementation of YAP1 dynamics on a lattice light-sheet microscope reveals heterogeneous cytoplasmic dynamics. (A) Schematic diagram of the lattice light-sheet imaging experimental design. (B) Representative image of a HaCaT cell expressing Zdk–Flag–mCherry–YAP1-WT imaged on lattice light-sheet microscope. Mitochondria (mito; red) were stained using MitoTracker Deep Red, and DNA (blue) was visualised using stable expression of an H2B–FP fusion. Scale bar: 5 μm. (C) Release and recovery experiment of Zdk–Flag–mCherry–YAP1-WT in 3D. Scale bar: 5 μm. (D) Quantification of fluorescence intensity during an optogenetic release and recovery experiment performed in a HaCaT cell expressing Zdk–Flag–mCherry–YAP1-WT. The curves show exemplar intensities from regions of the mitochondria, the cytoplasm and the nucleus. Blue boxes indicate periods of blue light illumination. (E) Quantification of intensities corresponding to five different cytoplasmic regions during the 3D optogenetic experiment in a HaCaT cell expressing Zdk–Flag–mCherry–YAP1-WT; right panel shows average post-blue light spike intensity profiles for different cytoplasmic regions combining the three technical replicate spikes. Symbols show the mean of the three temporal replicates and dashed lines show the s.d. (black dashed lines correspond to the grey triangles) for the different cytoplasm regions of one representative cell. Data in D and E are representative of n=36 cells from seven independent experiments for YAP1-WT. (F) Analysis of different cytoplasmic regions in HaCaT cells transiently transfected with Zdk–Flag–Venus (lilac) or Zdk–Flag–Venus–YAP1-WT (magenta) plotted as normalised fluorescence intensity. Each line represents a different region of a cell, with upward lines corresponding to cytoplasm regions of interest and downward lines corresponding to mitochondrial regions. Data derived from ≥14 cytoplasmic regions of three cells for each condition. (G) Heatmap and representative images showing the proportionate gain in fluorescence after blue light illumination of HaCaT cells transiently transfected with Zdk–Flag–Venus or Zdk–Flag–Venus–YAP1-WT; refer to Fig. 1C for images corresponding to the Zdk–Flag–Venus heatmap. Arrowheads indicate regions of high fluorescence signal gain in Zdk–Flag–Venus–YAP1-WT.