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
the 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

Comments on: Structural basis for kinesin-1:cargo recognition.

I evaluated the following article for F1000Prime. The original evaluation is published at http://f1000.com/prime/717995214 . This evaluation has a DOI of 10.3410/f.717995214.793476314

Structural basis for kinesin-1:cargo recognition.
S Pernigo, A Lamprecht, RA Steiner and MP Dodding (2013)
Science 340(6130):356-9
PMID: 23519214
DOI: 10.1126/science.1234264

This paper provides a basis to modulate cargo-motor interactions with a high degree of selectivity. The paper from Dodding and colleagues describes a crystal structure of the tetratricopeptide (TPR)-repeat domain of kinesin light chain (KLC) 2 in association with cargo. Conventional kinesin is a heterotetramer consisting of two each of heavy chain and light chain subunits. Cargo binds to the light chains by virtue of a “tryptophan-acidic motif”.

The new structure was obtained by engineering an in-frame fusion of the cargo, in this case SKIP (SifA-kinesin interacting protein) from the pathogen Salmonella, to the light chain. The structure reveals a number of intriguing features, including an apparent conformational shift in the TPR domain upon cargo binding to enclose the critical tryptophan determinant. This same binding site is also used by a very different cargo, calsyntenin, indicating that this site could serve as a contact site for all kinesin-interacting cargo that displays this tryptophan-acidic motif.

Furthermore, the authors propose a model in which those cargoes, including SKIP, which present two canonical KLC binding motifs might act to bridge the two light chains found in the kinesin tetramer. Such a model immediately proposes a means by which high-affinity cargo binding could transmit a large conformational change through the helical domain of the heavy chain. Further work to determine the validity of this model is clearly a high priority.

Comments on: Silencing of mammalian Sar1 isoforms reveals COPII-independent protein sorting and transport

Vicky Miller (Postdoc in the Stephens lab) and I (David) have written the following comments on this paper that also appear on the Faculty of 1000 site. Vicky is also on Twitter @Dr_VickyMiller

Silencing of mammalian Sar1 isoforms reveals COPII-independent protein sorting and transport.

Cutrona MB, Beznoussenko GV, Fusella A, Martella O, Moral P, Mironov AA.

Traffic 2013; DOI: 10.1111/tra.12060 PMID: 23433038

This paper describes studies of trafficking of secretory cargo from the ER in cells depleted of the small GTPase Sar1, an essential component of the COPII coat. A COPII-independent mechanism of secretory trafficking would mark a step-change in our understanding of secretory protein trafficking in cells. While we do not think that this paper requires such a re-evaluation of our thinking, it does provide some thought provoking data. By disrupting the normal mechanism of transport the authors seek to reveal COPII-independent mechanisms and challenge the primacy of COPII-dependent transport. The most important point to bear in mind when reading this paper is that siRNA knock-downs are unable to generate a complete depletion of any protein and that residual Sar1 may influence some of the results seen. Indeed in our opinion, one cannot conclusively draw the conclusion that COPII-independent ER export pathways exist in cells from this work alone.

The authors confirm that Sar1-depletion effectively inhibits COPII vesicle formation (demonstrated by EM and reduced immunofluorescence staining of COPII components). They see changes to both ER-Golgi intermediate compartment (ERGIC) formation (the compartment between the Golgi and the ER) and Golgi organisation (resulting in formation of mini-stacks), also in agreement with a reduction in secretory trafficking. Despite this, pulse-chase experiments show no reduction in the total amount of protein secretion in Sar1-depleted cells. From this, the authors conclude that alternative transport mechanisms are being used. The virus glycoprotein VSV-G also passes from the ER to the plasma membrane in Sar1-depleted cells, even when additionalCOPII subunits (Sec23A and B) are depleted in addition to Sar1A and B. Based on these and other experiments, the authors propose a COPI-dependent replacement pathway that takes VSV-G to the Golgi. Export of procollagen transport is inhibited by Sar1-depletion, but as collagens require specialized COPII-coated vesicles to accommodate their large size, they therefore are not representative of typical COPII trafficking (Jin et al., 2012). Furthermore, many other studies support the notion that procollagen secretion is exquisitely sensitive to perturbation of the early secretory pathway (Smits et al., 2010; Townley et al., 2008; Venditti et al., 2012). 

These data do support the concept of a “short-loop” ER-to-Golgi trafficking pathway that involves juxtanuclear ER and possible even ER-Golgi contact sites. It is as yet unclear how possible mechanisms such as kiss and run could retainsufficient selectivity to prevent non-selective transport. It remains possible however that the small remaining amount of COPII proteins in the RNAi experiments described here is sufficient to direct COPII-dependent selectivity.

In summary the paper presents some interesting findings. Complete removal of Sar1 from cells by gene deletion is required to examine fully the existence ofCOPII-independent trafficking pathways and the relative importance of COPII-trafficking in cells. 

References

Jin, L., K.B. Pahuja, K.E. Wickliffe, A. Gorur, C. Baumgärtel, R. Schekman, and M. Rape. 2012. Ubiquitin-dependent regulation of COPII coat size and function. Nature 482(7386):495-500. doi: 10.1038/nature10822

Smits, P., A.D. Bolton, V. Funari, M. Hong, E.D. Boyden, L. Lu, D.K. Manning, N.D. Dwyer, J.L. Moran, M. Prysak, B. Merriman, S.F. Nelson, L. Bonafe, A. Superti-Furga, S. Ikegawa, D. Krakow, D.H. Cohn, T. Kirchhausen, M.L. Warman, and D.R. Beier. 2010. Lethal skeletal dysplasia in mice and humans lacking the golginGMAP-210. N. Engl. J. Med. 362:206-216.

Townley, A.K., Y. Feng, K. Schmidt, D.A. Carter, R. Porter, P. Verkade, and D.J. Stephens. 2008. Efficient coupling of Sec23-Sec24 to Sec13-Sec31 drives COPII-dependent collagen secretion and is essential for normal craniofacial development. J. Cell Sci. 121:3025-3034.

Venditti, R., T. Scanu, M. Santoro, G. Di Tullio, A. Spaar, R. Gaibisso, G.V. Beznoussenko, A.A. Mironov, A. Mironov, Jr., L. Zelante, M.R. Piemontese, A. Notarangelo, V. Malhotra, B.M. Vertel, C. Wilson, and M.A. De Matteis. 2012. Sedlin controls the ER export of procollagen by regulating the Sar1 cycle. Science. 337:1668-1672.

       

Full disclosure: David is a member of the “Traffic” editorial board but had not role in the editing or reviewing of this paper.

These comments are also published on F1000 Prime

Comments on: The Microtubule-Binding Protein Ensconsin Is an Essential Cofactor of Kinesin-1.

I evaluated the following article for F1000 Prime.

The Microtubule-Binding Protein Ensconsin Is an Essential Cofactor of Kinesin-1.

Barlan K, Lu W, Gelfand VI.

Curr Biol 2013 PMID: 23394833 DOI: 10.1016/j.cub.2013.01.008

This paper defines the microtubule binding protein ensconsin as an obligate co-factor for kinesin-1 drive in motility in Drosophila. In a series of elegant experiments, the authors show that ensconsin is not required for the recruitment of kinesin to membranes but is in some way involved in activating the motor. Ensconsin does not seem to affect the amount of membrane-bound kinesin in cells so the authors sought to define its role in motor activation. Kinesin-1 normally exists in an auto-inhibited confirmation in cells. In a key experiment the authors showed that removal of this auto-inhibition within the kinesin-1 motor by mutation eliminates the requirement for ensoconsin in vivo. These data suggest a model in which ensconsin acts to relieve the auto-inhibition. An important point is that this function of ensconsin did not require its own microtubule binding activity. The authors postulate that spatial restriction of ensconsin to microtubules acts to refine the spatial activation of kinesin-1, adding to the diversity of mechanisms that control the spatial and temporal organization of microtubule motor activation.

Note: Ensconsin is also known as E-MAP-115 and MAP7.

These comments also appear on Faculty of 1000 Prime

Evaluation: A pseudoatomic model of the COPII cage

I evaluated this paper for F1000 Prime:

Noble AJ, Zhang Q, O’Donnell J, Hariri H, Bhattacharya N, Marshall AG, Stagg SM. (2013) A pseudoatomic model of the COPII cage obtained from cryo-electron microscopy and mass spectrometry. Nature Structural and Molecular Biology 20(2):167-73

This paper describes a technical tour de force that elucidates some of the finer detail of the molecular structure of the assembled COPII coat. The Stagg lab have obtained a 12-Å structure of the human COPII cage from cryo-electron microscopy and layered on top of this data from hydrogen deuterium exchange (HDX) experiments to define the flexible regions of the assembled structure. The structure was made possible in part by a neat gradient fixation protocol to isolate assembled cages from aggregated material (GraFix, described in (Kastner et al., 2008)). Molecular dynamics flexible fitting of the previous crystallographic structure of the Sec13-31 complex to the EM data provided clear insight into the formation of the vertex elements of the assembled coat. Specifically, the authors demonstrate that Sec13-Sec31 unit has an intrinsic “polarity” within the assembled coat with one end tightly packed and the pother more loosely integrated. This resulted in the identification of a further contact site at the vertex region that reveals a less significant role for Sec13 and a greater contact area through Sec31 than has been previously suggested (Stagg et al., 2008). This has the potential to explain data that suggest that the requirement for Sec13 in vivo is not as stringent as one might expect ((Copic et al., 2012; Townley et al., 2008)). The loose packing evident within the edge element of the Sec31 alpha-solenoid could flex to accommodate unusually large cargo. Overall, this paper is impressive from a technical perspective as well as for the insight it provides into COPII assembly.

Copic, A., C.F. Latham, M.A. Horlbeck, J.G. D’Arcangelo, and E.A. Miller. 2012. ER cargo properties specify a requirement for COPII coat rigidity mediated by Sec13p. Science. 335:1359-1362.

Kastner, B., N. Fischer, M.M. Golas, B. Sander, P. Dube, D. Boehringer, K. Hartmuth, J. Deckert, F. Hauer, E. Wolf, H. Uchtenhagen, H. Urlaub, F. Herzog, J.M. Peters, D. Poerschke, R. Luhrmann, and H. Stark. 2008. GraFix: sample preparation for single-particle electron cryomicroscopy. Nature methods. 5:53-55.

Stagg, S.M., P. LaPointe, A. Razvi, C. Gurkan, C.S. Potter, B. Carragher, and W.E. Balch. 2008. Structural basis for cargo regulation of COPII coat assembly. Cell. 134:474-484.

Townley, A.K., Y. Feng, K. Schmidt, D.A. Carter, R. Porter, P. Verkade, and D.J. Stephens. 2008. Efficient coupling of Sec23-Sec24 to Sec13-Sec31 drives COPII-dependent collagen secretion and is essential for normal craniofacial development. J. Cell Sci. 121:3025-3034.

Art competition entries 2012

I entered a few of my images into our Faculty Art Competition this year. They didn’t win but I thought I would post them here anyway.

Click on the images for a larger view.

You can see the winners here.

3D rendering of ciliated cells. LLC-PK1 (pig kidney epithelial) cells were grown on a round micropattern to constrain growth. Cilia are in green with the Golgi in magenta and nuclei in blue. The image is a 3D rendering of a deconvolved z-series acquired using widefield microscopy.

A pseudocoloured image of the jellyfish Aequorea victoria from which Green Fluorescent Protein was isolated. The pseudocolouring illustrates the diverse colour palette of GFP variants that we now have available.
The image is a photograph taken by me at Monterey Bay aquarium in 2003. There is only one jellyfish in the original photo, this image is a montage.

Article evaluation: Synthetic cell biology

I have evaluated the following article for F1000 Prime.

This article was published last year but has recently come to my attention again.

Lo Presti L, Martin SG. (2011) Shaping fission yeast cells by rerouting actin-based transport on microtubules. Curr Biol  Dec 20; 21(24):2064-9 PMID: 22137473 DOI: 10.1016/j.cub.

This is an elegant study that identifies remarkable plasticity in the cytoskeletal networks of fission yeast. S. pombe normally requires both actin filaments and microtubules for polarized growth. A key role of the actin network is the polarized delivery of the Rab11 orthologue Ypt3p. The authors generated a synthetic motor to re-route myosin V cargo (Ypt3p) to microtubules and showed that this chimera could restore polarized growth to cells lacking myosin V.

This paper showcases the utility of synthetic cell biology to further our understanding of fundamental cell biological processes. For those short of time, I also recommend the accompanying video abstract.