Phosphatidic Acid Dependent Recruitment of Microtubule Motors to Spherical Supported Lipid Bilayers forIn-vitroMotility Assays

Abstract

ABSTRACTA variety of membrane-bound vesicles are transported by the kinesin and dynein motor proteins respectively towards plus and minus ends of microtubules inside cells. How specific lipids, particularly rare lipids, bind and activate motors on the vesicle membrane and the biological consequences thereof are poorly understood. Here we preparesphericalsupported lipid bilayers (SLBs) consisting of a latex bead enclosed within a membrane that contains phosphatidic acid (PA). When incubated with motor-protein enriched fractions fromDictyosteliumor from Rat brain, PA-coated SLBs exhibit robust motion alongin-vitropolymerized microtubules. We quantify the activity of SLB-bound motors by measuring their force generation against an Optical trap on polarity-labelled microtubules. Such experiments reveal that PA recruits dynein-dynactin fromDictyostelium, but kinesin-1 from rat brain fractions to drive SLB motion. Kinesin-1 is able to dominate over dynein when dynactin, an activator of dynein, is deficient on SLBs. We demonstrate how inhibiting only kinesin can block both kinesin and dynein activity on a cargo, thus providing an explanation for the long-standing “paradox of co-dependence” in the field of intracellular transport. We also find that as the size of a motor-driven SLB increases, the SLB is able to reverse direction more easily along a microtubule. This observation provides clues as to how micron-sized cargoes are efficiently able to navigate the crowded cytoskeletal space inside cells.