Intracellular force comparison of pathogenic KIF1A, KIF5, and dynein by fluctuation analysis

In mammalian cells, there exist approximately 40 types of microtubule motor proteins that are assigned to specific cargo deliveries. For example, the kinesin-1 family motor KIF5 is the major motor responsible for anterograde mitochondrial transport, whereas the kinesin-3 family motor KIF1A is responsible for synaptic vesicle precursor transport. In contrast, cytoplasmic dynein is responsible for retrograde transport of nearly all cargos. The force and velocity of these microtubule motors have been investigated in in-vitro single-molecule experiments. In the present study, we compared the intracellular force and velocity of various types of motors in the mammalian neuronal axon obtained by non-invasive force measurement (fluctuation analysis) and extreme value analysis with those obtained by previous single-molecule experiments. As we found a high correlation between our results and the previous results, we next investigated synaptic vesicle precursor transport by hereditary spastic paraplegia-associated KIF1A variants (V8M, R350G, and A255V). KIF1A-V8M and KIF1A-A255V exhibited force and velocity impairment in mammalian neuronal axons, whereas the physical property of KIF1A-R350G was similar to that of the wild type. We believe that the development of new analytical techniques for investigating intracellular cargo transports is helpful to elucidate the molecular mechanism of KIF1A-associated neurological disorders. Statement of SignificanceRecent in-vitro single-molecule experiments have clearly revealed that microtubule motors only fully exert their functions when fully equipped with the proteins associated with cargo vesicle transport. This emphasizes the significance of intracellular physical measurements, in which the motors should fully function. In addition to motor force and velocity, the number of motors transporting a single cargo together is an important physical quantity to characterize cargo transport, but is difficult to estimate using in-vitro single-molecule experiments. In this study, we aimed to extract physical information on microtubule motors in the intracellular environment.