built-in function bwboundaries  (MathWorks Inc)

Journal: iScience

Article Title: RNA molecules display distinctive organization at nuclear speckles

doi: 10.1016/j.isci.2024.109603

Figure Lengend Snippet: SMLM imaging and analysis of intra-speckle organization of RNAs containing SRSF1 motifs in exon and hnRNPA1 motifs in intron (A) Schematic illustration of WT S1-H1, MUT S1-H1 and MUT S1-spacer-H1 constructs. (B) Representative SMLM image of MUT S1-H1 and MUT S1-spacer-H1 . FISH signals corresponding to hnRNPA1 (labeled with AF647) and SRSF1 (labeled with CF568) motifs in the RNAs are shown in magenta and green, respectively. Immunostaining of SRRM2 is shown in blue. Scale bar represents 5 μm (white) and 1 μm (black). (C) Calculation of the distribution of FISH signal as a function of the distance from the center of the nuclear speckle (i) and edge of the nuclear speckle (ii). Due to size differences among nuclear speckles, distances are all normalized from the center of the speckle (i) or the edge of the speckle (ii) to build the overlaid distribution. The black line represents the nuclear speckle edge; the pink and green lines are the intensity distributions of the RNA FISH signals of the hnRNPA1 and SRSF1 motifs. The speckle edge was determined using a MATLAB built-in function bwboundaries which identifies the pixels at the edge of the intensity mask of each speckle. (D) Plot of difference in absolute mean distance vs. separation in the RNA length (in the unit of nucleotide) between the probe targeting positions. (E and G) Population distribution of SRSF1 and hnRNPA1 motif signals for MUT S1-H1 as a function of the normalized distance from the center of the speckle (E) and edge of the speckle (G). (F and H) Population-weighted mean normalized distance of SRSF1 and hnRNPA1 signal from the center of speckle (F) and edge of speckle (H) for each speckle for MUT S1-H1 . (I and K) Population distribution of SRSF1 and hnRNPA1 motif signals for MUT S1-spacer-H1 as a function of the normalized distance from the center of the speckle (I) and edge of the speckle (K). (J and L) Population-weighted mean normalized distance of SRSF1 and hnRNPA1 signal from the center of speckle (J) and edge of speckle (L) for each speckle for MUT S1-spacer-H1. Error bars in the population vs. distance plots report the standard deviation (n = 2) from two replicates, each replicate containing at least 60–90 nuclear speckles collected from 4 to 6 cells. Scatterplots are generated by combining all nuclear speckles (120–180) from two replicates. Values in scatterplot represent mean ± standard error of mean (SEM). p values in the scatterplots are calculated with paired sample Wilcoxon signed rank test (one-sided), with ∗ p < 5e-2, ∗∗ p < 1e-2, ∗∗∗ p < 1e-3. Replicates are biological replicates collected starting from different dishes of cells and measured on different days. Also see Figures S4–S6 and .

Article Snippet: The speckle edge was determined using a MATLAB built-in function bwboundaries which identifies the pixels at the edge of the intensity mask of each speckle. (D) Plot of difference in absolute mean distance vs. separation in the RNA length (in the unit of nucleotide) between the probe targeting positions. (E and G) Population distribution of SRSF1 and hnRNPA1 motif signals for MUT S1-H1 as a function of the normalized distance from the center of the speckle (E) and edge of the speckle (G). (F and H) Population-weighted mean normalized distance of SRSF1 and hnRNPA1 signal from the center of speckle (F) and edge of speckle (H) for each speckle for MUT S1-H1 . (I and K) Population distribution of SRSF1 and hnRNPA1 motif signals for MUT S1-spacer-H1 as a function of the normalized distance from the center of the speckle (I) and edge of the speckle (K). (J and L) Population-weighted mean normalized distance of SRSF1 and hnRNPA1 signal from the center of speckle (J) and edge of speckle (L) for each speckle for MUT S1-spacer-H1.

Techniques: Imaging, Construct, Labeling, Immunostaining, Standard Deviation, Generated