Journal: eBioMedicine

Article Title: SLK is mutated in individuals with a neurodevelopmental disorder

doi: 10.1016/j.ebiom.2025.105725

Figure Lengend Snippet: Quantification of wound assay, Golgi appearance and microtubule distribution in patient-derived fibroblasts compared to control . (a) Representative images of sub-confluent fibroblast cells derived from either the index individual in Family 1 (IV:1) or control cell lines at 0 h of wound-induced cell migration assay. Scale bar: 1000 μm. (b) Wound closure was followed serially for 29 h during which a significant difference in cell migration was observed between the two cell lines. Scale bar: 1000 μm. (c) Percent wound closure was evaluated up to 29 h. (d) The remaining area of the wound was measured in μm 2 . The percent wound closure and area of the wound were measured using the ImageJ plugin Wound healing size tool image tool analysis ( https://github.com/AlejandraArnedo/Wound-healing-size-tool/wiki ). Data from 50 cells were collected and analysed. (e) Control cells showing perinuclear compact and polarized Golgi apparatus that are directed toward the migrating wound edge. (f) IV:1-derived fibroblast cells depicting a dispersed and non-polarized (oriented) Golgi apparatus towards the migrating/wound edge. (g) A representative image of normally polarized and parallel orientated microtubules in control cells. (h) A representative image of individual IV:1-derived fibroblast cells showing an un-polarized chaotic microtubule distribution; microtubules labelled with α-tubulin (green) and the centrosome is labelled with Pericentrin (orange) antibodies. (i) The total area of Golgi from 100 cells was measured using the ImageJ software. Error bars represent stdev. (j) The directional orientation of Golgi from 100 cells was assessed and presented as percent mean polarization with error bars representing stdev of 3 replicates. (k) The direction of orientation (paralellness) of microtubules was measured using the LPX ImageJ plugin ( https://lpixel.net/services/research/lpixel-imagej-plugins/ ) ; and data presented as mean and stdev of images of 100 cells. Data from 50 cells were collected and analysed and p-value was calculated based on Welch's t-test for (c, d, i, j and k). The red squares represent datapoints collected from control sample, the green circles represent datapoints collected from the patient sample (IV:1). The horizontal line represents the mean.

Article Snippet: Error bars represent stdev. (j) The directional orientation of Golgi from 100 cells was assessed and presented as percent mean polarization with error bars representing stdev of 3 replicates. (k) The direction of orientation (paralellness) of microtubules was measured using the LPX ImageJ plugin ( https://lpixel.net/services/research/lpixel-imagej-plugins/ ) ; and data presented as mean and stdev of images of 100 cells.

Techniques: Derivative Assay, Control, Cell Migration Assay, Migration, Software

Journal: Nature Communications

Article Title: NET4 and RabG3 link actin to the tonoplast and facilitate cytoskeletal remodelling during stomatal immunity

doi: 10.1038/s41467-023-41337-z

Figure Lengend Snippet: a , b , d , e , g , h Quantification of actin cytoskeletal patterns of Lifeact-mNeonGreen-expressing Col-0 WT and net4ab-c2 plants in guard cells. ImageJ analysis using the LPIXEL Inc LPX plugin set. a , b Mean angular difference ( a ) and parallelness ( b ) of the actin cytoskeleton in untreated guard cells. d , e Mean angular difference ( d ) and parallelness ( f ) of the actin cytoskeleton in guard cells treated for 1 h with flg22. g , h Mean angular difference ( g ) and parallelness ( h ) of the actin cytoskeleton in guard cells treated for 2 h with flg22. Box plots of the values are shown with whiskers from the 5th to 95th percentiles, the line in the box shows the median; 1 h flg22 treatment: WT Col-0 n = 73, net4ab-c2 n = 64 guard cells, p < 0.0001 Mann Whitney t -test; 2 h flg22 treatment: WT Col-0 n = 38, net4ab-c2 n = 23 guard cells, p = 0.0777 ns Mann Whitney t -test; Stomata opening buffer: WT Col-0 n = 10, net4ab-c2 n = 7 guard cells, - angle p = 0.3638, parallelness p = 0.1088 ns Mann Whitney t -test. c , f , i Representative super-resolution Airyscan confocal images of Lifeact-mNeonGreen-positive actin filaments in Col-0 WT and net4ab-c2 untreated and treated for 1 and 2 hrs with flg22, respectively. Scale bars = 10 µm. The experiments were repeated at least thrice with similar results.

Article Snippet: ImageJ analysis using the LPIXEL Inc LPX plugin set. a , b Mean angular difference ( a ) and parallelness ( b ) of the actin cytoskeleton in untreated guard cells. d , e Mean angular difference ( d ) and parallelness ( f ) of the actin cytoskeleton in guard cells treated for 1 h with flg22. g , h Mean angular difference ( g ) and parallelness ( h ) of the actin cytoskeleton in guard cells treated for 2 h with flg22.

Techniques: Expressing, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: Planar cell polarity induces local microtubule bundling for coordinated ciliary beating

doi: 10.1083/jcb.202010034

Figure Lengend Snippet: Daple regulates MT dynamics at the Fzd side of the AJC in tracheal MCCs. (A) Schematic drawing of ALI culture of MTECs for the lentivirus infection and live imaging. (B) Representative fluorescence images of EB1-mRuby3 (green) and centrin2 (blue) in GFP-centrin2–positive WT MTECs expressing EB1-mRuby3 via the lentivirus system. (C and D) Representative image for EB1-mRuby3 (red) in GFP-centrin2–positive WT MTECs expressing EB1-mRuby3 due to the lentivirus system (C) and time-lapse images of the boxed regions in C for EB1-mRuby3 at the Fzd and the other side of the AJC in tracheal MCCs (D). Red arrows indicate the EB1 comets that contact the cell membrane, whereas yellow arrows indicate the EB1 comets in the cytoplasm. (E) Representative fluorescence image for EB1-mRuby3 in GFP-centrin2–positive WT MTECs with boxes indicating the I and P regions. (F) EB1 speed at the I and P regions in MCCs of WT MTECs. Speed of EB1 comets showing spatial differences ( n = 10). Two-tailed Mann-Whitney U test; **, P < 0.01. (G) EB1 speeds in the I and P regions around the Fzd and the other sides of the AJC in MCCs of WT MTECs ( n = 10 cells). Kruskal-Wallis test with the Steel-Dwass multiple-comparison test; **, P < 0.01; n.s., P ≥ 0.05. (H) Schematic illustration of differences in EB1 dynamics around the Fzd and other side of the AJC in MCCs of GFP-centrin2–positive WT MTECs. The EB1 comets display a long residence time at the Fzd side of the AJC. Inter, I region; Peri, P region. (I) Representative image for mEGFP–α-tubulin (mEGFP-αtub; red) expressed in MCCs of WT MTECs (upper panel) and kymograph showing the motility of MTs in MCCs of WT MTECs (lower panel). Note the high motility of MTs as they move near the Vangl side of the AJC, as shown in the boxed region (yellow dashed lines). (J) Representative images of mEGFP–α-tubulin in MCCs of WT and Daple-KO MTECs (left panels) and analyses of MT live imaging using the LPX flow plugin (right panels). The movement speed of the α-tubulin signal is repressented by colors (red indicates high speed; blue indicates low speed). The arrow indicates a decrease in speed at the Fzd side of the AJC in WT MCCs. (K) Statistical analysis of the speed of MTs in WT MCCs ( n = 4 cells). ROIs were set at the Fzd and other side of the AJC. Two-tailed Mann-Whitney U test; *, P < 0.05. Scale bars represent 5 µm in B, C, E, I, and J and 1 µm in D.

Article Snippet: To evaluate the movement of MTs in MCCs, we analyzed the time-lapse images of mEGFP–α-tubulin using the LPX flow plugin (originally called KBI-Flow; ; https://lpixel.net/products/lpixel-imagej-plugins/ ) for ImageJ (National Institutes of Health; freely available at http://imagej.nih.gov/ij/index.html ).

Techniques: Infection, Imaging, Fluorescence, Expressing, Membrane, Two Tailed Test, MANN-WHITNEY, Comparison