Non-invasive force measurement reveals the number of active kinesins on a synaptic vesicle precursor in axonal transport regulated by ARL-8

Abstract

Kinesin superfamily protein UNC-104, a member of the kinesin-3 family, transports synaptic vesicle precursors (SVPs). In this study, the number of active UNC-104 molecules hauling a single SVP in axons in the worm Caenorhabditis elegans was counted by applying a newly developed non-invasive force measurement technique. The distribution of the force acting on a SVP transported by UNC-104 was spread out over several clusters, implying the presence of several force-producing units (FPUs). We then compared the number of FPUs in the wild-type worms with that in arl-8 gene-deletion mutant worms.ARL-8 is a SVP-bound arf-like small guanosine triphosphatase, and is known to promote unlocking of the autoinhibition of the motor, which is critical for avoiding unnecessary consumption of adenosine triphosphate when the motor does not bind to a SVP. There were fewer FPUs in the arl-8 mutant worms. This finding indicates that a lack of ARL-8 decreased the number of active UNC-104 motors, which then led to a decrease in the number of motors responsible for SVP transport.

Application of the fluctuation theorem for non-invasive force measurement in living neuronal axons

“…In other words, the degree of fluctuation χ can be used as a proxy for the force F in the case of cargo transport in neurons. The degree of fluctuation χ can be measured non-invasively even in living animals, and was shown to be very effective for the relative comparison of the force on the synaptic vesicles exerted by UNC-104 kinesin among mutant and wild type animals (Hayashi et al, 2018).…”

Physical parameters describing neuronal cargo transport by kinesin UNC-104

“…The C. elegans worms are anesthetized and fixed between cover glasses with highly viscous media prior to imaging in order to minimize the noise generated by fluctuations associated with their movement (Fig. 4a) (Hayashi et al 2018a;Niwa 2017;Niwa et al 2016). SVPs were labelled with GFPs ( Fig.…”

“…In this section, we summarize the assumptions and current model used to analyse the time courses of SVPs obtained by the fluorescence observation ( Fig. 4) (Hayashi et al 2018a;Hayashi et al 2018b). Note that time intervals exist, during which a single SVP can be tracked ( Fig.…”

“…6b), when the viscosity effect is high enough. This suggests that long time courses should be divided into several constant velocity segments (CVSs) when the number of motors transporting a cargo is an object to be measured (Hasegawa et al 2019;Hayashi 2018;Hayashi et al 2018a;Hayashi et al 2018b).…”

“…This section describes the physical parameters involved in SVP transport measured in the previous reports (Hayashi et al 2018a;Niwa et al 2016), and their comparison between WT and arl-8-deletion mutant C. elegans (Niwa et al 2016).…”