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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Other recent F1000 evaluations

I have not recently updated my blog to include my F1000 evaluations; I now include these missing evaluations here for the benefit of those who do not subscribe to F1000. All of the following were first published on F1000 Prime in the last 12 months.

The GTPase IFT27 is involved in both anterograde and retrograde intraflagellar transport.

Huet D, Blisnick T, Perrot S, Bastin P. elife 2014; 3:e02419 PMID: 24843028 DOI: 10.7554/eLife.02419.001

This fascinating paper exploits Trypanosomes as a model system to define a role for the intraflagellar transport (IFT) protein IFT27 in flagellar function. IFT27 is a known component of the IFT-B particle mediating transport from the flagellar base to tip (anterograde IFT); here, the authors show that the GTPase function of IFT27 is also required to load the retrograde IFT particle (IFT-A) and its associated motor (dynein-2) into cilia. As such, this work defines IFT27 as a critical control point not only for anterograde but also for retrograde IFT. A key point is that IFT27 is a small Rab-like GTPase, and the authors convincingly demonstrate a role for this enzyme activity in the transport process. In some ways this provides further analogy between IFT-mediated transport and canonical membrane trafficking steps in which Rab proteins have critical roles in controlling timing and directionality.

 

Proteins of the Ciliary Axoneme Are Found on Cytoplasmic Membrane Vesicles during Growth of Cilia.

Wood CR, Rosenbaum JL. Curr Biol 2014 May 19; 24(10):1114-20 PMID: 24814148 DOI: 10.1016/j.cub.2014.03.047

This paper provides good evidence that structural proteins of the ciliary axoneme are transported into cilia on vesicular structures. Using Chlamydomonas as a model system, the data show that axonemal components and intraflagellar transport (IFT) particle proteins are delivered to the base of cilia on the surface of vesicles. These then associate with transitional fibres at the point of contact with the cell membrane. The process is particularly evident during cilia formation when a burst of synthesis of the ciliary components must be matched by an upregulation of their delivery. The work suggests an important and ongoing association of axonemal proteins with membranes. This and other work also support the maintained association of IFT proteins with membranes during transport within the cilium. This is therefore a significant paper in that it provides an integrated model for the transport of both membrane and axonemal proteins into cilia.

 

SNARE and regulatory proteins induce local membrane protrusions to prime docked vesicles for fast calcium-triggered fusion.

Bharat TA, Malsam J, Hagen WJ, Scheutzow A, Söllner TH, Briggs JA. EMBO Rep 2014 Mar 1; 15(3):308-14 PMID: 24493260 DOI: 10.1002/embr.201337807

The paper provides strong supporting evidence in favour of a membrane deformation event early in soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent fusion. I concur entirely with what has already been said by Liangyi Chen and Junmei Fan and especially by Robert Burgoyne in his evaluation. Providing a mechanistic basis for some early electron microscopy (EM) is a lovely way to close the loop.

 

An Unconventional Secretory Pathway Mediates the Cilia Targeting of Peripherin/rds.

Tian G, Ropelewski P, Nemet I, Lee R, Lodowski KH, Imanishi Y. J Neurosci 2014 Jan 15; 34(3):992-1006 PMID: 24431457 DOI: 10.1523/JNEUROSCI.3437-13.2014

This paper provides some further evidence for the somewhat controversial idea of a non-conventional secretion pathway operating from the early secretory pathway to the cilium. Evidence on this originally came from the Witzgall lab looking at the trafficking of polycystin-2 {1}. Here, the authors use cell culture models and Xenopus to show that Peripherin-2/rds seems to be trafficked in a similar manner. Key evidence for this comes from glycosylation assays monitoring sensitivity to endoglycosidase H (EndoH). Canonical trafficking of glycoproteins results in an acquisition of EndoH resistance as a result of sugar chain modification in the Golgi. While there is some potential for slightly different interpretations, the data are broadly consistent with the idea of a non-canonical pathway operating in the delivery of cargo to the cilium.

References

Polycystin-2 takes different routes to the somatic and ciliary plasma membrane.

Hoffmeister H, Babinger K, Gürster S, Cedzich A, Meese C, Schadendorf K, Osten L, de Vries U, Rascle A, Witzgall R. J Cell Biol 2011 Feb 21; 192(4):631-45 PMID: 21321097 DOI: 10.1083/jcb.201007050

 

A Regulator of Secretory Vesicle Size, Kelch-Like Protein 12, Facilitates the Secretion of Apolipoprotein B100 and Very-Low-Density Lipoproteins.

Butkinaree C, Guo L, Ramkhelawon B, Wanschel A, Brodsky JL, Moore KJ, Fisher EA. Arterioscler Thromb Vasc Biol 2013 PMID: 24334870 DOI: 10.1161/ATVBAHA.113.302728

This article is important in that it extends our understanding of the molecular mechanisms governing the formation of large COPII vesicles at the endoplasmic reticulum (ER). Previously, the Rape lab has shown that the ubiquitin conjugation cofactor Klhl12 is required for the COPII-dependent formation of procollagen containing carriers {1}, likely through ubiquitylation of the outer layer COPII subunit Sec31. Here, Fisher and colleagues show that another atypically large cargo, apolipoprotein particles, also require Klhl12. While the paper does not fully explore the similarities or potential differences in mechanisms, the data do support a role for Klhl12 more generally in the export of unusually large cargo from the ER.

References

Ubiquitin-dependent regulation of COPII coat size and function.

Jin L, Pahuja KB, Wickliffe KE, Gorur A, Baumgärtel C, Schekman R, Rape M. Nature 2012 Feb 23; 482(7386):495-500 PMID: 22358839 DOI: 10.1038/nature10822

 

Microtubules that form the stationary lattice of muscle fibers are dynamic and nucleated at Golgi elements.

Oddoux S, Zaal KJ, Tate V, Kenea A, Nandkeolyar SA, Reid E, Liu W, Ralston E. J Cell Biol 2013 Oct 28; 203(2):205-13 PMID: 24145165 DOI: 10.1083/jcb.201304063

This article shows that static Golgi elements in flexor digitorum brevis (FDB) muscle fibers organize the microtubule network. The surprising finding is that this is not through a Golgi-nucleated microtubule mechanism involving proteins such as AKAP450 and GCC185 but instead through centrosomal proteins pericentrin and gamma-tubulin.
These experiments stemmed from the observation of microtubule bundles in these cells where an apparently stable microtubule lattice was seen to be decorated with dynamic EB3 puncta along their length. EB3 is known to only decorate growing microtubule ends. This was then explained through the good use of super-resolution microscopy (in this case gated stimulated emission depletion [GSTED]). Here, Oddoux and colleagues showed the presence of microtubule bundles emanating from clearly definable hubs. These hubs turned out to be Golgi elements, nucleating new microtubules which then grew alongside pre-existing ones. These in vitro data are also supported by in vivo data from intravital imaging. These experiments convincingly demonstrate the relevance of these findings in vivo.

The data are very carefully quantified and present a compelling case. The story adds nicely to our understanding of the relationship between the Golgi and centrosomal proteins in an interesting and important model system. The authors are now also well placed to determine the relevance of this organization and these mechanisms to Duchenne muscular dystrophy where loss of microtubule organization is thought to underlie the disease pathology {1}.

References

Microtubules underlie dysfunction in duchenne muscular dystrophy.

Khairallah RJ, Shi G, Sbrana F, Prosser BL, Borroto C, Mazaitis MJ, Hoffman EP, Mahurkar A, Sachs F, Sun Y, Chen YW, Raiteri R, Lederer WJ, Dorsey SG, Ward CW. Sci Signal 2012; 5(236):ra56  PMID: 22871609 DOI: 10.1126/scisignal.2002829

 

Asymmetric inheritance of centrosome-associated primary cilium membrane directs ciliogenesis after cell division.

Paridaen JTML, Wilsch-Bräuninger M, Huttner WB. Cell 2013 Oct 10; 155(2):333-44 PMID: 24120134 DOI: 10.1016/j.cell.2013.08.060

This is one of those papers that has such a simple underpinning observation that one wonders how it has not been seen before. The authors identify an Arl13b-positive membrane that is endocytosed from the primary cilium as the cell enters mitosis. This associates selectively with the mother centriole and gives this a head-start in terms of ciliogenesis and therefore in the ability to respond to sonic hedgehog signals. The paper demonstrates this in a number of different ways, both in vitro and in vivo, and shows some evidence for the importance of this asymmetric ciliogenesis in stem cell function.
Many questions remain as a result of this work. The evidence here strongly supports a model where this internalized Arl13b-positive membrane acts as a primer for ciliogenesis. Not only will it be important to define how this selective association occurs but whether and how selective biosynthetic trafficking to this compartment reinforces this process to drive subsequent cilia formation on exit from mitosis. I strongly recommend a look at the accompanying mini-review from Hoerner and Stearns as well {1}.

References

Remembrance of cilia past. Hoerner C, Stearns T. Cell 2013 Oct 10; 155(2):271-3 PMID: 24120128 DOI: 10.1016/j.cell.2013.09.027

 

Cerebral organoids model human brain development and microcephaly.

Lancaster MA, Renner M, Martin CA, Wenzel D … Homfray T, Penninger JM, Jackson AP, Knoblich JA.  Nature 2013 Sep 19; 501(7467):373-9 PMID: 23995685 DOI: 10.1038/nature12517

This is an outstanding manuscript that demonstrates not only the development of a system to model the human brain in vitro but also then applies this technique to prove that it can model complex human brain diseases. The authors develop what they term ‘cerebral organoids’ through differentiation of human pluripotent stem cells in a 3D culture system. They also develop organoids from patient-derived stem cells to show that the observed microcephaly caused by mutation in CDK5RAP2 is recapitulated in this system. The microcephaly phenotype is also evident after short hairpin RNA (shRNA) knockdown of CDK5RAP2, further demonstrating the applicability of this method. This paper provides an outstanding example for teaching purposes and of course will likely be widely applied to human developmental brain disorders.

 

A conserved role for atlastin GTPases in regulating lipid droplet size.

Klemm RW, Norton JP, Cole RA, Li CS … Farese RV Jr, Blackstone C, Guo Y, Mak HY.  Cell Rep 2013 May 30; 3(5):1465-75 PMID: 23684613 DOI: 10.1016/j.celrep.2013.04.015

The concept that atlastin is important for lipid metabolism raises new questions about the way that mutations in this protein cause human disease. The paper also points to a key link between endoplasmic reticulum morphology and lipid droplet biogenesis, which could have important implications for many other studies in this field.

 

ER exit sites are physical and functional core autophagosome biogenesis components.

Graef M, Friedman JR, Graham C, Babu M, Nunnari J. Mol Biol Cell 2013 PMID: 23904270 DOI: 10.1091/mbc.E13-07-0381

This paper convincingly identifies a key role for endoplasmic reticulum (ER) exit sites (ERESs) in autophagosome formation in Saccharomyces cerevisiae. These data show that autophagosomes form in close proximity to these specialised invaginations of the ER where COPII vesicles assemble.

Using a broad-ranging forward proteomics approach, Graef et al. defined multiple protein-protein interactions between autophagy proteins and vesicle trafficking proteins, most notably those that comprise the COPII coat. Key interactions were defined between COPII and proteins of the PI3-kinase and Atg9 complexes that initiate autophagosome formation. It is notable that the Sec23 subunit of the COPII coat appears to be at the hub of this network of interactions. This subunit also interacts with other key cellular machineries directing multiple functions of COPII and ensuring a directional flow to traffic (see {1}). Fluorescence imaging showed that early autophagosome structures formed adjacent to ERESs. Consistent with a key role for ERESs in this process, inhibiting ERES function using a temperature-sensitive form of Sec12 inhibited autophagic flux.

The authors conclude that ERESs act in the mechanical process of ERS formation and suggest that one role could be to tether the nascent autophagosome as it expands. While it is not yet clear exactly how ERESs function in autophagosome formation, the work places ERESs as acting downstream of Atg1 kinase complex localisation and speculate that ERESs provide membrane, and/or somehow scaffold, autophagosome assembly.

Interestingly, the authors extended their findings to higher eukaryotic cells, showing similar co-localisation between autophagy components and ERESs (labelled with mCherry-Sec16B) in Cos-7 cells. Exactly how ERESs direct autophagosome assembly, the identity of the proposed ERES-tether complex, and how the autophagosome is eventually released from its association with the ER remain to be defined.

References

Coordination of COPII vesicle trafficking by Sec23. Fromme JC, Orci L, Schekman R. Trends Cell Biol 2008 Jul; 18(7):330-6 PMID: 18534853 DOI: 10.1016/j.tcb.2008.04.006

 

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: Synergy between XMAP215 and EB1 increases microtubule growth rates to physiological levels

I made some comments on this article for F1000Prime. The original evaluation can be found at: http://f1000.com/prime/718009660
t
he DOI for the original post is 10.3410/f.718009660.793476858

M Zanic, PO Widlund, AA Hyman and J Howard (2013) Synergy between XMAP215 and EB1 increases microtubule growth rates to physiological levels.
Nat Cell Biol  DOI: 10.1038/ncb2744
PMID: 23666085

This article provides clear insight into how microtubule polymerization rates are achieved in vivo. Rates from assays of in vitro microtubule polymerization are typically slow. Here, the authors achieve in vitro rates of up to 20 μm .min¯¹, which is around the highest that is seen in live cells. The key seems to be an allosteric interaction between the microtubule polymerase XMAP215 and the plus-end binding protein EB1. It is interesting to see that this interaction is not a conventional EB1-directed plus-end binding event but is, in fact, allosteric with the combination of EB1 and XMAP215 promoting polymerization. Using mathematical modelling, the authors also suggest that this synergistic step is in the final addition of the tubulin dimer to the existing lattice, an isomerization step where a loosely bound dimer becomes tightly bound. This work also contrasts with other work reconstituting the Drosophila microtubule polymerization machinery which implicated another protein, Sentin, in this process of rapid microtubule polymerization {1}. The data presented in this paper do not support a role for Sentin in the vertebrate system described here. The definition of the minimal components needed for rapid microtubule growth now provides the opportunity to integrate other components of the regulatory network controlling microtubule dynamics to provide a complete picture of the control of this process.

References:
{1} Reconstitution of dynamic microtubules with Drosophila XMAP215, EB1, and Sentin.
Li W, Moriwaki T, Tani T, Watanabe T, Kaibuchi K, Goshima G.
J Cell Biol 2012 Nov 26; 199(5):849-62PMID: 23185033 DOI: 10.1083/jcb.201206101