Cholesterol in cargo membrane amplifies inhibitory effect of MAP tau on kinesin-1*

Here we combine advances in membrane biophysics with established single-molecule optical-trapping assays to characterize the transport of membrane-enclosed cargos in vitro. Our study employed the major microtubule-based kinesin-1 motor and tau, an important microtubule-associated protein (MAP) that inhibits kinesin-based transport of membrane-free cargos. Remarkably, we found that coupling kinesins via a biomimetic membrane reduces the inhibitory effect of tau, significantly enhancing the transport of membrane-enclosed cargos on tau-decorated microtubules. Further, adding cholesterol to the cargo membrane amplifies the inhibitory effect of tau on kinesin. Combing simulation and modeling approaches, we found that cholesterol hinders the ability of individual motors to search for available binding sites on the microtubule, thereby amplifying tau’s steric inhibition of transport. Our study establishes a direct link between cargo-membrane composition and MAP-based regulation of kinesin-1. The combination of experimental and theoretical approaches we developed is generally applicable for interrogating the regulation of motor proteins in a context directly relevant to in vivo scenarios.