Biology subjects

Fokin, A. I.

Publications and source records attributed to Fokin, A. I..

5 recordsLinked to original sources

bioRxiv2025

Identification of PKN2 and MOB4 as Coordinators of Collective Cell Migration

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.

cell biology↗

bioRxiv2023

Vinculin Antagonizes Branched Actin in Controlling Membrane Protrusion, Cell Migration and Cell Cycle Progression

Vinculin is a mechanotransducer that reinforces links between cell adhesions and linear arrays of actin filaments upon myosin-mediated contractility. Both adhesions to the substratum and neighboring cells, however, are initiated within membrane protrusions that originate from Arp2/3-nucleated branched actin networks. Vinculin has been reported to interact with the Arp2/3 complex, but the role of this interaction remains poorly understood. Here we compared the phenotypes of vinculin knock-out (KO) cells with those of knock-in (KI) cells, where the point mutation P878A that impairs the Arp2/3 interaction is introduced in the two vinculin alleles of MCF10A mammary epithelial cells. The interaction of vinculin with Arp2/3 inhibits actin polymerization at membrane protrusions and decreases migration persistence of single cells. In cell monolayers, vinculin recruits Arp2/3 and the vinculin-Arp2/3 interaction participates in cell-cell junction plasticity. Through this interaction, vinculin controls the decision to enter a new cell cycle as a function of cell density.

cell biology↗

bioRxiv2023

Inactivating Negative Regulators of Cortical Branched Actin Enhances Persistence of Single Cell Migration

The Rac1-WAVE-Arp2/3 pathway pushes the plasma membrane by polymerizing branched actin at the cell cortex and thereby powering membrane protrusions that mediate cell migration. Here, using knock-down (KD) or knock-out (KO), we combine the inactivation of the Arp2/3 inhibitory protein Arpin, the Arp2/3 subunit ARPC1A and the WAVE complex subunit, CYFIP2, that all enhance the polymerization of cortical branched actin (CBA). Inactivation of the 3 CBA negative regulators increases migration persistence of human breast MCF10A cells, and of endodermal cells in the zebrafish embryo, significantly more than any single or double inactivation. In the triple KO, but not triple KD cells, the "super-migrator" phenotype was associated with a heterogenous down-regulation of vimentin expression and a lack of coordination in collective behaviors, such as wound healing and acinus morphogenesis. Re-expression of vimentin in triple KO cells restored the normal persistence of single cell migration to a large extent, suggesting that vimentin down-regulation is one of the adjustments in gene expression through which the super-migrator phenotype is stably maintained in triple KO cells. Constant excessive production of branched actin at the cell cortex thus commits cells into a motile state through changes in gene expression.

cell biology↗

bioRxiv2021

Arpin regulates migration persistence by interacting with both tankyrases and the Arp2/3 complex

During cell migration, protrusion of the leading edge is driven by the polymerization of Arp2/3-dependent branched actin networks. Migration persistence is negatively regulated by the Arp2/3 inhibitory protein Arpin. To better understand Arpin regulation in the cell, we looked for interacting partners and identified both Tankyrase 1 and 2 (TNKS) using a yeast two hybrid screen and co-immunoprecipitation with full-length Arpin as a bait. Arpin interacts with ankyrin repeats of TNKS through a C-terminal binding site on its acidic tail overlapping with the Arp2/3 binding site. To uncouple the interactions of Arpin with TNKS and Arp2/3, we introduced point mutations in the Arpin tail and attempted to rescue the increased persistence of the Arpin knock-out using random plasmid integration or compensating knock-in at the ARPIN locus. Arpin mutations impairing either Arp2/3- or TNKS-interaction were insufficient to fully abolish Arpin activity. Only the mutation that affects both interactions rendered Arpin completely inactive, suggesting the existence of two independent pathways, by which Arpin controls migration persistence. Arpin was found to dissolve liquid-liquid phase separation of TNKS upon overexpression. Together these data suggest that TNKS might be mediating the function of Arpin rather than regulating Arpin.

cell biology↗

bioRxiv2020

The Arp1/11 Minifilament of Dynactin Primes the Endosomal Arp2/3 Complex

Dendritic actin networks develop from a first actin filament through branching by the Arp2/3 complex. At the surface of endosomes, the WASH complex activates the Arp2/3 complex and interacts with the Capping Protein for unclear reasons. Here we show that that the WASH complex interacts with Dynactin and uncaps it through its FAM21 subunit. In vitro, the uncapped Arp1/11 minifilament elongates an actin filament, which then primes the WASH-induced Arp2/3 branching reaction. In Dynactin-depleted cells or in cells where the WASH complex is reconstituted with a FAM21 mutant that cannot uncap Dynactin, formation of branched actin at the endosomal surface is impaired. Our results reveal the importance of the WASH complex in coordinating two complexes containing actin-related proteins. One Sentence SummaryDendritic actin networks grow in an autocatalytic manner starting from the uncapped minifilament of Dynactin.

cell biology↗