FHOD3 and DIAPH3 control cell migration and differentially shift the balance of parallel and perpendicular stress fibers

Cell morphology, dictated by the filamentous actin (F-actin) cytoskeleton, is fundamental to cell migration during wound healing and cancer metastasis. Cell morphology is shaped by the extracellular matrix (ECM), which provides mechanical cues in the form of ECM stiffness. These mechanical cues regulate the assembly of the F-actin cytoskeleton which in turn controls cell morphology and cell migration. Formins are key regulators of linear F-actin, assembling it into stress fibers, yet the specific roles of individual formins in controlling distinct stress fiber subpopulations to control cell morphology and migration remain poorly defined. Here, we characterize formin expression across different cell types and leverage the inherent expression and cell morphology differences to identify FHOD3 and DIAPH3 as strongly correlated with cell elongation. We demonstrate that these formins regulate complementary but distinct stress fiber networks. In contractile, but less motile cells, FHOD3 knock-down shifts the balance towards stress fibers oriented perpendicular to the long axis of the cell. In contrast, DIAPH3 knock-down shifts the balance towards stress fibers oriented parallel to the long axis of the cell. However, in less contractile and highly motile cells, knockdown of either formin significantly impairs cell migration speed, suggesting both F-actin fiber networks are necessary for cell migration. Our work establishes a model where FHOD3 and DIAPH3 function through non-overlapping mechanisms to control the F-actin architecture that governs cell shape and motility.