Subcellular and Dynamic Coordination between Src Activity and Cell Protrusion in Microenvironment

Abstract

Migration of endothelial cells is essential for wound healing and angiogenesis. Src kinase activity plays important roles at the protrusions of migrating endothelial cells. However, the spatiotemporal coordination between Src kinase activity and the protrusion of cell edge remains unclear. Therefore, we investigate these coordinated molecular events at the initiation of cell migration, by integrating microfabrication, fluorescence resonance energy transfer (FRET)-based biosensors, and automated computational image analysis. We demonstrate that the physical release of restrictive micropattern triggered a significant decrease of Src activity at the protrusive edge of endothelial cells. Computational cross-correlation analysis reveals that the decrease of Src activity occurred earlier in time, and was well-coordinated with the protrusion of cell edge in polarized cells, but not in non-polarized cells. These results suggest that the spatiotemporal control of Src kinase activity is well-coordinated with cell polarization and protrusion in endothelial cells upon the release of physical constraint, as that experienced by endothelial cells sprouting from stiff tumor micro-environment during angiogenesis. Therefore, our integrative approach enabled the discovery of a new model where Src is de-activated in coordination with membrane protrusion, providing important insights into the regulation of endothelial migration and angiogenesis.

KLF5 promotes cell migration by up‐regulating FYN in bladder cancer cells

“…We also found that KLF5 knockdown dramatically inhibited cell protrusion stimulated by EGF treatment (100 ng·mL −1 , 10 min) in 5637 cells (Fig. S1), suggesting that KLF5 involved in the regulation of the pathways related to cell protrusion, such as Src kinase family . Moreover, we calculated lamellipodia number and migration rate of 5637 cells in living cell imaging video, and confirmed that KLF5 knockdown decreased both lamellipodia number and migration rate of 5637 cells (Fig.…”

In-situ coupling between kinase activities and protein dynamics within single focal adhesions

“…S3a ) and evidenced in our previous publications 34 35 . Cross-correlation analysis 18 32 was performed to evaluate the temporal phase difference between FA structural changes and kinase activation ( Fig. 4e ).…”

“…While subcellularly targeted FRET biosensors enable the visualization of molecular signals with high resolution, computational analysis is indispensable for integrating multiple signals and deciphering regulatory parameters from live-cell images 18 23 31 32 . To elucidate the dynamic coupling between the biochemical kinase activities and structural FA dynamics we integrated the new FA-targeting biosensors, single-FA tracking, and cross-correlation analysis methods to quantitatively evaluate the temporal coordination of these molecular events at the single-FA level.…”

Coordinated cell motility is regulated by a combination of LKB1 farnesylation and kinase activity

“…The features for each ROI are the number of ROIs, the area of the ROI, angle of the ROI from major axis to the x-axis, and the duration time, which is the length in x-axis converted to time unit. Since there is no standard definition of a protrusion/retraction region, we asked multiple users to select the ROIs for the same heatmap, similar to refs 35,45,47 and 48. We then performed a one-way ANOVA test amongst all users to check reproducibility and found the p-value larger than 0.05, which indicated the selection among different users was similar.…”