Peer review of: Mitochondria-adaptor TRAK1 promotes kinesin-1 driven transport in crowded environments (2020)

Peer Review File 

Reviewers' comments first round: 
 
Reviewer #1 (Remarks to the Author): 
 
The authors investigate the activation of kinesin-1 by the TRAK1 cargo adapter molecule. These 
partners are known to interact and there are interesting disease phenotypes of TRAK1 and tau, but 
the mechanism underlying the TRAK-kinesin interaction are not clear. The authors clearly show 
that the partners bind, and that TRAK1 enhances the kinesin run-length through TRAK1 tethering 
the motor to the microtubule. They go on to show that TRAK1 enhances kinesin’s ability to 
traverse islands of tau and use purified mitochondria to show that TRAK1 and kinesin-1 cooperate 
in multi-motor transport of mitochondria. 
 
The single-molecule data are very solid and the analysis is correct. The work together makes a 
nice and pretty straightforward story that is relevant for kinesin-based intracellular cargo 
transport. I had a few comments to strengthen the manuscript, detailed below. 
 
Comments 
Top p6. “1.84 s (CI95 (1.64, 2.05) s, n = 534 molecules) in the absence of mCherry-TRAK1 to 
12.20 s (CI95 (8.54, 15.86) s, n = 221 molecules) in its presence (Figure 3f).” This is less than an 
order of magnitude 
 
Page 6. TRAK1 slows kinesin-1 down from 918 to 694 nm/s, which is significant. The authors 
postulate that this slowing is due to frictional drag from TRAK1. This point should be argued more 
rigorously, as follows. In Fig 1, the authors track the diffusion of isolated TRAK1. From this, they 
should be able to estimate the diffusion constant of TRAK1, and from there to measure the drag 
coefficient of TRAK1 along microtubules. Based on published force-velocity relationships of kinesin-
1, is the observed slowing consistent with the predicted drag coefficient? 
 
Page 11: “…it inhibits kinesin-dependent trafficking of mitochondria and thereby implicates 
Alzheimer’s disease”. This is a confusing sentence, “implicates” is not the right word here. 
 
The authors bring up some interesting points in the discussion related to tau and TRAK1 
interactions. I suggest the authors bring this point up in the introduction to set the stage better. 
And in general in the introduction, defining the questions more specifically, (rather than just 
seeing what it does) could significantly strengthen the impact of the results. For instance questions 
about the tail requirement, the activity on the mitochondria, and other points where experiments 
were performed but the questions were not introduced in the intro beforehand. 
 
 
 
Reviewer #2 (Remarks to the Author): 
 
With their work, the authors wanted to understand how kinesin-1 can function as efficient cargo 
transporter in crowded environments. The authors report that the kinesin-1-cargo adaptor TRAK1 
activates autoinhibited KIF5B and increases the processivity of KIF5B lacking its autoinhibiting C-
terminus. This effect is also detectable when microtubules are crowded with Tau protein islands 
both when using a pure in vitro assay and when imaging in extracts. Finally, the authors report 
that the KIF5Bdelta-TRAK1 complex transports mitochondria. TRAK1 reduces velocity and 
increases processivity – an effect that is amplified by the addition of mitochondria. Interestingly, 
when adding only TRAK1, the effects on the KIF5B motility parameters don’t seem to be caused by 
motor-bundling, but by the microtubule-binding activity of TRAK1 which was discovered in this 
work. 
I think the paper is an important step forward towards understanding full neuronal cargo transport 
complexes. A lot of these complexes are proposed based on classical cell biology approaches but 
are then difficult or impossible to build with pure components which indicates that it is not 
understood how these complexes are actually assembled and which factors are essential. 
 Major points: 
• TRAK1 and KIF5B were made in different expression systems at different temperatures which 
can affect fluorophore folding and maturation rate. For the fluorescence comparisons in Fig.2d, it 
would be important to understand, which fraction of the fluorophores are active. Could the authors 
measure the fluorescence intensities per amount of protein to test this? 
• If one TRAK1 dimer binds one KIF5 dimer, I would expect that two TRAK1 dimers would bind two 
KIF5 dimers and hence TRAK1 should cause KIF5 tetramerization to some extent. How do the 
authors explain that this is not observed? Or can TRAK1 bind a KIF5B dimer only as tetramer? 
• In Fig.3f, the interaction time of KIF5Bdelta-GFP and KIF5Bdelta-GFP+mCherrey-TRAK1 
complexes is compared and the mCherry-TRAK1 signal is used to report on KIF5 dwell time in the 
presence of TRAK1. Depending on imaging settings, GFP can bleach faster than mCherry which 
would artificially reduce the KIF5-GFP dwell times. It would be assuring to provide GFP and 
mCherry bleaching rates at the chosen imaging conditions. 
• The authors state that in control experiments lacking TRAK1 no processive movement of 
mitochondria is detectable (lines 284-286) but I could not find data supporting this statement. I 
think it is an important point because it shows that TRAK1 is sufficient for mitochondria transport 
by KIF5 in vitro. When KIF5Bdelta-mCherry alone is incubated with mitochondria-GFP - are no 
runs detectable? Or substantially less than with TRAK1? 
• The authors speculate that the lower velocity and higher processivity caused by adding 
mitochondria stems from the recruitment of multiple TRAK1-KIF5 complexes to one mitochondrion, 
but no data is provided to support this idea. It seems straightforward to provide intensity data 
supporting this idea. 
 
Minor points: 
• Is TRAK1 also a tetramer when it binds to microtubules without KIF5 or is KIF5 interaction 
triggering TRAK1 tetramerization? 
• As TRAK1 can bind microtubules – can it recruit mitochondria to microtubules or are both 
competing for TRAK1 interaction? 
• As TRAK1 promotes KIF5 movement through crowded microtubule regions, and TRAK1-
microtubule interactions were identified as the mechanism by which TRAK1 supports KIF5 motility, 
one would expect that the TRAK1-microtubule interaction is less affected by Tau compared to the 
KIF5-microtubule interaction. It would be very interesting to see if and to which extent TRAK1-
microtubule interactions change in the presence of Tau. 
• In line 287 the authors write “single-mitochondria”. How is it shown that we are looking at single 
mitochondria? 
 
 
 
Reviewer #3 (Remarks to the Author): 
 
In their manuscript, Henrichs et al. utilize in vitro reconstitution with purified proteins to show that 
the mitochondrial adaptor protein, TRAK1/Milton, directly interacts and activates kinesin-1 while 
also tethering it to the microtubule during stepping to increase its processivity both on naked 
microtubules as well as microtubules crowded with other “obstacles” such as the microtubule-
associated protein, tau. They then purify mitochondria from cultured cells and find that this cargo 
can be robustly transported in vitro by the kinesin-1-TRAK1 complex. Overall, this is a very nice 
story and is the first study to show a bona fide activator of full-length kinesin-1 in vitro, making it 
highly significant to a broad audience. If the authors address the below points, it will be acceptable 
for publication in Nature Communications. 
 
Major Points: 
 
1) They use a TRAK protein construct that lack its C-terminal microtubule-binding domain and find 
that it cannot tether or increase the processivity of the truncated, active kinesin-1. However, this 
experiment should be performed with full-length kinesin-1. If TRAK does indeed release the 
autoinhibition of full-length kinesin-1, it becomes an interesting question of whether TRAK does 
this allosterically or if it requires its microtubule-binding domain. There are two different 
mechanisms potentially at play, which do not need to be mutually exclusive, but should be 
examined more thoroughly. It could be that TRAK has two modes of action on kinesin-1: 1) it 
could allosterically release autoinhibition and 2) it could tether an active kinesin-1 motor as it 
moves processively along the lattice. The C-terminus of TRAK is obviously required for #2, but is it 
dispensable for #1? 
 
2) To date, TRAK is the first protein that can directly activate kinesin-1 using purified components 
in an in vitro system. This is incredibly exciting, but the authors should place this in the context of 
the field to highlight this novelty. For example, many proteins have been reported to activate 
kinesin-1 in vivo or using lysate experiments in vitro, but none of these proteins have been shown 
to directly activate kinesin-1 using purified components. In addition, the authors should reference 
the MAP7 work from my lab (Monroy et al., 2018), the Akhmanova lab (Hooikaas et al., 2019) and 
the Hendricks lab (Chaudhury et al., 2019) to highlight the contrast. MAP7 directly binds and 
recruits kinesin-1 to the microtubule, dramatically increasing the number of engaged motors, but 
does not activate it like TRAK does. Therefore, this is a unique and novel function of TRAK, and 
placing in in the context of other proposed adaptors will strengthen the significance. 
 
3) For the tau experiments, the authors hypothesize in the discussion that TRAK tethers and 
enables kinesin-1 to remain bound longer to the microtubule in order to displace tau from the 
protofilament so that it can move through a dense tau patch. This is interesting because they 
previously observed that a yeast kinesin can displace tau, and we've also seen that a non-
enzymatic MAP, MAP7, can do the same over a longer timescale (Monroy et al., 2018). Can the 
authors quantify the velocity of kinesin-1 before and after entry into a tau patch? It appears in 
their figure that the velocity may indeed slow down and this would provide more support for their 
hypothesis. 
 
4) The authors hypothesize on page 9 that “several” kinesin-TRAK complexes might be attached to 
a single mitochondrion, but the kymograph in Figure 5 shows a dimmer fluorescence intensity 
profile of the TRAK associated with the mitochondrion. Can the authors analyze the intensity of the 
TRAK in these runs to provide an estimate for the number of motors on the cargo? I realize that, 
based on Figure 1, there are most likely complexes that are not labeled, but if many kymographs 
are analyzed, it can still provide a general idea of motor number. Or, the authors could correlate 
run length with fluorescence intensity and compare the same three conditions as in Fig 5c. 
 
Minor Point: 
References need to be organized—some include the author/year while others are referenced with 
numbers (see second paragraph of the introduction as an example). 
 
Signed, Kassandra Ori-McKenney