Mitochondria-adaptor TRAK1 promotes kinesin-1 driven transport in crowded environments

Background

Motor proteins frequently encounter crowded environments in cells, which need to be traversed in order to ensure proper transport of cargoes, including organelles. While some motor proteins, such as cytoplasmic dynein 1, have the ability to switch to a neighbouring protofilament on the microtubule (Can et al., 2014) when faced with an obstacle, the plus-end directed kinesin-1 is notorious for its inability to remain moving on a crowded microtubule. How then is effective anterograde movement, such as that of mitochondria on neuronal microtubules, made possible?

 

Key Findings

In this work, Henrichs et al. provided evidence for a direct attachment of the kinesin-1 adaptor for mitochondrial trafficking, TRAK1 (Milton) to the microtubules. In the absence of the kinesin-1 motor, KIF5B, TRAK1 was observed to bind and diffuse on microtubules. This diffusional movement switched to processive plus-end directed movement upon addition of full-length KIF5B, illuminating a key role for TRAK1 in the activation of the motor. Further, analysis of the intensity histogram of TRAK1 spots that colocalized with processive KIF5B dimers indicated that at most two TRAK1 dimers were included in the transport complex.

By comparing with a constitutively active kinesin-1 mutant, KIF5BΔ, that lacked the autoinhibitory tail domain of KIF5B, but retained the ability to bind TRAK1, the authors demonstrated that the KIF5BΔ-TRAK1 complex had increased processivity (ability of the motor to take several steps on the microtubule before detachment) and interaction time with microtubules, than KIF5BΔ alone. The deletion of TRAK1’s microtubule binding domain (TRAK1Δ) abolished this increase, with KIF5BΔ-TRAK1Δ complexes exhibiting run lengths and interaction times similar to KIF5BΔ.

Next, the authors tested if the attachment of TRAK1 to KIF5BΔ was sufficient for the complex to achieve processive movement on the microtubule in crowded environments. First, a kinesin-1 rigor mutant was used, which binds tightly to microtubules due to its inability to hydrolyze ATP and therefore causes roadblocks on microtubules. The authors found that KIF5BΔ-TRAK1 had higher run lengths and interaction times on the microtubule that had roadblocks than KIF5BΔ alone. Then, the movement of KIF5BΔ-TRAK1 in the presence of cohesive tau islands on microtubules was monitored. While KIF5BΔ either detached at the boundary or moved only ~200nm into a tau island, only a small fraction of KIF5BΔ-TRAK1 detached at the boundary and the rest moved into the island for ~500nm before detaching from the microtubule. The latter was hypothesized to occur by sequential displacement of tau’s microtubule-binding repeats. Lastly, the authors extracted whole cell lysates from HEK293/T17 cells and added this to their in vitro stepping assay. Three different lysates were obtained: a native lysate, one from cells overexpressing TRAK1 and the last from cells overexpressing only the purification tag (halo lysate). Again, only the TRAK1 lysate exhibited processive movements on the microtubule.

Finally, the ability of TRAK1 to facilitate transport of mitochondria by KIF5BΔ was checked by isolating mitochondria from 4T1 cells. The isolated mitochondria lacked endogenous KIF5B or TRAK1, but had the transmembrane protein Miro, which links mitochondria to the transport complex. The authors observed that mitochondria transported by KIF5BΔ-TRAK1 exhibited median run lengths of >20µm, likely indicating that a single mitochondrion could be transported by several KIF5BΔ-TRAK1 complexes.

Taken together, TRAK1 functions as an additional microtubule tether of kinesin-1 to microtubules (à la dynactin for dynein (Culver-Hanlon et al., 2006)), thereby allowing kinesin-1 to run longer on microtubules even in the presence of obstacles on the microtubule.

 

What I like about this work

While previous research has identified that TRAK1 binds mitochondria to kinesin-1 (e.g. (Brickley and Stephenson, 2011)), I like that this work provides clean evidence for the role of TRAK1 in regulating kinesin-1 activity. I also like the fact that the paper is very well written, and so can be read and understood by a non-expert without too much difficulty. The question of how motors overcome crowding on the microtubule has been long-standing in the field. The authors have combined several elegant methods to introduce crowding on the microtubule and have clearly shown TRAK1’s influence on the processivity of kinesin-1 in all instances. This work opens up the field to test how other motor proteins might cope with the issue of roadblocks on the microtubule.

 

Questions to the authors

• Most of the experiments have been performed with the constitutively active KIF5BΔ mutant. Would you expect to see a similar behavior of full-length KIF5B when coupled with TRAK1?
• Do you see instances where a run is abrogated due to detachment of TRAK1 from full-length KIF5B, i.e. what are the kinetics of attachment and detachment between KIF5B and TRAK1?
• Would disabling the dimerization of TRAK1 have any effect on the transport complex?
• How does microtubule crowding affect mitochondrial transport by KIF5BΔ-TRAK1?
• Could you employ photobleaching experiments (similar to those used in Fig. S2) to ascertain that multiple KIF5BΔ-TRAK1 complexes transport mitochondria?
• Perhaps some questions for future work: (i) How do the KIF5B-TRAK1 complexes function in a tug-of-war situation with cytoplasmic dynein? (ii) How do differential post-translational modifications of tubulin alter the processivity of KIF5B-TRAK1?

 

References

Brickley, K. and Stephenson, F. A. (2011). Trafficking Kinesin Protein (TRAK)-mediated Transport of Mitochondria in Axons of Hippocampal Neurons. J. Biol. Chem. 286, 18079–18092.

Can, S., Dewitt, M. A. and Yildiz, A. (2014). Bidirectional helical motility of cytoplasmic dynein around microtubules. Elife 3,.

Culver-Hanlon, T. L., Lex, S. A., Stephens, A. D., Quintyne, N. J. and King, S. J. (2006). A microtubule-binding domain in dynactin increases dynein processivity by skating along microtubules. Nat. Cell Biol. 8, 264–270.

Henrichs, V., Grycova, L., Barinka, C., Nahacka, Z., Neuzil, J., Diez, S., Rohlena, J., Braun, M. and Lansky, Z. (2020). Mitochondria-adaptor TRAK1 promotes kinesin-1 driven transport in crowded environments. bioRxiv.

 

doi: https://doi.org/10.1242/prelights.17071

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