F1000Prime eval: Dynein and SQSTM1

I recommended this article on F1000 Prime, comments appended below.

Interaction of SQSTM1 with the motor protein dynein: SQSTM1 is required for normal dynein function and trafficking.
L Calderilla-Barbosa, ML Seibenhener, Y Du, MT Diaz-Meco, J Moscat, J Yan, MW Wooten and MC Wooten
J Cell Sci 2014 Jul 11 DOI: 10.1242/jcs.152363

Here, Calderilla-Barbosa and colleagues define an interaction between the cytoplasmic dynein motor protein complex and the SQSTM1 protein (also known as p62) a key component of the machinery that removes aggregated proteins from the cell.

This work defines the binding site on SQSTM1 and is consistent with a direct interaction with the dynein intermediate chain subunit. The data suggest that the binding site for SQSTM1 on dynein could be shared with that of the histone deacetylase HDAC6. The complex interplay between these three components remains to be defined in any real depth. However, models are proposed within the paper where the dynamic interplay of HDAC6-mediated microtubule acetylation/deacetylation, SQSTM1-mediated binding of aggregated cargo, and dynein-based motility acts to control the removal of aggregated proteins from within cells.

While much of the data rely on immunofluorescence of dynein, which must always be treated with caution owing to the difficulty of detecting specific dynein immunolabelling in cells, it is clear from this work that dynein and SQSTM1 have an interdependent relationship in the removal of aggregated proteins from cells.

These comments were originally posted at:

http://f1000.com/prime/718489227

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Our first BioRxiv preprint uploaded

I have just uploaded our first paper to the BioRxiv preprint server:

Figure from Brown-et-al

“OPPOSING MICROTUBULE MOTORS CONTROL MOTILITY, MORPHOLOGY, AND CARGO SEGREGATION DURING ER-TO-GOLGI TRANSPORT.”

Anna K Brown, Sylvie D Hunt, David J Stephens
doi: 10.1101/001743

The paper is a follow up to our Journal of Cell Science paper from 2013 in which we showed similar outcomes for motors in the endosomal system.

This paper has been long in the making and was in fact our starting point for the project. Despite us thinking that this would prove  a more tractable system (with a defined end-point for trafficking etc) the endosomal system provided a better model for this work. In this project we have also really been trying to find membrane anchors for dynein that direct ER-to-Golgi transport. I’ll admit that we held off on publishing early in the hope that we did and that we could incorporate these assays into that work. Unfortunately it didn’t work out that way. We also could have published this some time ago before Anna (first author) left for maternity leave. I retained the hope of extending the study through other work even while she was away. This didn’t happen (but we still have hopes). We left these data out of Sylvie’s paper for clarity. That paper remained entirely focussed on the endosomal network.So now, we have decided to publish as a follow-up to Sylvie’s paper on sorting nexins and motors from last year, we had to choose what to do. This in our view did not have the necessary detail (“mechanistic insight”??) for somewhere like Molecular Biology of the Cell or Journal of Cell Science. This choice coincided with the opening of BioRxiv as a preprint server for Biology.

[Declaration: I (David) am an “Affiliate” of BioRxiv]

This seemed like a very good option for this work to get it in the public domain ASAP. We will also submit this to a journal for full peer review as I believe that this remains an important part of the process. As such posting on BioRxiv is not the end point on this journey just an extra step for us that makes our data (and interpretation) freely available to all immediately. We don’t have to wait for peer review, revision, formatting and hosting. I cannot see that as anything but a good thing. The preprint has a DOI so can be cited and easily referred to. We can now decide where to submit this for full peer review. Many journals already allow posting (including EMBO and PLOS) and likely others will soon change their editorial policies to follow suit. Some won’t and that is their prerogative. Would we still send our future work to one of the journals that does not allow BioRxiv posting first? Yes, if we thought it was the most appropriate place, we certainly would. 

Our funder for this work (the UK Medical Research Council or MRC) mandates open access and I think BioRxiv is a good part of this process. I also chose the CC-BY license option as the most liberal in terms of re-use, data mining etc. This is also consistent with our funder policy.

For further info on BioRxiv I recommend you take a look. The common questions are readily answered on the BioRxiv website.

Alfonso Martinez-Arias has written on his blog on why he supports the preprint server system and I see no point in reiterating comments that I agree with! I strongly recommend that you read this if you are still wondering whether this is a good idea or not.

Comments on “Structural basis for microtubule binding and release by dynein”.

I evaluated the following article for F1000 Prime:

Redwine WB, Hernández-López R, Zou S, Huang J, Reck-Peterson SL, Leschziner AE. (2012) Structural basis for microtubule binding and release by dynein. Science 337, 1532-6. PMID: 22997337 DOI: 10.1126/science.1224151

My comments build on those from Terrence Frey (San Diego State University) which provide a nice description of the structural data in this paper.

I entirely agree with the thoughts of Terrence Frey here. The concept of a sub-maximal dynein constrained by virtue of an intramolecular salt bridge is a very intriguing one. The possibility that this provides dynein-1 with a greater “dynamic range” seems quite likely. I very much like the proposed explanation that cytoplasmic dynein-2, which is involved in long range unidirectional transport in cilia and flagella, is not constrained in this way because it lacks the possibility to form such a salt bridge. This provides a nice explanation for the need to maintain two distinct cytoplasmic dynein heavy chains.

 

Dynein alone?

I evaluated the following article for F1000:

Dynein light chain 1 and a spindle-associated adaptor promote dynein asymmetry and spindle orientation.
AK Dunsch, D Hammond, J Lloyd, L Schermelleh, U Gruneberg and FA Barr J Cell Biol. 2012 Sep 17; 198(6): 1039-54
PMID: 22965910 DOI: 10.1083/jcb.201202112

The thing that fascinates me the most about this is the possibility that dynein containing light chain-1 acts without dynactin…..hence the crap title for the post.

This paper is highly intriguing from a dynein motor perspective as it strongly suggests that, in mitosis, the binding of dynein light chain 1 (DYNLL1) and the major dynein regulator dynactin are mutually exclusive. The work details very clearly how the adaptor proteins CHICA and HMMR target dynein to the mitotic spindle. This serves to control spindle positioning in cells, at least in part through promoting dynein asymmetry. It will be fascinating to determine whether DYNLL1 and dynactin can bind to dynein at the same time in interphase cells; this would of course have major implications for those studying dynein and dynactin function.

A Golgi anchor for dynein

I started my lab in 2001 with the aim of defining the mechanisms that link endomembranes, particularly those operating in membrane trafficking between the ER and Golgi, to the microtubule cytoskeleton. This month in Developmental Cell, Adam Linstedt’s lab published the following paper:

Golgin160 Recruits the Dynein Motor to Position the Golgi Apparatus.

Smita Yadav, Manojkumar A. Puthenveedu, Adam D. Linstedt

Developmental Cell

Volume 23, Issue 1, 17 July 2012, Pages 153–165 http://dx.doi.org/10.1016/j.devcel.2012.05.023,

I must say that I am very impressed with this paper – it is precisely the one that I wish we had published from my lab. Of course there is much more to do but this is a definitive answer to a question. Nicely done.

Yadav and colleagues set out by defining a role for golgin160 (also known as golgin-160 in the literature) in minus end directed movement of Golgi membranes using siRNA depletion. They also showed that dynein is no longer recruited to Golgi membranes in the absence of golgin160 using an antibody against the dynein light chain Tctex-1. This is no mean feat and immuno-detection of dynein is notoriously difficult and unreliable. They also showed that golgin160 interacts through its own C-terminal coiled coil domain with the dynein intermediate chain subunit. This interaction is direct with a relatively weak (~1 micromolar) affinity.

Golgin160 is a peripheral membrane protein which of course leaves open the question of how it is recruited to membrane to subsequently drive recruitment of dynein. The answer turned out to be Arf1, a small GTP binding protein that operates in ER-to-Golgi transport, primarily to recruit the COPI complex. COPI drives retrograde trafficking from the Golgi to the ER. The N-terminal domain of Golgin160 (i.e. not that which binds dynein) binds preferentially to active GTP-bound Arf1. Consistent with this hierarchical mechanism, golgin160 is not required for the recruitment of GBF1, the main Arf1-activator at this point in the cell1.

Finally the authors show that Golgin160 dissociates from Golgi membranes during mitosis. This is perhaps not surprising given that it is well known that COPI-dependent membrane traffic is largely arrested during mitosis2, 3. However, it does provide a nice mechanism to drive Golgi disassembly during mitosis. Inhibition (or at least down-regulation) of Arf1 activation would lead to a loss of golgin160 from membranes which would in turn lead to a failure to recruit dynein. This would mean reduced minus end motility and consequently a failure to maintain the Golgi as a juxtanuclear organelle.

These data are very clear and convincing. There are also some nice nuances for aficionados. Importantly the fact that this process relies on Arf1 activation at the start suggests that golgin160 might indeed be a more universal membrane anchor for dynein operating at the Golgi itself as well as in ER-to-Golgi trafficking. My own lab showed previously that the COPII coat4 can recruit dynactin, a key accessory factor to dynein that is also required for ER-to-Golgi transport5. However, the COPII complex is only involved very locally at ER exit sites6 and so another factor must be involved as early as the ER-Golgi intermediate compartment (ERGIC). This factor looks likely to be golgin160. In agreement with this, Yadav et al find that dynactin can be identified in complex with golgin160 and dynein. Whether there is an additional membrane anchor for dynactin, or whether this role is performed by spectrin for example7, remains to be defined. Golgin160 is also cleaved during apoptosis by caspase-28 so these new data suggest an obvious mechanism by which this would cause rapid dissociation of the Golgi.

Other questions do remain. How does this golgin160 pathway relate to what we know about the role of Cdc42 and the COPI coat in the recruitment of dynein9, 10.

References:

1.             Szul T, et al. Traffic (2005);6:374-385.

2.             Altan-Bonnet N, et al. Mol Biol Cell (2006);17:990-1005.

3.             Altan-Bonnet N, et al. Proc Natl Acad Sci U S A (2003);100:13314-13319.

4.             Jensen D, Schekman R. J Cell Sci (2011);124:1-4.

5.             Watson P, et al. Nat Cell Biol (2005);7:48-55.

6.             Budnik A, Stephens DJ. FEBS Lett (2009);583:3796-3803.

7.             Holleran EA, et al. J Cell Biol (1996);135:1815-1829.

8.             Mancini M, et al. The Journal of cell biology (2000);149:603-612.

9.             Hehnly H, et al. Traffic (2010);11:1067-1078.

10.          Chen JL, et al. J Cell Biol (2005);169:383-389.

Evaluation of ciliobrevins paper

I have evaluated the following paper for Faculty of 1000:

Small-molecule inhibitors of the AAA+ ATPase motor cytoplasmic dynein.
AJ Firestone, JS Weinger,…, TM Kapoor, JK Chen Nature 2012 Mar 18
PMID 22425997 DOI 10.1038/nature10936
http://www.nature.com/nature/journal/vaop/ncurrent/full/nature10936.html

The F1000 evaluation can be found here (£/€/$): http://f1000.com/14237961

This paper identifies and characterizes a series of small molecule inhibitors of the dynein motor. This work is significant as this represents only the second example of selective targeting of a member of the AAA+ ATPase family by a selective small molecule inhibitor, the first being inhibition of p97 by DBeQ {1}. These ‘ciliobrevins’ are benzoyl dihydroquinazolinone derivatives and are identified as inhibitors of dynein in an assay of Hedgehog (Hh) signalling, which occurs through primary cilia. The small molecules effectively block Hh signalling and localization of Arl13b to primary cilia (an assay for primary cilia formation). When cilia are formed, these ciliobrevins cause an accumulation of cargo (such as IFT88) at the distal tip, consistent with a defect in retrograde transport within the cilia. Dynein-2 is known to be the motor for retrograde transport along the ciliary axoneme. Together, these data indicate effective inhibition of dynein-2 function. These reagents have the potential to elucidate the role of dynein-2 in the very early stages of cilia formation. The authors also demonstrate that the ciliobrevins inhibit the more widely used motor dynein-1, effectively validating this in assays of mitotic spindle function, organelle motility, and in vitro assays. Further data are entirely consistent with the ciliobrevins competing for ATP binding and thereby inhibiting the ATPase activity of the motor. Specificity, it is shown by comparative analysis with a number of other AAA+ ATPases. Given that crystallography has achieved high resolution structures of p97 and the dynein motor, one might also expect some key atomic structures that could define why these compounds are selective in each case.

Please note that this is a distinct nomenclature to that of ‘ciliabrevin’, used previously for another inhibitor of cilia function {2}.

References:
{1} Chou et al. Proc Natl Acad Sci U S A 2011, 108:4834-9 [PMID:21383145].
{2} Engel et al. Cytoskeleton 2011, 68:188-203 [PMID:21360831].

Small molecule inhibitors of dynein: how selective is EHNA?

Recent work in my lab has used siRNA-mediated depletion to intefere with dynein motor function in intact cells. We are now developing other ways of blocking dynein function that could act more quickly to provide rapid readouts in live cell imaging assays. It would also be nice if this inhibition was reversible. There are of course many ways in which this might work – we are trying a “knock-sideways” approach as exploited by Scottie Robinson to explore adaptor complex function. What I describe could of course easily be done using a small molecule inhibitor and if one does a quick search then it is evident that two “dynein inhibitors” are available: vanadate and EHNA. My exploration of the true specificity of EHNA was driven by its seemingly increasing naming as a “selective dynein inhibitor”. For examples of this see this recent paper (which incidentally I really liked and have even recently evaluated for F1000 – which I will post soon): Kaplan and Reiner (2011) Journal of Cell Science http://jcs.biologists.org/content/124/23/3989.

The use of vanadate as a dynein inhibitor was first described in 1978 by Kobayashi et al: http://www.sciencedirect.com/science/article/pii/0006291X78912792

It seems pretty clear to me at least that vanadate is not going to be selective in this context. Our knowledge of its use as a protein phosphatase inhibitor means that applying this to live cells is going to have all sorts of implications for protein phosphorylation. I would of course be interested in any contrary opinions.

So what about EHNA? EHNA, like vanadate, was first described as a dynein inhibitor based on work on flagellar movement in sperm:

Penningroth et al. (1982) Dynein ATPase is inhibited selectively in vitro by erythro-9-[3-2-(hydroxynonyl)]adenine. Biochem Biophys Res Comm. http://www.sciencedirect.com/science/article/pii/0006291X82919647

It does indeed appear to have some selectivity for the dynein motor and, at least in vitro, is a good tool to probe dynein function relative to other ATP-dependent motors such as myosins. However, it also causes significant morphological and functional changes to the actin cytoskeleton – see Schliwa et al (1984) erythro-9-[3-(2-Hydroxynonyl)]adenine is an effective inhibitor of cell motility and actin assembly.   http://www.pnas.org/content/81/19/6044.full.pdf.

Both vanadate and EHNA were used by Beckerle and Porter in their paper in Nature in 1983 (http://www.nature.com/nature/journal/v295/n5851/abs/295701a0.html) showing that dynein was required for motility of pigment granules in squirrelfish erythropores. Where this paper excels is that is shows very clearly that the effect of EHNA on granule motility is not due to inhibition of another EHNA target, protein carboxyl-methylase. This was done by microinjecting cells with S-adenosyl homocysteine. This had previously been shown to block monocyte chemotaxis (perhaps explaining some of the effects on the actin cytoskeleton in the PNAS paper from Schliwa et al above) again showing some pretty clear evidence for selectivity.

There are plenty of other papers that have used EHNA and shown some very clear dynein-dependent functions are perturbed. I am not citing these here – its a blog, not a review (but Google “EHNA dynein” for examples).

So I guess my conclusion here is that EHNA might indeed be a useful tool to study dynein activity in cells. My concern is really over the use of the label “selective inhibitor of dynein” when applied to EHNA. I would urge anyone using this in the hope of exploring dynein function in any way to read the Schliwa paper mentioned above (http://www.pnas.org/content/81/19/6044.full.pdf).

I guess one simple solution is to get some and for us to compare this to some of our own experiments. Maybe I’ll post some info on this in the future. In the meantime, if anyone has any experience with EHNA in particular, or comments to make on its use and/or the use of such “selective inhibitors” in general I would welcome them.