Naoi, T. | Search bioRxiv

bioRxiv2021

Extreme value analysis of intracellular cargo transport by motor proteins

Extreme-value analysis (EVA) deals with deviations in the data from the median of the probability distributions. EVA serves various purposes such as predicting disasters and analyzing sports records. Herein, we extended the use of EVA to investigate the motility functions of nanoscale motor proteins in neurons of the living worm Caenorhabditis elegans (C. elegans). Motor proteins, such as kinesin and dynein, move along microtubules anterogradely and retrogradely, respectively, to deliver the cargo-containing materials needed for the cells. Although the essential difference between the two motors could not be inferred from the mean velocity values, the return-level EVA plots obtained from the velocity data revealed a difference. Shape parameters of the generalized extreme value distribution of EVA{xi} < 0 for anterograde transport and{xi} [≥] 0 for retrograde transport. Our findings extend the possibility and applicability of EVA for analyzing motility data of nanoscale proteins in vivo.

biophysics↗

bioRxiv2021

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.

biophysics↗