Cellular adhesome screen identifies critical modulators of focal adhesion dynamics, cellular traction forces and cell migration behaviour

Michiel Fokkelman,Erik H J Danen,Janna E Klip,Hayri E Balcıoğlu,Fons J Verbeek,Kuan Yan,Bob Van De Water

doi:10.1038/srep31707

Michiel Fokkelman, Erik H J Danen + Show 5 more

Open Access

https://doi.org/10.1038/srep31707

Copy DOI

Abstract

Cancer cells migrate from the primary tumour into surrounding tissue in order to form metastasis. Cell migration is a highly complex process, which requires continuous remodelling and re-organization of the cytoskeleton and cell-matrix adhesions. Here, we aimed to identify genes controlling aspects of tumour cell migration, including the dynamic organization of cell-matrix adhesions and cellular traction forces. In a siRNA screen targeting most cell adhesion-related genes we identified 200+ genes that regulate size and/or dynamics of cell-matrix adhesions in MCF7 breast cancer cells. In a subsequent secondary screen, the 64 most effective genes were evaluated for growth factor-induced cell migration and validated by tertiary RNAi pool deconvolution experiments. Four validated hits showed significantly enlarged adhesions accompanied by reduced cell migration upon siRNA-mediated knockdown. Furthermore, loss of PPP1R12B, HIPK3 or RAC2 caused cells to exert higher traction forces, as determined by traction force microscopy with elastomeric micropillar post arrays, and led to considerably reduced force turnover. Altogether, we identified genes that co-regulate cell-matrix adhesion dynamics and traction force turnover, thereby modulating overall motility behaviour.

Highlights

All 3D environments and cells show a high level of plasticity allowing them to switch between different modes of migration in 3D16,17
We identified myosin phosphatase target 2 (PPP1R12B), Homeodomain Interacting Protein Kinase 3 (HIPK3) and Ras-related C3 botulinum toxin substrate 2 (RAC2) to be involved in the organization and dynamics of traction forces, the cellular adhesion machinery, and cell motility induced by growth factor signalling
Our current findings provide a strong correlation between cellular traction force generation and dynamics, cell-matrix adhesion turnover and cell migration

Summary

Introduction

All 3D environments and (tumour) cells show a high level of plasticity allowing them to switch between different modes of migration in 3D16,17. We aimed to understand the underlying machinery of tumour cell migration and its relation to adhesion turnover and cellular traction forces. For this purpose we performed a siRNA screen targeting cell adhesion and actin dynamics associated genes, and evaluated their role in cell-matrix adhesion size and/or dynamics. We tested the relationship with tumour cell migration as well as cellular traction forces for the most relevant candidate genes. We identified myosin phosphatase target 2 (PPP1R12B), Homeodomain Interacting Protein Kinase 3 (HIPK3) and Ras-related C3 botulinum toxin substrate 2 (RAC2) to be involved in the organization and dynamics of traction forces, the cellular adhesion machinery, and cell motility induced by growth factor signalling

Objectives

Methods

Results

Conclusion