Using single-cell tracing technique to elucidate SLK-ROCK interaction in endothelial and cancer cell migration

[Speaker] Cheng-Yu Fan:1
[Co-author] Tsai Chia-Jung:5,6, Lin Hsuan-Chao:2, Yu Ling-Yea:1, Hsiao Yung-Jen:1, Chang Ching-Cheng:3, Tsai Feng-Chiao:1,4
1:Dept. of Pharmacology, National Taiwan University College of Medicine, Taiwan, 2:Dept. of Immunology, National Taiwan University College of Medicine, Taipei, Taiwan, 3:Dept. of Medicine, National Taiwan University College of Medicine, Taipei, Taiwan, 4:Dept. of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan, 5:Ph. D. Program in Human Biology, School of Integrative and Global Majors, University of Tsukuba, Tsukuba, Japan, 6:International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, Tsukuba, Japan

Cell migration is a fundamental process in embryonic development, tissue repair and cancer metastasis. Previous studies have identified many genes regulating cell migration, but how these genes interact with each other during cell migration remains unclear. We therefore conducted a "two-hit" screen using shRNAs combined with small molecule inhibitors and scratch wound healing assays in endothelial cells, to identify potential synergistic or antagonistic effects among signaling molecules during cell migration. The screen identified Ste20-like kinase (SLK), a serine-threonine kinase. Its knockdown together with ROCK inhibitor Y27632 drastically increased HUVEC migration. We therefore hypothesized that SLK interacts with ROCK signaling during cell migration.
Surprisingly, in contrast to HUVEC cells, both knockdown and over-expression of SLK in cancer line SAS decreased its migration speed, indicating that SLK did not regulate cell migration directly through motility modulation. We thus performed single-cell tracing techniques to analyze individual moving cells in scratch wound healing assays, showing that SLK knockdown disrupted the migration polarity of SAS cells. Moreover, SLK knockdown rescued the destroyed cell-cell coordination by shCTNNA1, suggesting the importance of cell-cell interactions on the determination of cell migration polarity. Such results also explain why SLK synergistically works with ROCK, which predominantly affects cell adhesion and motility, during cell migration. Through these work we will elucidate how SLK interacts with ROCK signaling during cell migration and develop new pharmacological strategies to treat cell migration-related diseases.

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