The mammalian class IX myosins Myo9a and Myo9b use different cellular actin assemblies for force production and...

Myosins exert directed mechanical force along actin filaments. However, little is known about how myosins select particular cellular actin assemblies for their diverse physiological functions. The mammalian class IX myosins, Myo9a and Myo9b, share homologous motor and RhoGAP domains, but it remains unclear whether they target the same actin filament assemblies and thereby serve redundant functions in cells. We showed previously that Myo9b localizes to dynamic actin filament networks in extending lamellipodia and that its motor activity is both necessary and sufficient for this localization. We now show that both motor activity and additionally a predicted four-helix bundle motif in the tail region are required for the accumulation of Myo9b at the tips of filopodia. Interestingly, the class IX loop 2 insertion in the motor region is dispensable. In contrast, Myo9a does not localize to either lamellipodia or filopodia tips. However, the head domain of Myo9a alone targets actin stress fibers, while constructs that also include the neck and tail domains exhibit reduced or negligible targeting. This suggests that the head domain is sterically hindered by a folded conformation. In conclusion, Myo9a and Myo9b target different subcellular sites and actin filament assemblies, implying that they perform different physiological functions.