Motor Clustering Enhances Kinesin-driven Vesicle Transport

Rui Jiang, Qingzhou Feng, Daguan Nong, You Jung Kang, David Sept, William O. Hancock

Posted on: 16 November 2024

Preprint posted on 27 October 2024

Teamwork makes the dream work- Motor organisation influences the efficiency of cargo transport in cells.

Selected by Sharvari Pitke

Background and hypothesis

Motor proteins are responsible for the transport of cargo in cells by associating with the cellular cytoskeleton elements such as microtubules. Microtubule-associated dynein and kinesin motors are typically involved in such transport across the cell with multiple of these motors attached to the cargo. Previous in vitro reconstitution studies suggest that numerous individual motors may be required for the long-range transport of cargo as opposed to the limited number available in vivo.

This preprint hypothesises that the organisation of motors can be key to resolving this observed inconsistency and that clustering of molecular motors can improve the transport efficiency of cargo transport. By reconstituting kinesin-1 motors in vitro, visualising the transport of liposomes, and inducing clustering of the motors, the authors address the discrepancy in the motor number requirements between in vivo and in vitro studies. They further emphasise the importance of motor organisation. The findings of this study reveal that motor clustering is required for the long-range transport of cargo and that the organisation of these motors impacts the efficiency of cargo transport.

Key findings

A large number of Kinesin-1 motors are required for liposome transport in vitro: The authors of the preprint utilised the truncated Drosophila kinesin-1 construct (K406GFP-SNAP) and attached it to the liposomes with a biotinylated DNA oligonucleotide. The run length of liposomes driven by multiple kinesin-1 motors was found to increase in a logarithmic manner compared to single motors. This result suggests that if the motors are distributed randomly on the surface of the cargo, a greater number of individual motors are required to drive long-range transport.

Clustering improves the efficiency of liposome transport: DNA scaffolds were used to cluster precisely three motors. The authors then identified liposomes with clustered motors with Cy3 labelling. Motor clustering resulted in a marked increase in run length compared to single motors. Clustering of motors may also regulate the directionality of intracellular transport.

Figure 1: Reproduced from the preprint. Liposomes associated with clusters of kinesins are transported across longer distances.

What do I like most about the preprint

What I like about the preprint is that it explains the discrepancy in motor copy numbers while commenting on how motor organisation can affect transport efficiency. I found the role of motor organisation in regulating bidirectional transport particularly fascinating as it gives an idea of how motor clustering can be controlled.

Significance of the study

This preprint- apart from highlighting the regulatory role clustering might play in governing the directionality of bidirectional transport- sets up the need to investigate molecular mechanisms responsible for the formation of motor clusters on cargos. The results from the preprint can help identify the molecular underpinnings of diseases arising from defective intracellular transport.

Tags: intracellular transport, kinesin, motors

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Author's response

Rui Jiang shared

Many microtubule post-translational modifications are known to modulate the propensity of binding of certain types of kinesin motors. Can these PTMs affect motor clustering?

Although microtubule PTMs likely don’t directly determine motor organization on the cargo membrane, they can indirectly influence motor clustering by modulating motor binding on microtubules. PTMs that enhance motor affinity (e.g., acetylation promoting kinesin-1 binding (Reed et al., 2006)) can increase local motor concentration at the cargo-microtubule interface, potentially facilitating recruitment of clustering factors. Stable motor clusters may subsequently help motors navigate regions with less favourable PTMs. Additionally, PTMs may impact motor clustering by regulating motor affinity through microtubule-associated proteins (MAPs).

Are there any instances where motor clustering may not be favoured for the transport of the cargo?

Clustering is most beneficial for motors with slow microtubule attachment rates and limited run lengths, like kinesin-1 (Jiang et al., 2019). For highly processive motors with fast attachment rates, such as kinesin-3 (Soppina & Verhey, 2014), clustering may not aid and can potentially hinder transport by causing motor crowding and competition for binding sites. Additionally, dynein clustering has been shown to switch phagosome transport from bidirectional to minus-end-directed motion (Rai et al., 2016), so improper motor clustering could lead to cargo stalling or misdirected transport.

How can molecular mechanisms that regulate clustering drive motor organization in tug-of-war scenarios?

Increasing evidence suggests that clustering of one motor type can regulate the net directionality of bidirectional vesicle transport by kinesin and dynein teams. For example, dynein clustering in cholesterol-rich lipid microdomains shifts phagosome motility from bidirectional to minus-end-directed transport (Rai et al., 2016), while Septin 9, a scaffold for dynein-dynactin, localizes to lysosomal subdomains and promotes perinuclear lysosome localization (Kesisova, Robinson, & Spiliotis, 2021). As emerging evidence suggests that cargo adaptors can recruit both kinesin and dynein motors, open questions remain about whether opposite motors are excluded from clusters, co-cluster but stay inactive, or if the clustered motor type switches as cargo directionality changes.

References:

Jiang, R., Vandal, S., Park, S., Majd, S., Tuzel, E., & Hancock, W. O. (2019). Microtubule binding kinetics of membrane-bound kinesin-1 predicts high motor copy numbers on intracellular cargo. Proc Natl Acad Sci U S A, 116(52), 26564-26570. doi:10.1073/pnas.1916204116

Kesisova, I. A., Robinson, B. P., & Spiliotis, E. T. (2021). A septin GTPase scaffold of dynein-dynactin motors triggers retrograde lysosome transport. J Cell Biol, 220(2). doi:10.1083/jcb.202005219

Rai, A., Pathak, D., Thakur, S., Singh, S., Dubey, A. K., & Mallik, R. (2016). Dynein Clusters into Lipid Microdomains on Phagosomes to Drive Rapid Transport toward Lysosomes. Cell, 164(4), 722-734. doi:10.1016/j.cell.2015.12.054

Reed, N. A., Cai, D., Blasius, T. L., Jih, G. T., Meyhofer, E., Gaertig, J., & Verhey, K. J. (2006). Microtubule acetylation promotes kinesin-1 binding and transport. Curr Biol, 16(21), 2166-2172. doi:10.1016/j.cub.2006.09.014

Soppina, V., & Verhey, K. J. (2014). The family-specific K-loop influences the microtubule on-rate but not the superprocessivity of kinesin-3 motors. Mol Biol Cell, 25(14), 2161-2170. doi:10.1091/mbc.E14-01-0696

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