BPS2026 – Visualization of Myosin-9b processivity in human cells

Using two-color, live-cell super-resolution SORA confocal microscopy, we visualized Myo9b processive motility in cells for the first time. Myosin-9b (Myo9b) is a motorized RhoA-specific GAP that localizes to sites of actin polymerization including ruffles, junctions, and the apical cortex of polarized cells, where it suppresses RhoA-ROCK signaling and non-muscle myosin-2 contractility. Loss of Myo9b impairs intestinal barrier function in mice and cell models, while human variants contribute to celiac disease and Irritable Bowel syndrome. Despite decades of work, Myo9b motility has not previously been observed in cells. In epithelial models, we captured fluorescently tagged Myo9b moving toward actin bundle plus-ends at microvilli tips. Endogenous Myo9b localization at brush border microvilli tips in intestinal tissue validated this finding. We quantified motility and found that while purified single-headed Myo9b moves at ∼20 nm/s as a single motor in vitro, in cells, Myo9b particles travel at 295 ± 78 nm/s with run lengths exceeding 1 μm, limited by microvilli length. Pull-down assays revealed endogenous Myo9b co-associates with tagged Myo9b, suggesting a multi-motor complex underlies these enhanced properties. A point mutation in the motor ATPase salt bridge abolished motility, confirming its dependence on motor activity. To probe function, we generated the first Myo9b CRISPR knockout in human epithelial cells and observed microvillar defects by electron and super-resolution microscopy. Finally, using a spatiotemporal RhoA biosensor, we demonstrated that Myo9b locally regulates actin organization, microvilli architecture, and NM2 contractility. Together, these results redefine the model by which Myo9b acts as a processive motor in cells to link its motility and RhoA regulatory activities.