Journal: Nature Communications

Article Title: A Rho GTPase-effector ensemble governs cell migration behavior

doi: 10.1038/s41467-025-64635-0

Figure Lengend Snippet: a Scheme of Phase-separated condensates for detecting P-P interactions. Schematic was created in BioRender. Heo, W. (2025) https://BioRender.com/2718swf . b Condensate formation efficiency was measured by counting the number of co-localized condensate-formed cells in total co-transfected cells with FRB-CFP-FT and FKBP-RFP (mStrawberry, n = 150 cells, mOrange, n = 150 cells, dTomato, n = 150 cells, LanRFP, n = 173 cells, DsRed, n = 393 cells, N = 3 independent experiments, one-way ANOVA). Means and SD are shown. c Fluorescence intensity (RFP) changes before and after the addition of rapamycin (500 nM). After rapamycin addition, cytosolic dispersed FKBP-DsRed was redistributed into clusters ( n = 13 cells, N = 3 independent experiments). d HeLa cells showing activity-dependent interaction between HRas and RBD Raf1 . Cells were co-expressed with DsRed-HRas (S17N or Q61L) and RBD Raf1 -CFP-FT. Clusters were only detected when HRas was active (HRasQ61L). Line profiles corresponding to the yellow lines indicated in merged images. The Pearson correlation coefficients (r) with corresponding two-tailed P -values are shown. e Table shows the calculation of the condensate formation efficiency (0–100%, top), signal-to-noise ratio (SNR, 0–1, middle), and protein interaction index (PI, 0–1, bottom). f Tables showing the Ras family and Raf1 interactions, as evaluated using condensate formation efficiency (0–100%, top), signal-to-noise ratio (SNR, 0–1, middle), and a protein interaction index (PI, 0–1, bottom). g heatmap of the PIs between the Rho small GTPases and effectors. For each GTPase, the PIs with the effectors within a gene family are reduced, and the maximum values are shown. Hierarchical clustering (with Euclidean distance and complete linkage) was performed for GTPases and effectors, respectively. The number of optimal clusters was determined based on pvclust (Pvclust: an R package for assessing the uncertainty in hierarchical clustering). h Representative images of HeLa cells expressing DsRed-RhoAQ63L (ΔCAAX), -Rac1Q61L (ΔPB and ΔCAAX), -Cdc42Q61L (ΔCAAX) with GBD ROCK1 -CFP-FT. The insets show an enlarged image of the boxed areas. The line scan profiles were obtained from the yellow lines in each image. The Pearson correlation coefficients (r) with corresponding two-tailed P -values are shown. Scale bars, 20 μm. i Table displays the values for efficiency, SNR and the product of efficiency and SNR. These raw values are then normalized by dividing each by the maximum value in the respective column to yield a scaled PI.

Article Snippet: mOrange (Addgene plasmid no. 29723), mStrawberry (Addgene plasmid no. 21076), dTomato (Addgene plasmid no. 18725), and LanRFP (Genescript) were amplified and then introduced into AgeI and BsrGI sites of EGFP-N1 vector to generate mOrange-N1, mStrawberry-N1, dTomato-N1, and LanRFP-N1.

Techniques: Transfection, Fluorescence, Activity Assay, Two Tailed Test, Expressing