Identification of PKN2 and MOB4 as Coordinators of Collective Cell Migration

ABSTRACT In animals, collective cell migration is critical during development and adult life for repairing organs. It remains, however, poorly understood compared with single cell migration. The polymerization of branched actin by the RAC1-WAVE-Arp2/3 pathway is established to power membrane protrusions at the front of migrating cells, but also to maintain cell junctions in epithelial monolayers. Here we have identified novel regulators of collective cell migration using a two-pronged approach: candidates were extracted from publicly available RAC1-WAVE-Arp2/3 dependency maps and screened in a second step using CRISPR/Cas9 genetic inactivation. In a wound healing assay, PKN2 knockout (KO) MCF10A cells display decreased collective migration due to destabilization of adherens junctions, whereas MOB4 KO cells display increased collective migration with a loss of migration orientation. Upon wound healing, PKN2 relocalizes to lateral junctions and maintains coordinated migration in the monolayer, whereas MOB4 relocalizes to the front edge of leader and follower cells collectively migrating towards the wound. The role of MOB4 in controlling collective migration requires YAP1, since MOB4 KO cells fail to activate YAP1 and their phenotype is rescued by constitutively active YAP1. Together, these findings reveal two complementary activities required for coordinating cells in collective migration. Competing Interest Statement The authors have declared no competing interest. Footnotes -Qualification of the MOB4 KO phenotype: streamlines in Fig.6A introduced to avoid using swirling -Inclusion of a new migration parameter: velocity correlation length in Fig.2D, 5B, 7D -Confocal microscopy to localize PKN2 and MOB4 at cell-cell junctions (Fig.3 and 4) -Important control: no effect of the PKN2 and MOB4 KO on the actin cytoskeleton at cell-cell junctions (Fig.4C) -Inclusion of averaged and single fields of view heat maps for all collective migration results (Fig. S3, S12, S14) -Assays of single cell migration (Fig.S5)