Phosphatidic acid-dependent recruitment of microtubule motors to spherical supported lipid bilayers for in vitro motility assays

Abstract

Summary: Motor proteins transport diverse membrane-bound vesicles along microtubules inside cells. How specific lipids, particularly rare lipids, on the membrane recruit and activate motors is poorly understood. To address this, we prepare spherical supported lipid bilayers (SSLBs) consisting of a latex bead enclosed within a membrane of desired lipid composition. SSLBs containing phosphatidic acid recruit dynein when incubated with Dictyostelium fractions but kinesin-1 when incubated with rat brain fractions. These SSLBs allow controlled biophysical investigation of membrane-bound motors along with their regulators at the single-cargo level in vitro. Optical trapping of single SSLBs reveals that motor-specific inhibitors can “lock” a motor to a microtubule, explaining the paradoxical arrest of overall cargo transport by such inhibitors. Increasing their size causes SSLBs to reverse direction more frequently, relevant to how large cargoes may navigate inside cells. These studies are relevant to understand how unidirectional or bidirectional motion of vesicles might be generated.

Keywords

• CP: Molecular biology