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.

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). 

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. 

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

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.

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.

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 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.

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.

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.

Experimental detail:

Cells plated 24h before transfection and imaged 18h after that (so a total of 42h from plating to imaging).

Transfection was done using 1 microgram of DNA and Lipofectamine 2000 (standard protocol, premix LF2000 with Optimem for 5 mins then mix with DNA and 15 mins later drop carefully on to cells). GFP-Rab8a cDNA was a kind gift from Johan Peränen (Helsinki, Finland; construct described in Hattula, K., Furuhjelm, J., Tikkanen, J., Tanhuanpaa, K., Laakkonen, P., and Peranen, J., 2006. Characterization of the Rab8-specific membrane traffic route linked to protrusion formation. J. Cell Sci. 119, 4866-4877).

Cells were grown in complete medium throughout and imaged in DMEM supplemented with HEPES and sodium bicarbonate but in the absence of serum or phenol red. Over these short times, serum withdrawal does not seem to change ciliation of these cells – they grow cilia in complete medium on reaching confluence. Imaging was done on a wide-field microscope and hopefully these movies include scale bars and time stamps.

The videos are hosted by YouTube and are available through the the links below.

Movies:

1. Tubular network visible below the primary cilium.

Is this a subset of endosomes? Part of the ER? Part of the cilium itself e.g. an expanded pre-ciliary vesicle? I wonder if this could be an expanded Rab11/Rab8 compartment as implicated by many papers including those of Chris Westlake and colleagues (. ).

2. Unusual looping out from base of cilia.

This looks odd, 3D reconstructions suggests that this “object” is indeed emerging from the base of the cilium and then seemingly heading back in again. Could this be something related to intraflagellar transport (IFT)? I need to read more on the way that Rab8 is trafficked to, and within, the cilium.

The next three are more just observations of things that one might expect to see.

3. Long wavy cilia.

This is a maximum intensity projection of a z-stack where the cilium is showing some substantial movements – not entirely sure how to describe it – we aren’t doing anything to these cells so this is really just diffusive/random motion. These are of course non-motile cilia so it isn’t beating. Just looks good! A key caveat is of course the speed of imaging versus the speed of movement.

4. 3-part cilia?

This video shows some retraction of a ling cilium, presumably induced by us imaging it (i.,e. photodamage). The interesting parts are the apparent 3-part labelling of the structure at the start of the movie suggesting a difference in thickness or accumulation of GFP-Rab8a along the length. The final structure is something we often see around the dish of cells – presumably “damaged” cilia. They are long and fragile in these in vitro cultures.

5. Cilia wrapping around one another

Here we see several cilia on adjacent cells interacting with one another. This seems similar to what the Lippincott-Schwartz lab has recently described in the journal Cilia:

Primary cilia utilize glycoprotein-dependent adhesion mechanisms to stabilize long-lasting cilia-cilia contacts Carolyn Ott, Natalie Elia, Suh Jeong, Christine Insinna, Prabuddha Sengupta, Jennifer Lippincott-Schwartz Cilia 2012, 1:3

It is hard from our video to define individual cilia or to determine the extent or effect of any interaction. As always it is hard to capture this to video – looking down the microscope, these structures clearly look like cilia and are certainly on top of the cells so do not represent the tubular network seen in video 1.

So, thoughts please. Comments here preferred but I’ll check YouTube as well.

PS Any alternative to this method of posting or problems people encounter would be welcome. Would this be better on Figshare? I haven’t explored that yet and these aren’t really figures, just observations on which I would welcome feedback.

If you got this far, thanks for checking my first contribution to the brave new world of Open Science also known as “crowd sourcing better understanding”!

David