Motor Coordination in Long-Distance Transport in Axons

Abstract

Retrograde transport of nerve growth factor signaling endosomes by microtubular motors, from the axon terminals to cell bodies, is vital for the survival of neurons. The robustness of this fast long-distance axonal transport and biased directionality could be attributed to the cooperative mechanics of multiple motors and/or intracellular regulation mechanisms. Here, we present a comprehensive motion analysis of retrograde nerve growth factor (NGF)-endosome trajectories in axons to show that cooperative motor mechanics and intracellular motor regulation are both important factors determining the endosome directionality. We used quantum dot (QD) to fluorescently label NGF and acquired trajectories of retrograde QD-NGF-endosomes with < 20 nm accuracy at 32 Hz, using pseudo-total internal reflection fluorescence imaging. Using a combination of transient motion analysis and Bayesian parsing, we segregated the trajectories into sustained periods of retrograde (dynein-driven) motion, constrained pauses and brief anterograde reversals. Mean square displacement analysis and the temperature dependence of transient reversals confirm that motors of opposite polarities (dyneins and kinesins) are both active on the endosomes during retrograde transport. Stochastic multi-motor model simulations show that the biased directionality as well as several statistical metrics of NGF-endosome transport can only be simulated reasonably by assuming that the microtubule-binding affinity of kinesin is down-regulated. Specifically, the simulations suggest that the NGF-endosomes are driven on average by 4-7 active dyneins and 1-3 down-regulated kinesins. These observations are corroborated by the dynamics of endosomes detaching under load in axons; showcasing the cooperativity of multiple dyneins and the subdued activity of kinesins. We discuss the ramifications of our results for intracellular transport regulation, in conjunction with recent studies on cellular cargo in a wide range of motility (bidirectional to unidirectional) regimes.