Cooperative Mechanics of Multi-Motor Axonal Transport Revealed by Novel Nanomanipulation in Live Neurons

Despite remarkable advances in characterizing molecular motors and microtubular transport in vitro, our understanding of intracellular cargo transport is rudimentary. The intracellular mechanochemical properties and cooperative mechanics of multiple motors, sharing load and coordinating cargo direction, are fundamental in this regard. To elucidate these, we developed two novel approaches for manipulating the transport of axonal endosomes, loaded with nanoprobes (<100nm) by receptor-mediated endocytosis, in microfluidic DRG cultures.Firstly, we show that by culturing neurons in a novel microfluidic-magnetic device, with axons aligned along high magnetic gradient zones, we can exert pN forces on axonal endosomes carrying magnetic nanoparticles (MNP∼100nm). TIRF imaging of axonal MNP-endosome transport under external load reveals that mechanical means alone can transiently reverse endosome directionality. This suggests that mechanical force balance between kinesins and dyneins is critical for the regulation of endosome direction.Secondly, we show that laser-absorption by a gold nanoparticle (GNP∼80nm) in the endosome, stochastically results in the endosome being elastically tethered in axon (mechanism will be discussed). Motors driving the tethered endosome gradually come to a stall (like beads held by an optical trap) before detaching from the microtubule. The instantaneous recoil velocity of the endosome following lead-motor(s) detachment is proportional to the multi-motor stall force. We captured the recoil velocity distribution of dynein-driven retrograde and kinesin-driven anterograde endosomes using darkfield imaging at <2ms time, <5nm spatial resolutions. The peak structure of these distributions reflects discrete multi-motor stall forces and the peak-separation represents single-motor detachment velocity. Surprisingly, we find the single-motor detachment velocity for dynein comparable to that for kinesin suggesting comparable single-motor stall forces in this case. Further high-resolution motion analyses and stochastic modeling of GNP-endosome trajectories reveal several key signatures of cooperative mechanics, multi-motor force profiles and detachment kinetics.