The class VIII myosin ATM2 stabilizes actin–plasma membrane contacts in tobacco pollen tubes by locally promoting PtdIns(4,5)P2 formation

The intersection of actin filaments with the plasma membrane (PM) in plant cells involves lipid–protein interactions by the Arabidopsis class VIII myosin ATM2 that are currently not understood. Using pollen tube cells as a model, we describe how ATM2 enables actin–PM contacts by modulating membrane lipid nano-organization. ATM2 bound anionic PM lipids, including phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ), with no apparent preference for any particular lipid species. Overexpression of ATM2 unexpectedly stabilized actin dynamics and altered pollen tube morphology. Actin dynamics remained unaltered upon overexpression of a lipid-binding-defective ATM2 variant. ATM2 effects on actin dynamics required PtdIns(4,5)P 2 , as artificial PtdIns(4,5)P 2 depletion in pollen tubes abolished the effects of full-length ATM2 on actin dynamics despite its retained PM association. ATM2 colocalized and interacted with the PI4P 5-kinase PIP5K2, which resides at actin–PM contacts and forms PtdIns(4,5)P 2 nanodomains facilitating rho-of-plants-dependent actin stabilization. Upon ATM2 overexpression, a fluorescent PtdIns(4,5)P 2 biosensor decorated an extended PM region in vivo, indicating a promoting effect of ATM2 on PtdIns(4,5)P 2 abundance. The catalytic activity of purified recombinant PIP5K2 protein was enhanced upon coincubation with a purified carboxy-terminal ATM2 fragment in vitro, suggesting that ATM2 contributes to the intrinsic regulation of PtdIns(4,5)P 2 formation. Actin dynamics are thus stabilized at the actin–PM interface by ATM2 locally promoting the formation of PtdIns(4,5)P 2 nanodomains, thereby self-reinforcing ATM2 recruitment while also facilitating rho of plants activation to stabilize membrane-proximal actin filaments.