Journal: Molecular Systems Biology

Article Title: A dynamic, spatially periodic, micro‐pattern of HES5 underlies neurogenesis in the mouse spinal cord

doi: 10.15252/msb.20209902

Figure Lengend Snippet: Schematic of extracting kymograph information from tissue data by averaging Venus::HES5 intensities observed in E10.5 heterozygous spinal cord slices to generate one intensity profile in the dorsal–ventral axis per timepoint (see Materials and Methods). Representative kymograph data showing spatiotemporal Venus::HES5 expression profile along ventral–dorsal direction in a 15 μm wide apical region and observed over 14 h; local bands of 20 μm width in D‐V; region of interest markers indicate: *low to high, **high to low and ***re‐occurring high/low activity in the same area. Hierarchical clustering of apical Venus::HE5 expression from one representative experiment showing behaviour in the same area over time; columns represent fluctuations in Venus::HES5 intensity in small local areas (bands) obtained by dividing the spatial signal into non‐overlapping 20 μm regions and normalising to the mean and standard deviation of each region over time (z‐scoring); data have been subject to a Gaussian blur pre‐processing step (see Appendix Fig S2B and Materials and Methods). Persistence of Venus::HES5 in 20 μm regions expressed as continuous time intervals when signal in the band is high or low compared with its mean (see Materials and Methods); individual datapoints (grey) indicate quantification of high and low persistence time obtained from over 300 thin bands collected from multiple tissues with 2 z‐stacks per tissue and two repeats (left and right of ventricle) per z‐stack; dots indicate paired medians of five independent experiments; statistical test is paired t ‐test of median per experiment with two‐tail significance and P = 0.7171. Persistence of Venus::HES5 levels in high and low states taken from 60 tracked single cells collected from three independent experiments; paired t ‐test not significant P = 0.0533. Relative distance between cell pairs computed from relative 3D Euclidean distance between nuclei over 12–15 h; dots indicate median distance over tracking period; horizontal lines show mean and SD of 14 cell pairs from three experiments. Spearman correlation coefficients computed in the same cell pairs from Venus::HES5 and H2B::mCherry (control) nuclear intensity timeseries; markers in each condition indicate pairs; black dots indicate median correlation coefficients per experiment (four pairs, three pairs and seven pairs); lines show median of 14 pairs from three experiments; paired t ‐test with significance P = 0.0058. Representative example timeseries of Venus::HES5 in cells pairs identified as remaining in close proximity; r ‐values indicate Spearman correlation coefficients between time traces over all co‐existing timepoints. Detrended Venus::HES5 fluorescent intensity timeseries (after z‐scoring) corresponding to examples in (H); red arrows indicate in‐phase peaks. Density phase plots from instantaneous Hilbert phase reconstruction at multiple timepoints over a 12–14 h period; dots indicate the phase angle in Cell 1 and Cell 2 from 14 pairs collected from three experiments; colormap indicates probability density showing accumulation of phase values predominantly along the (0,0) and (2π, 2π) diagonal; light colours indicate most frequent. Graphic representation of a neuroepithelial tissue with nuclei colour‐coded to indicate clusters of high or low HES5 expression. The tissue is illustrated at three different time points to depict how clusters of cells can dynamically switch from high to low or low to high while the periodic spatial pattern is maintained. In the example time traces (corresponding to the three grey and one red highlighted nuclei), synchronised ultradian oscillations are shown as being overlayed on the slow‐varying higher‐amplitude switching dynamics. Source data are available online for this figure.

Article Snippet: To account for any single‐cell movement in DV, we applied a 2 μm Gaussian blur filter onto the kymograph data using the MATLAB routine imgaussfilt.m prior to extracting timeseries per region.

Techniques: Expressing, Activity Assay, Standard Deviation, Control

Journal: Communications Biology

Article Title: PIEZO1 and PECAM1 interact at cell-cell junctions and partner in endothelial force sensing

doi: 10.1038/s42003-023-04706-4

Figure Lengend Snippet: HUVEC data. a – f Cells cultured in a confluent monolayer treated with a control (Ctrl) or b PIEZO1 siRNA and subjected to extracellular Ca 2+ switch assay: pre-treatment (normal Ca 2+ ); −calcium (30 min Ca 2+ -depletion); and recovery (+calcium (post); 30 min after restoring Ca 2+ ). After treatments, cells were stained with anti-CDH5 antibody (αCDH5) (green). The scale bars are 100 µm. c Images are enlarged from the white boxes in a , b +calcium (post). To the right are line-intensity plots (black) with a Gaussian fit (red) used to calculate peak width at half maximum for the superimposed grey lines shown in the enlarged images. d , e As for a and b but showing box plots for +calcium pre-treatment ( n = 3, P = 0.92988) and +calcium (post) ( n = 3, *** P = 8.86242 ×10 −5 ) conditions. Superimposed data points are measurements from individual cell-cell junctions for +calcium (pre) (Ctrl si = 89, PIEZO1 si = 90) and +calcium (post) (Ctrl si = 87, PIEZO1 si = 88). f Enlarged and merged images of CDH5 (green, from a , b ) and F-actin (phalloidin) (magenta, from Supplementary Fig. ) staining for the calcium switch recovery (+calcium (post); 30 min after restoring Ca 2+ ) conditions in Ctrl or PIEZO1 siRNA treated HUVECs. Scale bars are 25 µm g Schematic representation of F-actin and CDH5 with (+) and without (−) PIEZO1 or PIEZO1 activation by mechanical force, based on the data of a – f and Supplementary Fig. . In the +PIEZO1 condition, there is suggested to be junctional remodelling with more radial actin and wider (less tight and more leaky) junctions.

Article Snippet: Images were prepared for particle analysis by using the Gaussian Blur filter (Sigma radius = 0.015 µm in scaled units).

Techniques: Cell Culture, Staining, Activation Assay