Multiple kinesins induce tension for smooth cargo transport.
Ontology highlight
ABSTRACT:
SUBMITTER: Tjioe M
PROVIDER: S-EPMC6904222 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The number of microtubule motors attached to vesicles, organelles, and other subcellular commodities is widely believed to influence their motile properties. There is also evidence that cells regulate intracellular transport by tuning the number and/or ratio of motor types on cargos. Yet, the number of motors responsible for cargo motion is not easily characterized, and the extent to which motor copy number affects intracellular transport remains controversial. Here, we examined the load-dependent properties of structurally defined motor assemblies composed of two kinesin-1 molecules. We found that a group of kinesins can produce forces and move with velocities beyond the abilities of single kinesin molecules. However, such capabilities are not typically harnessed by the system. Instead, two-kinesin assemblies adopt a range of microtubule-bound configurations while transporting cargos against an applied load. The binding arrangement of motors on their filament dictates how loads are distributed within the two-motor system, which in turn influences motor-microtubule affinities. Most configurations promote microtubule detachment and prevent both kinesins from contributing to force production. These results imply that cargos will tend to be carried by only a fraction of the total number of kinesins that are available for transport at any given time, and provide an alternative explanation for observations that intracellular transport depends weakly on kinesin number in vivo.
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Project description:Consistent with their diverse intracellular roles, the processivity of N-terminal kinesin motors varies considerably between different families. Kinetics experiments on isolated motor domains suggest that differences in processivity result from differences in the underlying biochemistry of the catalytic heads. However, the length of the flexible neck linker domain also varies from 14 to 18 residues between families. Because the neck linker acts as a mechanical element that transmits interhead tension, altering its mechanical properties is expected to affect both front and rear head gating, mechanisms that underlie processive walking. To test the hypothesis that processivity differences result from family-specific differences in neck linker mechanics, we systematically altered the neck linker length in kinesin-1, -2, -3, -5, and -7 motors and measured run length and velocity in a single-molecule fluorescence assay. Shortening the neck linkers of kinesin-3 (Unc104/KIF1A) and kinesin-5 (Eg5/KSP) to 14 residues enhanced processivity to match kinesin-1, which has a 14-residue neck linker. After substituting a single residue in the last alpha helix of the catalytic core, kinesin-7 (CENP-E) exhibited this same behavior. This convergence of processivity was observed even though motor speeds varied over a 25-fold range. These results suggest that differences in unloaded processivity between diverse kinesins is primarily due to differences in the lengths of their neck linker domains rather than specific tuning of rate constants in their ATP hydrolysis cycles.
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Project description:Motor-dependent anterograde transport, a process that moves cytoplasmic components from sites of biosynthesis to sites of use within cells, is crucial in neurons with long axons. Evidence has emerged that multiple anterograde kinesins can contribute to some transport processes. To test the multi-kinesin possibility for a single vesicle type, we studied the functional relationships of axonal kinesins to dense core vesicles (DCVs) that were filled with a GFP-tagged neuropeptide in the Drosophila nervous system. Past work showed that Unc-104 (a kinesin-3) is a key anterograde DCV motor. Here we show that anterograde DCV transport requires the well-known mitochondrial motor Khc (kinesin-1). Our results indicate that this influence is direct. Khc mutations had specific effects on anterograde run parameters, neuron-specific inhibition of mitochondrial transport by Milton RNA interference had no influence on anterograde DCV runs, and detailed colocalization analysis by superresolution microscopy revealed that Unc-104 and Khc coassociate with individual DCVs. DCV distribution analysis in peptidergic neurons suggest the two kinesins have compartment specific influences. We suggest a mechanism in which Unc-104 is particularly important for moving DCVs from cell bodies into axons, and then Unc-104 and kinesin-1 function together to support fast, highly processive runs toward axon terminals.
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