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.

PhD studentship opportunity in our lab

We have a potential PhD studentship available to start in October 2013.

This studentship is part of the MRC Doctoral Training Programme at the University of Bristol. As such you apply to the Programme and the selection process is in competition with all other advertised projects. You can find full details on the programme, including the other available projects here:

MRC Doctoral Training Programme at the University of Bristol.

This is an exciting inter-disciplinary project bridging ongoing work in Biochemistry and Physics. The project would suit a graduate in biochemistry, cell biology, or biophysics. Full training will be provided and the training elements can be tailored to the interests of the successful candidate.

Please note the eligibility criteria: Only applicants from the UK/EU are eligible for this programme.

Title: Analysis Of Integration Between Membrane And Cytoskeleton Dynamics Using Advanced Light Microscopy

Supervisors: Professor David Stephens (Biochemistry) & Dr Henkjan Gersen (Physics)

To apply for this project please select ‘Faculty of Medical and Veterinary Sciences’ and ‘Biochemistry (PhD)(4-yr)’. Please also identify ‘MRCDTG’ as your fee payer in the Funding section of the online application.

The intricate relationship between endomembranes and cytoskeletal filaments governs the spatial organization, morphology, and function or organelles. Multiple cellular functions that coalesce around Golgi membranes are governed by small GTPases of the Rho family, Cdc42 being the most significant Rho GTPase at the Golgi (1). Recent years have seen the emergence of the septins as a critical component of this system; Cdc42 is known to dictate septin filament organization (2). Septin filaments act in concert with microtubules to direct trafficking around the Golgi (3). Septins also dictate the formation and function of primary cilia, a “cellular antenna” that integrates key signalling pathways essential to normal organism development and tissue function (4, 5, 6). Through selective disruption of Cdc42, Golgi, or septin function, we will define how the classical structure of the Golgi apparatus is defined by septin filaments and vice versa.

Septins adopt a highly conserved structural organization within filaments that can be detected by polarization fluorescence microscopy (7, 8), allowing the subunit architecture of septin filaments to be analysed in an intact cell context. This advanced bioimaging approach will form a core training aspect of the work and would suit a biomedical science graduate with a keen interest in imaging or a biophysics graduate with a strong interest in cell biology. The project bridges the Biochemistry and Physics departments at the University of Bristol. You would be based in the Stephens lab in the School of Biochemistry within newly refurbished cell biology laboratories and the project will involve considerable mammalian cell biology using gene silencing and advanced light microscopy. The Gersen lab, located a short distance away, will provide training in development and application of novel optical microscopy methods, notably fluorescence polarization. Successful PhD training is ensured through links to existing cell biology and nanoscience students in both labs as well as international collaboration.

Informal enquires to David Stephens (david.stephens@bristol.ac.uk) or Henkjan Gersen (H.Gersen@bristol.ac.uk) are welcome.

For further details see:

http://www.bristol.ac.uk/biochemistry/stephens/index.html

http://www.bristol.ac.uk/physics/people/henkjan-gersen/index.html

References

• S. Etienne-Manneville, Cdc42–the centre of polarity. J. Cell Sci. 117, 1291 (Mar 15, 2004).
• G. Joberty et al., Borg proteins control septin organization and are negatively regulated by Cdc42. Nat. Cell Biol. 3, 861 (Oct, 2001).
• E. T. Spiliotis, Regulation of microtubule organization and functions by septin GTPases. Cytoskeleton 67, 339 (Jun, 2010).
• Q. Hu et al., A septin diffusion barrier at the base of the primary cilium maintains ciliary membrane protein distribution. Science 329, 436 (Jul 23, 2010).
• J. R. Bowen, D. Hwang, X. Bai, D. Roy, E. T. Spiliotis, Septin GTPases spatially guide microtubule organization and plus end dynamics in polarizing epithelia. J. Cell Biol. 194, 187 (Jul 25, 2011).
• E. T. Spiliotis, S. J. Hunt, Q. Hu, M. Kinoshita, W. J. Nelson, Epithelial polarity requires septin coupling of vesicle transport to polyglutamylated microtubules. J. Cell Biol. 180, 295 (Jan 28, 2008).
• B. S. DeMay et al., Septin filaments exhibit a dynamic, paired organization that is conserved from yeast to mammals. The Journal of cell biology 193, 1065 (Jun 13, 2011).
• S. A. Rosenberg, M. E. Quinlan, J. N. Forkey, Y. E. Goldman, Rotational Motions of Macromolecules by Single-Molecule Fluorescence Microscopy, . Accounts of Biochemical Research 38, 583 (2005).

Potential applicants are encouraged to contact David when applying.

The deadline for applications is Wednesday 16th January 2013 and interviews are likely to be in the weeks of 11th February and 18th February 2013.

Evalutaion of “SUMOylation of the small GTPase ARL-13 promotes ciliary targeting of sensory receptors”

I evaluated this article for F1000 Prime

Y Li, Q Zhang, Q Wei, Y Zhang, K Ling and J Hu (2012)
SUMOylation of the small GTPase ARL-13 promotes ciliary targeting of sensory receptors.
J Cell Biol. 2012 Nov 12; 199(4): 589-98. PMID: 23128241 DOI: 10.1083/jcb.201203150

This paper is intriguing because it shows that SUMOylation of a very small pool of the small GTPase Arl13 is required for trafficking of some receptors into primary cilia. Arl13 is essential for cilia function, with mutations in Arl13 leading the Joubert syndrome. Arl13 is required for the formation of primary cilia (ciliogenesis) and also for the trafficking of certain receptors into the cilium. Here, the authors show that SUMOylation of Arl13 is not required for ciliogenesis itself, but is required for the trafficking of cargo, including polycystin-2, to the cilium once formed. The mechanistic basis of this role remains to be determined. The fact that only a very small pool of Arl13 is SUMOYlated at any one time will complicate analysis, but also hints at either a highly specialized pathway or, perhaps more likely, a very dynamic SUMOylation/deSUMOylation pathway being in operation.

Evaluated: The Structure of Sec12 Implicates Potassium Ion Coordination in Sar1 Activation.

The Structure of Sec12 Implicates Potassium Ion Coordination in Sar1 Activation.
McMahon C, Studer SM, Clendinen C, Dann GP, Jeffrey PD, Hughson FM. (2012)
J Biol Chem. in press. PMID: 23109340 DOI: 10.1074/jbc.M112.420141

This article caught my eye for two reasons: first, it maps at high resolution the likely interface between the guanine nucleotide exchange factor Sec12 and the small GTP binding protein Sar1. This interaction is critical for Sar1 activation, the essential precursor to COPII vesicle formation at the endoplasmic reticulum. Second, and where this paper is really worth a look is in the finding that this “K loop” on Sec12 that contacts Sar1 also binds a potassium ion. Furthermore, this K+ ion is essential for the catalytic activity of Sec12 towards Sar1. This paper provides a great example of the value of crystallography on proteins for which there is a very strongly predicted structure.

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 mitosis^{2, 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 dynein^{9, 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.